Modular armadillo repeat proteins

Abstract

The present invention relates to Armadillo Repeat proteins with a specificity for a particular amino acid or a particular amino-acid-motif.

Description

[0001] Modular Armadillo Repeat Proteins

[0002] This application claims the right of priority of European Patent Application EP23200965.4 filed 29 September 2023, which is incorporated by reference herein.

[0003] Field

[0004] The present invention relates to Armadillo Repeat proteins with a specificity for a particular amino acid or a particular amino-acid-motif.

[0005] Specific binding proteins are a central pillar of biological and biomedical research due to their application as detection agents in a wide variety of different assays, ranging from western blots, ELISA, immunohistochemistry assays, to affinity chromatography and many more. Historically, antibodies are the most commonly used agents, and the majority of them outside of therapy are still created by immunization. Polyclonal antibodies suffer from batch-to- batch variability, but even monoclonal antibodies show cross-reactivities, clones can be lost, and they are usually not molecularly defined by the sequence. Besides recombinant antibodies that are selected from libraries, over the last decade many specially engineered scaffolds have been brought forward that by design do not contain disulfides or glycosylation sites and can thus be expressed in E. coli inexpensively, and are by definition sequence-defined and monoclonal, thereby greatly enhancing binder production and quality. However, even with these new scaffolds, binders have to be selected de novo individually for each new target, significantly limiting the throughput and speed at which those binders can be generated to new targets. To overcome this remaining issue, we propose the use of modular binders.

[0006] Modular binders are built from custom modules that recognize subsections of an epitope and are assembled to generate a binder specific for the full epitope. Given a pre-existing set of modules, binders can be rapidly assembled according to the desired target sequence. The concept of modular binding is especially well suited to generate binders against linear epitopes, since the prediction of secondary or tertiary structure elements at an atomic level is still difficult and the diversity of structured epitopes renders a modular approach impractical. Nevertheless, in many of the desired applications, the target proteins will by default be detected in a denatured state (e.g. WBs, Proteomics) and, considering that also correctly folded proteins often contain unstructured parts in loops or termini, the range of possible applications for linear peptide binders is very broad. It was also found over the last decade that up to 15-40 % of all cellular interactions are actually protein-peptide interactions, being involved in essential tasks such as signaling, regulation of cell adhesion or protein trafficking. For simplicity, we will refer to linear epitopes as peptides, even if they are part of a folded protein.

[0007] Repeat proteins represent a viable starting point for the design of modular peptide binders. The binding interface of designed Armadillo repeat proteins (dArmRPs) is formed by the juxtaposition of its repetitive subunits (modules), naturally resulting in a modular interaction with its extended peptide ligand. Iterative cycles of engineering and design based on a consensus-derived initial scaffold resulted in a designed ArmRP (dArmRP), with generic internal modules retaining all the peptide-binding features while being “stackable” to form binders of varying length. Furthermore, the consensus dArmRP scaffold has been shown to bind its designated (KR)ntarget peptide in a highly modular fashion. Moreover, the overall affinity can be adjusted over many orders of magnitude by altering the length of the protein and the peptide.

[0008] Based on the above-mentioned state of the art, the objective of the present invention is to provide individual amino-acid-residue-binding pockets as part of an Armadillo repeat protein. This objective is attained by the subject-matter of the independent claims of the present specification, with further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification.

[0009] Summary of the Invention

[0010] A first aspect of the invention relates to a polypeptide sequence which comprises armadillo repeat protein units, and which confers a certain binding specificity to a particular amino acid residue.

[0011] A further aspect of the invention relates to an armadillo repeat protein comprising the polypeptide sequence according to the first aspect.

[0012] A further aspect of the invention relates to a polypeptide domain comprising the polypeptide sequence according to the first aspect.

[0013] A further aspect of the invention relates to a binding motif comprising the polypeptide sequence according to the first aspect.

[0014] Terms and definitions

[0015] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control. The terms “comprising”, “having”, “containing”, and “including”, and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open-ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of’ or “consisting of.”

[0016] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0017] Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

[0018] As used herein, including in the appended claims, the singular forms “a”, “or” and “the” include plural referents unless the context clearly dictates otherwise.

[0019] "And / or" where used herein is to be taken as specific recitation of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, organic synthesis). Standard techniques are used for molecular, genetic, and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.

[0021] Any patent document cited herein shall be deemed incorporated by reference herein in its entirety. Sequences

[0022] Sequences similar or homologous (e.g., at least about 70% sequence identity) to the sequences disclosed herein are also part of the invention. In some embodiments, the sequence identity at the amino acid level can be about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. At the nucleic acid level, the sequence identity can be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Alternatively, substantial identity exists when the nucleic acid segments will hybridize under selective hybridization conditions (e.g., very high stringency hybridization conditions), to the complement of the strand. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.

[0023] In the context of the present specification, the terms sequence identity and percentage of sequence identity refer to a single quantitative parameter representing the result of a sequence comparison determined by comparing two aligned sequences position by position. Methods for alignment of sequences for comparison are well-known in the art. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988) or by computerized implementations of these algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information (http: / / blast.ncbi.nlm.nih.gov / ).

[0024] One example for comparison of amino acid sequences is the BLASTP algorithm that uses the default settings: Expect threshold: 10; Word size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence 11 , Extension 1 ; Compositional adjustments: Conditional compositional score matrix adjustment. One such example for comparison of nucleic acid sequences is the BLASTN algorithm that uses the default settings: Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match / Mismatch Scores: 1.-2; Gap costs: Linear. Unless stated otherwise, sequence identity values provided herein refer to the value obtained using the BLAST suite of programs (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) using the above identified default parameters for protein and nucleic acid comparison, respectively.

[0025] Reference to identical sequences without specification of a percentage value implies 100% identical sequences (i.e. the same sequence).

[0026] General Biochemistry: Peptides, Amino Acid Sequences

[0027] The term polypeptide in the context of the present specification relates to a molecule consisting of 50 or more amino acids that form a linear chain wherein the amino acids are connected by peptide bonds. The amino acid sequence of a polypeptide may represent the amino acid sequence of a whole (as found physiologically) protein or fragments thereof. The term "polypeptides" and "protein" are used interchangeably herein and include proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences.

[0028] The term peptide in the context of the present specification relates to a molecule consisting of up to 50 amino acids, in particular 8 to 30 amino acids, more particularly 8 to 15 amino acids, that form a linear chain wherein the amino acids are connected by peptide bonds.

[0029] Amino acid residue sequences are given from amino to carboxyl terminus. Capital letters for sequence positions refer to L-amino acids in the one-letter code (Stryer, Biochemistry, 3rded. p. 21). Lower case letters for amino acid sequence positions refer to the corresponding D- or (2R)-amino acids. Sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (lie, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Vai, V). In certain embodiments, reference to “any” amino acid means that this amino acid is a proteinogenic amino acid.

[0030] The term armadillo repeat protein refers to a polypeptide comprising at least one armadillo repeat, wherein an armadillo repeat is characterized by helices a, b, and c which are connected via loop regions.

[0031] Detailed Description of the Invention

[0032] Objective of the invention

[0033] The aim of the modular binder technology is to provide on-demand binders for user specified epitopes. This could significantly enhance the scientific output in biology and medicine- related fields. A modular Armadillo repeat scaffold has been built, where the modularity of the binder matches the target peptide sequence. Individual binding pockets are being designed to be specific for individual amino acids on the target that can later be combined. Thus, with an existing set of binding pockets in place it will be possible to assemble an affinity reagent for a specific target sequence in a very short time. Apart from slight adaptations at the pocket interfaces no further experimental selections and computational optimizations are necessary during the assembly of new sequence specific binding proteins. This fundamentally new concept allows one to bind linear target sequences in an unfolded state. In contrast to conventional approaches, a modular binder can be designed in silica from individual modules to, in principle, recognizing any desired linear epitope.

[0034] Armadillo structure

[0035] A first aspect of the invention relates to a polypeptide sequence which comprises armadillo repeat protein units, and which confers a certain binding specificity to a particular amino acid residue. Each repeat unit comprises from N-terminus to C-terminus three helices a, b, and c. The helices a and b are connected via a loop a / b, and the helices b and c are connected via a loop b / c, and wherein two armadillo repeats are connected via a loop c / a.

[0036] X residues

[0037] Tyr-binding pocket

[0038] In a first sub-aspect of the invention, the polypeptide sequence is capable of providing Tyr- residue-binding specificity to an armadillo repeat protein sequence, and the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below, with X not included in calculating identity (which means that zero, or one or two amino acids of the variable B residues and of the constant residues may be exchanged for a different amino acid):

[0039] • helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 001) with B1, B2, B3, B4, and B5being variable amino acids as defined below, I being isoleucine, V being valine, A being alanine;

[0040] • loop a1 / b1consists of a single glycine G;

[0041] • helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 002) with B6, B7, B8, and B9being variable amino acids as defined below, A being alanine, L being leucine, V being valine;

[0042] • loop b1 / c1consists of a sequence serine-proline SP;

[0043] • helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 003) with B10, B11, B12, and B13being variable amino acids as defined below, I being isoleucine, V being valine, A being alanine, and X11, X12, X13, X14, and X15conferring binding specificity and being defined inside the table below; and

[0044] • loop c1 / a2consists of 1 to 20 (particularly proteinogenic) amino acids;

[0045] • helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 004);

[0046] • loop a2 / b2consists of a single glycine G;

[0047] • helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 005);

[0048] • loop b2 / c2consists of a sequence serine-proline SP; • helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ ID NO 006);

[0049] • loop c2 / a3consists of 1 to 20 (particularly proteinogenic) amino acids; wherein X11-X25have the following definition (one line (row) is an alternative definition): wherein optionally, 0, 1 , or 2 (particularly 0 or 1) amino acids of X12, X14, X15, X21, X22, X23, X24may be exchanged for a different amino acid; wherein X11, X13, and X25each independently is any amino acid which allows for specific binding of a selected residue adjacent to the Tyr residue.

[0050] In certain embodiments, the polypeptide sequence is restricted to a sequence which is able to bind to a Tyr residue with a specificity factor of at least 2 in comparison to binding a different amino acid. The assay for determining the specificity factor is given in “Affinity determination by fluorescence polarization”.

[0051] Trp-binding pocket

[0052] In a second sub-aspect of the invention, the polypeptide sequence is capable of providing Trp-residue-binding specificity to an armadillo repeat protein sequence, and the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below, with X not included in calculating identity (which means that zero, or one or two amino acids of the variable B residues and of the constant residues may be exchanged for a different amino acid):

[0053] • helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 007);

[0054] • loop a1 / b1consists of a single glycine G;

[0055] • helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 008);

[0056] • loop b1 / c1consists of a sequence serine-proline SP;

[0057] • helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 009); and

[0058] • loop c1 / a2consists of 1 to 20 (particularly proteinogenic) amino acids • helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 010);

[0059] • loop a2 / b2consists of a single glycine G;

[0060] • helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 011);

[0061] • loop b2 / c2consists of a sequence serine-proline SP;

[0062] • helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ ID NO 012);

[0063] • loop c2 / a3consists of 1 to 20 (particularly proteinogenic) amino acids; wherein X11-X25have the following definition (one line (row) is an alternative definition): wherein optionally, 0, 1 , or 2 (particularly 0 or 1) amino acids of X12, X14, X15, X21, X22, X23, X24may be exchanged for a different amino acid; wherein X11, X13, and X25each independently is any amino acid which allows for specific binding of a selected residue adjacent to the Trp residue.

[0064] In certain embodiments, the polypeptide sequence is restricted to a sequence which is able to bind to a Trp residue with a specificity factor of at least 2 in comparison to binding a different amino acid. The assay for determining the specificity factor is given in “Affinity determination by fluorescence polarization”.

[0065] His-binding pocket

[0066] In a third sub-aspect of the invention, the polypeptide sequence is capable of providing His- residue-binding specificity to an armadillo repeat protein sequence, and the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below, with X not included in calculating identity (which means that zero, or one or two amino acids of the variable B residues and of the constant residues may be exchanged for a different amino acid):

[0067] • helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 013);

[0068] • loop a1 / b1consists of a single glycine G;

[0069] • helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 014);

[0070] • loop b1 / c1consists of a sequence serine-proline SP; • helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 015); and

[0071] • loop c1 / a2consists of 1 to 20 (particularly proteinogenic) amino acids

[0072] • helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 016);

[0073] • loop a2 / b2consists of a single glycine G;

[0074] • helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 017);

[0075] • loop b2 / c2consists of a sequence serine-proline SP;

[0076] • helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ ID NO 018);

[0077] • loop c2 / a3consists of 1 to 20 (particularly proteinogenic) amino acids; wherein X11-X25have the following definition (one line (row) is an alternative definition): wherein optionally, 0, 1 , or 2 (particularly 0 or 1) amino acids of X12, X14, X15, X21, X22, X23, X24may be exchanged for a different amino acid; wherein X11, X13, and X25each independently is any amino acid which allows for specific binding of a selected residue adjacent to the His residue.

[0078] In certain embodiments, the polypeptide sequence is restricted to a sequence which is able to bind to a His residue with a specificity factor of at least 2 in comparison to binding a different amino acid. The assay for determining the specificity factor is given in “Affinity determination by fluorescence polarization”. lie-binding pocket

[0079] In a fourth sub-aspect of the invention, the polypeptide sequence is capable of providing lle- residue-binding specificity to an armadillo repeat protein sequence, and the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below, with X not included in calculating identity (which means that zero, or one or two amino acids of the variable B residues and of the constant residues may be exchanged for a different amino acid):

[0080] • helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 019);

[0081] • loop a1 / b1consists of a single glycine G;

[0082] • helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 020);

[0083] • loop b1 / c1consists of a sequence serine-proline SP;

[0084] • helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 021); and

[0085] • loop c1 / a2consists of 1 to 20 (particularly proteinogenic) amino acids

[0086] • helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 022);

[0087] • loop a2 / b2consists of a single glycine G;

[0088] • helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 023);

[0089] • loop b2 / c2consists of a sequence serine-proline SP;

[0090] • helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ ID NO 024);

[0091] • loop c2 / a3consists of 1 to 20 (particularly proteinogenic) amino acids; wherein X11-X25have the following definition (one line (row) is an alternative definition): wherein optionally, 0, 1 , or 2 (particularly 0 or 1) amino acids of X12, X14, X15, X21, X22, X23, X24may be exchanged for a different amino acid; wherein X11, X13, and X25each independently is any amino acid which allows for specific binding of a selected residue adjacent to the lie residue. In certain embodiments, the polypeptide sequence is restricted to a sequence which is able to bind to an lie residue with a specificity factor of at least 2 in comparison to binding a different amino acid. The assay for determining the specificity factor is given in “Affinity determination by fluorescence polarization”.

[0092] Glu-binding pocket

[0093] In a fifth sub-aspect of the invention, the polypeptide sequence is capable of providing Glu- residue-binding specificity to an armadillo repeat protein sequence, and the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below, with X not included in calculating identity (which means that zero, or one or two amino acids of the variable B residues and of the constant residues may be exchanged for a different amino acid):

[0094] • helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 025) with B1 , B2 ... being variable amino acids as defined herein, I being isoleucine, V valine, A alanine;

[0095] • loop a1 / b1consists of a single glycine G;

[0096] • helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 026);

[0097] • loop b1 / c1consists of a sequence serine-proline SP;

[0098] • helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 027); and

[0099] • loop c1 / a2consists of 1 to 20 (particularly proteinogenic) amino acids

[0100] • helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 028);

[0101] • loop a2 / b2consists of a single glycine G;

[0102] • helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 029);

[0103] • loop b2 / c2consists of a sequence serine-proline SP;

[0104] • helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ ID NO 030);

[0105] • loop c2 / a3consists of 1 to 20 (particularly proteinogenic) amino acids; wherein X11-X25have the following definition (one line (row) is an alternative definition): wherein optionally, 0, 1 , or 2 (particularly 0 or 1) amino acids of X12, X14, X15, X21, X22, X23, X24may be exchanged for a different amino acid; wherein X11, X13, and X25each independently is any amino acid which allows for specific binding of a selected residue adjacent to the Glu residue.

[0106] In certain embodiments, the polypeptide sequence is restricted to a sequence which is able to bind to a Glu residue with a specificity factor of at least 2 in comparison to binding a different amino acid. The assay for determining the specificity factor is given in “Affinity determination by fluorescence polarization”.

[0107] Leu-binding pocket

[0108] In a sixth sub-aspect of the invention, the polypeptide sequence is capable of providing Leu- residue-binding specificity to an armadillo repeat protein sequence, and the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below, with X not included in calculating identity (which means that zero, or one or two amino acids of the variable B residues and of the constant residues may be exchanged for a different amino acid):

[0109] • helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 025) with B1 , B2 ... being variable amino acids as defined herein, I being isoleucine, V valine, A alanine;

[0110] • loop a1 / b1consists of a single glycine G;

[0111] • helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 026);

[0112] • loop b1 / c1consists of a sequence serine-proline SP;

[0113] • helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 027); and

[0114] • loop c1 / a2consists of 1 to 20 (particularly proteinogenic) amino acids

[0115] • helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 028);

[0116] • loop a2 / b2consists of a single glycine G;

[0117] • helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 029);

[0118] • loop b2 / c2consists of a sequence serine-proline SP;

[0119] • helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ ID NO 030); loop c2 / a3consists of 1 to 20 (particularly proteinogenic) amino acids; wherein X11-X25have the following definition (one line (row) is an alternative definition): wherein optionally, 0, 1 , or 2 (particularly 0 or 1) amino acids of X12, X14, X15, X21, X22, X23, X24may be exchanged for a different amino acid; wherein X11, X13, and X25each independently is any amino acid which allows for specific binding of a selected residue adjacent to the Leu residue.

[0120] In certain embodiments, the polypeptide sequence is restricted to a sequence which is able to bind to a Leu residue with a specificity factor of at least 2 in comparison to binding a different amino acid. The assay for determining the specificity factor is given in “Affinity determination by fluorescence polarization”.

[0121] Tyr-Z-Tyr-binding pocket

[0122] In a sixth sub-aspect of the invention, the polypeptide sequence is capable of providing Tyr- Z-Tyr-motif-binding specificity to an armadillo repeat protein sequence, and the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below, with X not included in calculating identity (which means that zero, or one or two amino acids of the variable B residues and of the constant residues may be exchanged for a different amino acid):

[0123] • helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 031);

[0124] • loop a1 / b1consists of a single glycine G;

[0125] • helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 032);

[0126] • loop b1 / c1consists of a sequence serine proline SP;

[0127] • helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 033); and

[0128] • loop c1 / a2consists of 1 to 20 (particularly proteinogenic) amino acids • helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 034);

[0129] • loop a2 / b2consists of a single glycine G;

[0130] • helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 035);

[0131] • loop b2 / c2consists of a sequence serine proline SP; • helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ

[0132] ID NO 036);

[0133] • loop c2 / a3consists of 1 to 20 (particularly proteinogenic) amino acids;

[0134] • helix a3consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 037); • loop a3 / b3consists of a single glycine G;

[0135] • helix b3consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 038);

[0136] • loop b3 / c3consists of a sequence serine proline SP;

[0137] • helix c3consists of a sequence B10B11X31B12B13X32X33ALX34ALX35NIA (SEQ ID NO 039); • loop c3 / a4consists of 1 to 20 (particularly proteinogenic) amino acids;

[0138] • wherein X11-X35have the following definition (one line (row) is an alternative definition): wherein optionally, 0, 1 , 2, 3 or 4 (particularly 0, 1 or 2, more particularly 0 or 1) amino acids of X12, X14, X15, X21, X22, X23, X24, X25, X31, X32, X33, X34may be exchanged for a different amino acid, wherein X11, X13, and X35each independently is any amino acid which allows for specific binding of a selected residue adjacent to the Tyr-Z-Tyr motif.

[0139] In certain embodiments, the polypeptide sequence is restricted to a sequence which is able to bind to a Tyr-Z-Tyr motif with a specificity factor of at least 4 in comparison to binding a different motif. The assay for determining the specificity factor is given in “Affinity determination by fluorescence polarization”.

[0140] Trp-Z-Trp-binding pocket

[0141] In a sixth sub-aspect of the invention, the polypeptide sequence is capable of providing Trp- Z-Trp -motif-binding specificity to an armadillo repeat protein sequence, and the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below, with X not included in calculating identity (which means that zero, or one or two amino acids of the variable B residues and of the constant residues may be exchanged for a different amino acid):

[0142] • helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 040);

[0143] • loop a1 / b1consists of a single glycine G;

[0144] • helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 041);

[0145] • loop b1 / c1consists of a sequence serine proline SP;

[0146] • helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 042); and

[0147] • loop c1 / a2consists of 1 to 20 (particularly proteinogenic) amino acids helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 043); • loop a2 / b2consists of a single glycine G;

[0148] • helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 044);

[0149] • loop b2 / c2consists of a sequence serine proline SP;

[0150] • helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ ID NO 045);

[0151] • loop c2 / a3consists of 1 to 20 (particularly proteinogenic) amino acids;

[0152] • helix a3consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 046);

[0153] • loop a3 / b3consists of a single glycine G;

[0154] • helix b3consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 047);

[0155] • loop b3 / c3consists of a sequence serine proline SP;

[0156] • helix c3consists of a sequence B10B11X31B12B13X32X33ALX34ALX35NIA (SEQ ID NO 048);

[0157] • loop c3 / a4consists of 1 to 20 (particularly proteinogenic) amino acids;

[0158] • wherein X11-X35have the following definition (one line (row) is an alternative definition): wherein optionally, 0, 1 , 2, 3 or 4 (particularly 0, 1 or 2, more particularly 0 or 1) amino acids of X12, X14, X15, X21, X22, X23, X24, X25, X31, X32, X33, X34may be exchanged for a different amino acid, wherein X11, X13, and X35each independently is any amino acid which allows for specific binding of a selected residue adjacent to the Trp-Z-Trp motif.

[0159] In certain embodiments, the polypeptide sequence is restricted to a sequence which is able to bind to a Trp-Z-Trp motif with a specificity factor of at least 4 in comparison to binding a different motif. The assay for determining the specificity factor is given in “Affinity determination by fluorescence polarization”. lle-Z-lle-binding pocket

[0160] In a sixth sub-aspect of the invention, the polypeptide sequence is capable of providing lle-Z- I le-motif-binding specificity to an armadillo repeat protein sequence, and the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below, with X not included in calculating identity (which means that zero, or one or two amino acids of the variable B residues and of the constant residues may be exchanged for a different amino acid):

[0161] • helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 040);

[0162] • loop a1 / b1consists of a single glycine G;

[0163] • helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 041);

[0164] • loop b1 / c1consists of a sequence serine proline SP;

[0165] • helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 042); and

[0166] • loop c1 / a2consists of 1 to 20 (particularly proteinogenic) amino acids

[0167] • helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 043);

[0168] • loop a2 / b2consists of a single glycine G;

[0169] • helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 044);

[0170] • loop b2 / c2consists of a sequence serine proline SP;

[0171] • helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ ID NO 045);

[0172] • loop c2 / a3consists of 1 to 20 (particularly proteinogenic) amino acids;

[0173] • helix a3consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 046);

[0174] • loop a3 / b3consists of a single glycine G;

[0175] • helix b3consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 047);

[0176] • loop b3 / c3consists of a sequence serine proline SP;

[0177] • helix c3consists of a sequence B10B11X31B12B13X32X33ALX34ALX35NIA (SEQ ID NO 048);

[0178] • loop c3 / a4consists of 1 to 20 (particularly proteinogenic) amino acids;

[0179] • wherein X11-X35have the following definition (one line (row) is an alternative definition): wherein optionally, 0, 1 , 2, 3 or 4 (particularly 0, 1 or 2, more particularly 0 or 1) amino acids of X12, X14, X15, X21, X22, X23, X24, X25, X31, X32, X33, X34may be exchanged for a different amino acid, wherein X11, X13, and X35each independently is any amino acid which allows for specific binding of a selected residue adjacent to the lle-Z-lle motif.

[0180] In certain embodiments, the polypeptide sequence is restricted to a sequence which is able to bind to a lle-Z-lle motif with a specificity factor of at least 4 in comparison to binding a different motif. The assay for determining the specificity factor is given in “Affinity determination by fluorescence polarization”.

[0181] Residues X11, X13, andX25 / X35

[0182] X11, X13, and X25or X35confer binding specificity to an armadillo repeat protein with respect to the residue adjacent to the claimed binding residue. Thus, X11, X13, and X25or X35can, in principle, be any amino acid. X11, X13, and X25or X35each independently is any amino acid which allows for specific binding of a selected residue adjacent to claimed amino acid or amino acid motif. The residue in the peptide that is two residues upstream or two residues downstream in the peptide sequence, and thus structurally adjacent on either side of the peptide residue whose binding pocket is being considered, can therefore restrict the choice of X11, X13, and X25or X35. In other words, an amino acid side chain that is two residues downstream in the peptide occupies the adjacent pocket upstream in the protein, because of the antiparallel binding orientation. The side chains defining this latter pocket will therefore define the identity of X11and X13. In an analogous manner, the identity of X15or X35, respectively, is defined by the requirements of the adjacent pocket downstream in the protein sequence, that binds the side chain upstream in the peptide sequence. Affinity determination by fluorescence polarization:

[0183] Affinities of dArmRPs containing the specified binding pockets to peptides containing the respective target amino acids fused to sfGFP (or mNeonGreen) were determined by fluorescence anisotropy on a Tecan Safire II plate reader equipped with a fluorescence polarization module. A fixed amount of 2 nM peptide-sfGFP was titrated with dArmRP in four replicates with 24 dilutions ranging from 160 pM to 20 pM. Excitation and emission wavelengths were set to 470 and 510 nm, respectively, using a bandwidth of 10 nm. The averages of four replicates were subtracted with the anisotropy obtained with the lowest dArmRP concentration and were fit to equation 1: where FAP is the fraction of bound peptide, CA is the concentration of dArmRP, CP is the fixed concentration of peptide, Kd is the dissociation constant, and m is the anisotropy amplitude between unbound and bound peptide.

[0184] B residues

[0185] The residues designated with “B” are residues which allow for a broader sequence variation while retaining the overall armadillo repeat protein structure. This is mainly due to solvent exposure of these residues. For all pockets described above, the B residues have one of the following definitions:

[0186] In certain embodiments, B1-B13have the following definition:

[0187] - B1: is selected from the group of K, D, E, N, S, P, Q;

[0188] - B2: is selected from the group of Q, N, E, D, P, S, A;

[0189] - B3: is selected from the group of K, Q, D, L, A, M;

[0190] - B4: is selected from the group of S, T, A, V, I, K, Y, L;

[0191] - B5: is selected from the group of N, D, Q, E, L;

[0192] - B6: is selected from the group of P, E, Q, K, S;

[0193] - B7: is selected from the group of N, A, V, I, M, P, R, K, L, T;

[0194] - B8: is selected from the group of K, R, Q, E, N;

[0195] - B9: is selected from the group of K, R, Q, S, T, G, D;

[0196] - B10: is selected from the group of N, D, E, K, R, V, S;

[0197] - B11: is selected from the group of E, N, V, F, W, P;

[0198] - B12: is selected from the group of I, N, V, L;

[0199] - B13: is selected from the group of L, I, V, Q, T.

[0200] In certain embodiments, B1-B13have the following definition:

[0201] - B1: is selected from the group of K, D, E, N, S, P, Q; - B2: is selected from the group of Q, N, E, D, P, S, A;

[0202] - B3: is selected from the group of K, Q, D, L, A, M;

[0203] - B4: is selected from the group of S, T, A, V, I, K, Y, L;

[0204] - B5: is selected from the group of N, D, Q, E, L;

[0205] - B6: is selected from the group of P, E, Q, K, S;

[0206] - B7: is selected from the group of N, A, V, I, M, P, R, K, L;

[0207] - B8: is selected from the group of K, R, Q, E, N;

[0208] - B9: is selected from the group of K, R, Q, S, T, G, D;

[0209] - B10: is selected from the group of N, D, E, K, R, V, S;

[0210] - B11: is selected from the group of E, N, V, F, W, P;

[0211] - B12: is selected from the group of I, N, V, L;

[0212] - B13: is selected from the group of L, I, V, Q, T.

[0213] In certain embodiments, B1-B13have the following definition:

[0214] - B1: is selected from the group of K, D, E, N, S, P, Q;

[0215] - B2: is selected from the group of Q, N, E, D, P, S, A;

[0216] - B3: is selected from the group of K, Q, D, L, A, M;

[0217] - B4: is selected from the group of S, T, A, V, I, K, Y;

[0218] - B5: is selected from the group of N, D, Q, E, L;

[0219] - B6: is selected from the group of P, E, Q, K, S;

[0220] - B7: is selected from the group of N, A, V, I, M, P, R, K;

[0221] - B8: is selected from the group of K, R, Q, E, N;

[0222] - B9: is selected from the group of K, R, Q, S, T, G;

[0223] - B10: is selected from the group of N, D, E, K, R, V, S;

[0224] - B11: is selected from the group of E, N, V, F, W, P;

[0225] - B12: is selected from the group of I, N, V, L;

[0226] - B13: is selected from the group of L, I, V, Q, T.

[0227] In certain embodiments, B1-B13have the following definition

[0228] B1: is selected from the group of K, D, E, N, P, Q;

[0229] B2: is selected from the group of Q, N, E, A, D;

[0230] B3: is selected from the group of Q, D, L, A;

[0231] B4: is selected from the group of S, T, A, V, I, L;

[0232] B5: is selected from the group of N, D, Q, E;

[0233] B6: is selected from the group of P, E, Q, S;

[0234] B7: is selected from the group of N, A, R, K, L;

[0235] B8: is selected from the group of K, R, Q, E, N;

[0236] B9: is selected from the group of Q, S, D, T, G;

[0237] B10: is selected from the group of N, D, E, S;

[0238] B11: is selected from the group of E, N, P; - B12: is selected from the group of I, V, L;

[0239] - B13: is selected from the group of L, I, V, Q.

[0240] In certain embodiments, B1-B13have the following definition

[0241] - B1: is selected from the group of D, E, N, P; - B2: is selected from the group of Q, N, E, A;

[0242] - B3: is selected from the group of Q, L, A;

[0243] - B4: is selected from the group of S, T, A, V;

[0244] - B5: is selected from the group of N, D;

[0245] - B6: is selected from the group of P, E, Q; - B7: is selected from the group of N, A, R, K;

[0246] - B8: is selected from the group of K, R, Q, E;

[0247] - B9: is selected from the group of S, T, G;

[0248] - B10: is selected from the group of N, D, E, S;

[0249] - B11: is selected from the group of E, N, P; - B12: is selected from the group of I, V, L;

[0250] - B13: is selected from the group of L, I, V.

[0251] In certain embodiments, B1-B13have the following definition (one line (row) is an alternative definition):

[0252] Constant residues

[0253] In certain embodiments, the polypeptide sequence comprises one or two substitutions according to the rules below. In certain embodiments, the polypeptide sequence comprises one substitution according to the rules below: helix a:

[0254] - I (at position 3 of helix a) may be exchanged for: T, P, L;

[0255] - V (at position 6 of helix a) may be exchanged for: I, L, N;

[0256] - I (at position 7 of helix a) may be exchanged for: V, L;

[0257] - A (at position 9 of helix a) may be exchanged for: S, C; loop a / b:

[0258] - G (of loop a / b) may be exchanged for: N, S, Q; helix b:

[0259] - A (at position 1 of helix b) may be exchanged for: G, V, I;

[0260] - L (at position 2 of helix b) may be exchanged for: I, V, P;

[0261] - L (at position 5 of helix b) may be exchanged for: I, F, M;

[0262] - V (at position 6 of helix b) may be exchanged for: L, I, C;

[0263] - L (at position 8 of helix b) may be exchanged for: I, F, C;

[0264] - L (at position 9 of helix b) may be exchanged for: I, M, S; loop b / c:

[0265] - SP (of loop b / c) may be exchanged for: HA, KN, PD, SN, YP, KD, SG, particularly exchanged for HA, KN, PD, SN, YP, KD; helix c:

[0266] - A (at position 8 of helix c) may be exchanged for: T, C, S;

[0267] - L (at position 9 of helix c) may be exchanged for: T, A, V, I;

[0268] - A (at position 11 of helix c) may be exchanged for: T, V, G, 0;

[0269] - L (at position 12 of helix c) may be exchanged for: I, V;

[0270] - N (at position 14 of helix c) may not be exchanged;

[0271] - I (at position 15 of helix c) may be exchanged for: L, V, A; - A (at position 16 of helix c) may be exchanged for: T, S, V, L.

[0272] In certain embodiments, the constant and loop residues have the following definition (one line (row) is an alternative definition):

[0273] Loop c / a

[0274] Loop c / a confers binding specificity to an armadillo repeat protein with respect to the residue which is present at an opposite site of the armadillo repeat protein (see Fig. 1A). In addition, loop c / a is solvent-exposed and has no defined secondary structure. Thus, loop c / a can be of variable length and variable sequence. Indeed, in natural armadillo repeat proteins, loops of various lengths are found (Table 5 and 6). This large diversity found in natural armadillo repeat proteins underlines the stability of the armadillo repeat framework and its ability to accommodate such insertions.

[0275] In certain embodiments, loop c1 / a2and loop c2 / a3and loop c3 / a4consist each of 1 to 12 amino acids. In certain embodiments, loop c1 / a2and loop c2 / a3and loop c3 / a4consist each of 3 to 12 amino acids. In certain embodiments, loop c1 / a2and loop c2 / a3and loop c3 / a4consist each of 3 to 9 amino acids. In certain embodiments, loop c1 / a2and loop c2 / a3and loop c3 / a4consist each of a sequence selected from Table 5 or Table 6.

[0276] Table 5. Structural plasticity and variety in loops c / a illustrated with a non-exhaustive list of naturally occurring loop sequences (with respective PDB ID given) in proteins retrieved from the PDB, where the helices superimpose with an rmsd of less than 2 A with the corresponding helices c, a, and b in dArmRPs.

[0277] Table 6. Structural plasticity and variety in loops c / a illustrated with a non-exhaustive list of naturally occurring loop sequences (with respective PDB ID) in proteins retrieved from the PDB, where the helices superimpose with an rmsd of less than 2 A with the corresponding helices c and a in dArmRPs. Further aspects

[0278] A further aspect of the invention relates to an armadillo repeat protein comprising the polypeptide sequence according to the first aspect.

[0279] A further aspect of the invention relates to a polypeptide domain comprising the polypeptide sequence according to the first aspect.

[0280] A further aspect of the invention relates to a binding motif comprising the polypeptide sequence according to the first aspect.

[0281] In some embodiments, the polypeptide sequence according to the invention can be employed as part of an armadillo repeat sequence. In other embodiments, the polypeptide sequence according to the invention can be employed as an isolated polypeptide.

[0282] Items:

[0283] 1. A polypeptide sequence providing Tyr-residue-binding specificity to an armadillo repeat protein sequence, said polypeptide sequence comprising a sequence consisting of two repeat units 1 and 2, wherein each repeat unit comprises three helices a, b, and c, wherein the helices a and b are connected via a loop a / b, and the helices b and c are connected via a loop b / c, and wherein two armadillo repeats are connected via a loop c / a; wherein the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below, with X not included in calculating identity:

[0284] • helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 001);

[0285] • loop a1 / b1consists of a single glycine G;

[0286] • helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 002);

[0287] • loop b1 / c1consists of a sequence serine-proline SP;

[0288] • helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 003); and

[0289] • loop c1 / a2consists of 1 to 20 amino acids

[0290] • helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 004);

[0291] • loop a2 / b2consists of a single glycine G;

[0292] • helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 005);

[0293] • loop b2 / c2consists of a sequence serine-proline SP;

[0294] • helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ ID NO 006); loop c2 / a3consists of 1 to 20 amino acids; wherein X11-X25have the following definition: wherein optionally, 1 or 2 amino acids of X12, X14, X15, X21, X22, X23, X24may be exchanged for a different amino acid, wherein X11, X13, and X25each independently is any amino acid; wherein B1-B13have the following definition:

[0295] - B1: is selected from the group of K, D, E, N, S, P, Q;

[0296] - B2: is selected from the group of Q, N, E, D, P, S, A;

[0297] - B3: is selected from the group of K, Q, D, L, A, M;

[0298] - B4: is selected from the group of S, T, A, V, I, K, Y, L;

[0299] - B5: is selected from the group of N, D, Q, E, L;

[0300] - B6: is selected from the group of P, E, Q, K, S;

[0301] - B7: is selected from the group of N, A, V, I, M, P, R, K, L;

[0302] - B8: is selected from the group of K, R, Q, E, N;

[0303] - B9: is selected from the group of K, R, Q, S, T, G, D;

[0304] - B10: is selected from the group of N, D, E, K, R, V, S;

[0305] - B11: is selected from the group of E, N, V, F, W, P;

[0306] - B12: is selected from the group of I, N, V, L;

[0307] - B13: is selected from the group of L, I, V, Q, T. . A polypeptide sequence providing Trp-residue-binding specificity to an armadillo repeat protein sequence, said polypeptide sequence comprising a sequence consisting of two repeat units 1 and 2, wherein each repeat unit comprises three helices a, b, and c, wherein the helices a and b are connected via a loop a / b, and the helices b and c are connected via a loop b / c, and wherein two armadillo repeats are connected via a loop c / a; wherein the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below, with X not included in calculating identity:

[0308] • helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 007);

[0309] • loop a1 / b1consists of a single glycine G;

[0310] • helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 008);

[0311] • loop b1 / c1consists of a sequence serine-proline SP;

[0312] • helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 009); and

[0313] • loop c1 / a2consists of 1 to 20 amino acids

[0314] • helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 010);

[0315] • loop a2 / b2consists of a single glycine G;

[0316] • helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 011);

[0317] • loop b2 / c2consists of a sequence serine-proline SP;

[0318] • helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ ID NO 012);

[0319] • loop c2 / a3consists of 1 to 20 amino acids; wherein X11-X25have the following definition: wherein optionally, 1 or 2 amino acids of X12, X14, X15, X21, X22, X23, X24may be exchanged for a different amino acid, wherein X11, X13, and X25each independently is any amino acid; wherein B1-B13have the following definition:

[0320] - B1: is selected from the group of K, D, E, N, S, P, Q;

[0321] - B2: is selected from the group of Q, N, E, D, P, S, A;

[0322] - B3: is selected from the group of K, Q, D, L, A, M;

[0323] - B4: is selected from the group of S, T, A, V, I, K, Y. L;

[0324] - B5: is selected from the group of N, D, Q, E, L;

[0325] - B6: is selected from the group of P, E, Q, K, S;

[0326] - B7: is selected from the group of N, A, V, I, M, P, R, K, L;

[0327] - B8: is selected from the group of K, R, Q, E, N; - B9: is selected from the group of K, R, Q, S, T, G, D;

[0328] - B10: is selected from the group of N, D, E, K, R, V, S;

[0329] - B11: is selected from the group of E, N, V, F, W, P;

[0330] - B12: is selected from the group of I, N, V, L;

[0331] - B13: is selected from the group of L, I, V, Q, T. . A polypeptide sequence providing His-residue-binding specificity to an armadillo repeat protein sequence, said polypeptide sequence comprising a sequence consisting of two repeat units 1 and 2, wherein each repeat unit comprises three helices a, b, and c, wherein the helices a and b are connected via a loop a / b, and the helices b and c are connected via a loop b / c, and wherein two armadillo repeats are connected via a loop c / a; wherein the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below, with X not included in calculating identity:

[0332] • helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 013);

[0333] • loop a1 / b1consists of a single glycine G;

[0334] • helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 014);

[0335] • loop b1 / c1consists of a sequence serine-proline SP;

[0336] • helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 015); and

[0337] • loop c1 / a2consists of 1 to 20 amino acids

[0338] • helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 016);

[0339] • loop a2 / b2consists of a single glycine G;

[0340] • helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 017);

[0341] • loop b2 / c2consists of a sequence serine-proline SP;

[0342] • helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ ID NO 018);

[0343] • loop c2 / a3consists of 1 to 20 amino acids; wherein X11-X25have the following definition: wherein optionally, 1 or 2 amino acids of X12, X14, X15, X21, X22, X23, X24may be exchanged for a different amino acid, wherein X11, X13, andX25each independently is any amino acid; wherein B1-B13have the following definition:

[0344] - B1: is selected from the group of K, D, E, N, S, P, Q;

[0345] - B2: is selected from the group of Q, N, E, D, P, S, A;

[0346] - B3: is selected from the group of K, Q, D, L, A, M;

[0347] - B4: is selected from the group of S, T, A, V, I, K, Y, L;

[0348] - B5: is selected from the group of N, D, Q, E, L;

[0349] - B6: is selected from the group of P, E, Q, K, S;

[0350] - B7: is selected from the group of N, A, V, I, M, P, R, K, L;

[0351] - B8: is selected from the group of K, R, Q, E, N;

[0352] - B9: is selected from the group of K, R, Q, S, T, G, D;

[0353] - B10: is selected from the group of N, D, E, K, R, V, S;

[0354] - B11: is selected from the group of E, N, V, F, W, P;

[0355] - B12: is selected from the group of I, N, V, L;

[0356] - B13: is selected from the group of L, I, V, Q, T. . A polypeptide sequence providing lle-residue-binding specificity to an armadillo repeat protein sequence, said polypeptide sequence comprising a sequence consisting of two repeat units 1 and 2, wherein each repeat unit comprises three helices a, b, and c, wherein the helices a and b are connected via a loop a / b, and the helices b and c are connected via a loop b / c, and wherein two armadillo repeats are connected via a loop c / a; wherein the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below, with X not included in calculating identity:

[0357] • helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 019); • loop a1 / b1consists of a single glycine G;

[0358] • helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 020);

[0359] • loop b1 / c1consists of a sequence serine-proline SP;

[0360] • helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 021); and

[0361] • loop c1 / a2consists of 1 to 20 amino acids

[0362] • helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 022);

[0363] • loop a2 / b2consists of a single glycine G;

[0364] • helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 023);

[0365] • loop b2 / c2consists of a sequence serine-proline SP;

[0366] • helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ ID NO 024);

[0367] • loop c2 / a3consists of 1 to 20 amino acids; wherein X11-X25have the following definition: wherein optionally, 1 or 2 amino acids of X12, X14, X15, X21, X22, X23, X24may be exchanged for a different amino acid, wherein X11, X13, and X25each independently is any amino acid; wherein B1-B13have the following definition:

[0368] - B1: is selected from the group of K, D, E, N, S, P, Q;

[0369] - B2: is selected from the group of Q, N, E, D, P, S, A;

[0370] - B3: is selected from the group of K, Q, D, L, A, M;

[0371] - B4: is selected from the group of S, T, A, V, I, K, Y, L;

[0372] - B5: is selected from the group of N, D, Q, E, L;

[0373] - B6: is selected from the group of P, E, Q, K, S;

[0374] - B7: is selected from the group of N, A, V, I, M, P, R, K, L;

[0375] - B8: is selected from the group of K, R, Q, E, N;

[0376] - B9: is selected from the group of K, R, Q, S, T, G, D;

[0377] - B10: is selected from the group of N, D, E, K, R, V, S; - B11: is selected from the group of E, N, V, F, W, P;

[0378] - B12: is selected from the group of I, N, V, L;

[0379] - B13: is selected from the group of L, I, V, Q, T;

[0380] 5. A polypeptide sequence providing Glu-residue-binding specificity to an armadillo repeat protein sequence, said polypeptide sequence comprising a sequence consisting of two repeat units 1 and 2, wherein each repeat unit comprises three helices a, b, and c, wherein the helices a and b are connected via a loop a / b, and the helices b and c are connected via a loop b / c, and wherein two armadillo repeats are connected via a loop c / a; wherein the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below, with X not included in calculating identity:

[0381] • helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 025);

[0382] • loop a1 / b1consists of a single glycine G;

[0383] • helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 026);

[0384] • loop b1 / c1consists of a sequence serine-proline SP;

[0385] • helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 027); and

[0386] • loop c1 / a2consists of 1 to 20 amino acids

[0387] • helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 028);

[0388] • loop a2 / b2consists of a single glycine G;

[0389] • helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 029);

[0390] • loop b2 / c2consists of a sequence serine-proline SP;

[0391] • helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ ID NO 030);

[0392] • loop c2 / a3consists of 1 to 20 amino acids; wherein X11-X25have the following definition: wherein optionally, 1 or 2 amino acids of X12, X14, X15, X21, X22, X23, X24may be exchanged for a different amino acid, wherein X11, X13, and X25each independently is any amino acid; wherein B1-B13have the following definition:

[0393] - B1: is selected from the group of K, D, E, N, S, P, Q;

[0394] - B2: is selected from the group of Q, N, E, D, P, S, A;

[0395] - B3: is selected from the group of K, Q, D, L, A, M;

[0396] - B4: is selected from the group of S, T, A, V, I, K, Y, L;

[0397] - B5: is selected from the group of N, D, Q, E, L;

[0398] - B6: is selected from the group of P, E, Q, K, S;

[0399] - B7: is selected from the group of N, A, V, I, M, P, R, K, L;

[0400] - B8: is selected from the group of K, R, Q, E, N;

[0401] - B9: is selected from the group of K, R, Q, S, T, G, D;

[0402] - B10: is selected from the group of N, D, E, K, R, V, S;

[0403] - B11: is selected from the group of E, N, V, F, W, P;

[0404] - B12: is selected from the group of I, N, V, L;

[0405] - B13: is selected from the group of L, I, V, Q, T.

[0406] 6. A polypeptide sequence providing Leu-residue-binding specificity to an armadillo repeat protein sequence, said polypeptide sequence comprising a sequence consisting of two repeat units 1 and 2, wherein each repeat unit comprises three helices a, b, and c, wherein the helices a and b are connected via a loop a / b, and the helices b and c are connected via a loop b / c, and wherein two armadillo repeats are connected via a loop c / a; wherein the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below, with X not included in calculating identity:

[0407] • helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 025);

[0408] • loop a1 / b1consists of a single glycine G;

[0409] • helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 026);

[0410] • loop b1 / c1consists of a sequence serine-proline SP;

[0411] • helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 027); and loop c1 / a2consists of 1 to 20 amino acids

[0412] • helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 028);

[0413] • loop a2 / b2consists of a single glycine G;

[0414] • helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 029);

[0415] • loop b2 / c2consists of a sequence serine-proline SP;

[0416] • helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ ID NO 030);

[0417] • loop c2 / a3consists of 1 to 20 amino acids; wherein X11-X25have the following definition: wherein optionally, 1 or 2 amino acids of X12, X14, X15, X21, X22, X23, X24may be exchanged for a different amino acid, wherein X11, X13, and X25each independently is any amino acid; wherein B1-B13have the following definition:

[0418] - B1: is selected from the group of K, D, E, N, S, P, Q;

[0419] - B2: is selected from the group of Q, N, E, D, P, S, A;

[0420] - B3: is selected from the group of K, Q, D, L, A, M;

[0421] - B4: is selected from the group of S, T, A, V, I, K, Y, L;

[0422] - B5: is selected from the group of N, D, Q, E, L;

[0423] - B6: is selected from the group of P, E, Q, K, S;

[0424] - B7: is selected from the group of N, A, V, I, M, P, R, K, L;

[0425] - B8: is selected from the group of K, R, Q, E, N;

[0426] - B9: is selected from the group of K, R, Q, S, T, G, D;

[0427] - B10: is selected from the group of N, D, E, K, R, V, S;

[0428] - B11: is selected from the group of E, N, V, F, W, P;

[0429] - B12: is selected from the group of I, N, V, L;

[0430] - B13: is selected from the group of L, I, V, Q, T. A polypeptide sequence providing Tyr-Z-Tyr-motif-binding specificity to an armadillo repeat protein sequence, said polypeptide sequence comprising a sequence consisting of three repeat units 1 , 2, and 3, wherein each repeat unit comprises three helices a, b, and c, wherein the helices a and b are connected via a loop a / b, and the helices b and c are connected via a loop b / c, and wherein two armadillo repeats are connected via a loop c / a; wherein the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below with X not included in calculating identity:

[0431] • helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 031);

[0432] • loop a1 / b1consists of a single glycine G;

[0433] • helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 032);

[0434] • loop b1 / c1consists of a sequence serine proline SP;

[0435] • helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 033); and

[0436] • loop c1 / a2consists of 1 to 20 amino acids

[0437] • helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 034);

[0438] • loop a2 / b2consists of a single glycine G;

[0439] • helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 035);

[0440] • loop b2 / c2consists of a sequence serine proline SP;

[0441] • helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ ID NO 036);

[0442] • loop c2 / a3consists of 1 to 20 amino acids;

[0443] • helix a3consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 037);

[0444] • loop a3 / b3consists of a single glycine G;

[0445] • helix b3consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 038);

[0446] • loop b3 / c3consists of a sequence serine proline SP;

[0447] • helix c3consists of a sequence B10B11X31B12B13X32X33ALX34ALX35NIA (SEQ ID NO 039);

[0448] • loop c3 / a4consists of 1 to 20 amino acids;

[0449] • wherein X11-X35have the following definition: wherein optionally, 1 , 2, 3 or 4 amino acids of X12, X14, X15, X21, X22, X23, X24, X25, X31, X32, X33, X34may be exchanged for a different amino acid, wherein X11, X13, and X35each independently is any amino acid; wherein B1-B13have the following definition:

[0450] - B1: is selected from the group of K, D, E, N, S, P, Q;

[0451] - B2: is selected from the group of Q, N, E, D, P, S, A;

[0452] - B3: is selected from the group of K, Q, D, L, A, M;

[0453] - B4: is selected from the group of S, T, A, V, I, K, Y, L; - B5: is selected from the group of N, D, Q, E, L;

[0454] - B6: is selected from the group of P, E, Q, K, S;

[0455] - B7: is selected from the group of N, A, V, I, M, P, R, K, L;

[0456] - B8: is selected from the group of K, R, Q, E, N;

[0457] - B9: is selected from the group of K, R, Q, S, T, G, D;

[0458] - B10: is selected from the group of N, D, E, K, R, V, S;

[0459] - B11: is selected from the group of E, N, V, F, W, P;

[0460] - B12: is selected from the group of I, N, V, L;

[0461] - B13: is selected from the group of L, I, V, Q, T. A polypeptide sequence providing Trp-Z-Trp-motif-binding specificity to an armadillo repeat protein sequence, said polypeptide sequence comprising a sequence consisting of three repeat units 1 , 2, and 3, wherein each repeat unit comprises three helices a, b, and c, wherein the helices a and b are connected via a loop a / b, and the helices b and c are connected via a loop b / c, and wherein two armadillo repeats are connected via a loop c / a; wherein the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below with X not included in calculating identity:

[0462] • helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 040);

[0463] • loop a1 / b1consists of a single glycine G;

[0464] • helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 041);

[0465] • loop b1 / c1consists of a sequence serine proline SP;

[0466] • helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 042); and

[0467] • loop c1 / a2consists of 1 to 20 amino acids

[0468] • helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 043);

[0469] • loop a2 / b2consists of a single glycine G;

[0470] • helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 044);

[0471] • loop b2 / c2consists of a sequence serine proline SP;

[0472] • helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ ID NO 045);

[0473] • loop c2 / a3consists of 1 to 20 amino acids;

[0474] • helix a3consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 046);

[0475] • loop a3 / b3consists of a single glycine G; • helix b3consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 047);

[0476] • loop b3 / c3consists of a sequence serine proline SP;

[0477] • helix c3consists of a sequence B10B11X31B12B13X32X33ALX34ALX35NIA (SEQ ID NO 048);

[0478] • loop c3 / a4consists of 1 to 20 amino acids;

[0479] • wherein X11-X35have the following definition: wherein optionally, 1 , 2, 3 or 4 amino acids of X12, X14, X15, X21, X22, X23, X24, X25, X31, X32, X33, X34may be exchanged for a different amino acid, wherein X11, X13, and X35each independently is any amino acid; wherein B1-B13have the following definition:

[0480] - B1: is selected from the group of K, D, E, N, S, P, Q;

[0481] - B2: is selected from the group of Q, N, E, D, P, S, A;

[0482] - B3: is selected from the group of K, Q, D, L, A, M;

[0483] - B4: is selected from the group of S, T, A, V, I, K, Y, L;

[0484] - B5: is selected from the group of N, D, Q, E, L;

[0485] - B6: is selected from the group of P, E, Q, K, S;

[0486] - B7: is selected from the group of N, A, V, I, M, P, R, K, L;

[0487] - B8: is selected from the group of K, R, Q, E, N;

[0488] - B9: is selected from the group of K, R, Q, S, T, G, D;

[0489] - B10: is selected from the group of N, D, E, K, R, V, S;

[0490] - B11: is selected from the group of E, N, V, F, W, P;

[0491] - B12: is selected from the group of I, N, V, L;

[0492] - B13: is selected from the group of L, I, V, Q, T.

[0493] 9. A polypeptide sequence providing I le-Z-l le-motif-binding specificity to an armadillo repeat protein sequence, said polypeptide sequence comprising a sequence consisting of three repeat units 1 , 2, and 3, wherein each repeat unit comprises three helices a, b, and c, wherein the helices a and b are connected via a loop a / b, and the helices b and c are connected via a loop b / c, and wherein two armadillo repeats are connected via a loop c / a; wherein the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below with X not included in calculating identity:

[0494] • helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 040);

[0495] • loop a1 / b1consists of a single glycine G;

[0496] • helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 041);

[0497] • loop b1 / c1consists of a sequence serine proline SP;

[0498] • helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 042); and

[0499] • loop c1 / a2consists of 1 to 20 amino acids

[0500] • helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 043);

[0501] • loop a2 / b2consists of a single glycine G;

[0502] • helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 044);

[0503] • loop b2 / c2consists of a sequence serine proline SP;

[0504] • helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ ID NO 045);

[0505] • loop c2 / a3consists of 1 to 20 amino acids;

[0506] • helix a3consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 046);

[0507] • loop a3 / b3consists of a single glycine G;

[0508] • helix b3consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 047);

[0509] • loop b3 / c3consists of a sequence serine proline SP;

[0510] • helix c3consists of a sequence B10B11X31B12B13X32X33ALX34ALX35NIA (SEQ ID NO 048);

[0511] • loop c3 / a4consists of 1 to 20 amino acids;

[0512] • wherein X11-X35have the following definition: wherein optionally, 1 , 2, 3 or 4 amino acids of X12, X14, X15, X21, X22, X23, X24, X25, X31, X32, X33, X34may be exchanged for a different amino acid, wherein X11, X13, and X35each independently is any amino acid; wherein B1-B13have the following definition:

[0513] - B1: is selected from the group of K, D, E, N, S, P, Q;

[0514] - B2: is selected from the group of Q, N, E, D, P, S, A;

[0515] - B3: is selected from the group of K, Q, D, L, A, M;

[0516] - B4: is selected from the group of S, T, A, V, I, K, Y, L;

[0517] - B5: is selected from the group of N, D, Q, E, L;

[0518] - B6: is selected from the group of P, E, Q, K, S;

[0519] - B7: is selected from the group of N, A, V, I, M, P, R, K, L;

[0520] - B8: is selected from the group of K, R, Q, E, N;

[0521] - B9: is selected from the group of K, R, Q, S, T, G, D;

[0522] - B10: is selected from the group of N, D, E, K, R, V, S;

[0523] - B11: is selected from the group of E, N, V, F, W, P;

[0524] - B12: is selected from the group of I, N, V, L;

[0525] - B13: is selected from the group of L, I, V, Q, T.

[0526] 10. The polypeptide sequence according to any one of the preceding items, wherein B1-

[0527] B13have the following definition

[0528] - B1: is selected from the group of K, D, E, N, P, Q;

[0529] - B2: is selected from the group of Q, N, E, A, D;

[0530] - B3: is selected from the group of Q, D, L, A;

[0531] - B4: is selected from the group of S, T, A, V, I, L;

[0532] - B5: is selected from the group of N, D, Q, E;

[0533] - B6: is selected from the group of P, E, Q, S;

[0534] - B7: is selected from the group of N, A, R, K, L;

[0535] - B8: is selected from the group of K, R, Q, E, N; - B9: is selected from the group of Q, S, D, T, G;

[0536] - B10: is selected from the group of N, D, E, S;

[0537] - B11: is selected from the group of E, N, P;

[0538] - B12: is selected from the group of I, V, L;

[0539] - B13: is selected from the group of L, I, V, Q. The polypeptide sequence according to any one of the preceding items, wherein B1- B13have the following definition

[0540] - B1: is selected from the group of K, D, E, N, S, P, Q;

[0541] - B2: is selected from the group of Q, N, E, D, P, S, A;

[0542] - B3: is selected from the group of K, Q, D, L, A, M;

[0543] - B4: is selected from the group of S, T, A, V, I, K, Y;

[0544] - B5: is selected from the group of N, D, Q, E, L;

[0545] - B6: is selected from the group of P, E, Q, K, S;

[0546] - B7: is selected from the group of N, A, V, I, M, P, R, K;

[0547] - B8: is selected from the group of K, R, Q, E, N;

[0548] - B9: is selected from the group of K, R, Q, S, T, G;

[0549] - B10: is selected from the group of N, D, E, K, R, V, S;

[0550] - B11: is selected from the group of E, N, V, F, W, P;

[0551] - B12: is selected from the group of I, N, V, L;

[0552] - B13: is selected from the group of L, I, V, Q, T. The polypeptide sequence according to any one of the preceding items, wherein B1- B13have the following definition

[0553] - B1: is selected from the group of D, E, N, P;

[0554] - B2: is selected from the group of Q, N, E, A;

[0555] - B3: is selected from the group of Q, L, A;

[0556] - B4: is selected from the group of S, T, A, V;

[0557] - B5: is selected from the group of N, D;

[0558] - B6: is selected from the group of P, E, Q;

[0559] - B7: is selected from the group of N, A, R, K;

[0560] - B8: is selected from the group of K, R, Q, E;

[0561] - B9: is selected from the group of S, T, G;

[0562] - B10: is selected from the group of N, D, E, S;

[0563] - B11: is selected from the group of E, N, P;

[0564] - B12: is selected from the group of I, V, L;

[0565] - B13: is selected from the group of L, I, V. 13. The polypeptide sequence according to any one of the preceding items, wherein B1- B13have the following definition:

[0566] 14. The polypeptide sequence according to any one of the preceding items, wherein B1- B13have the following definition:

[0567] 15. The polypeptide sequence according to any one of the preceding items, wherein the polypeptide sequence comprises one or two substitutions according to the rules below: helix a:

[0568] - I (at position 3 of helix a) may be exchanged for: T, P, L;

[0569] - V (at position 6 of helix a) may be exchanged for: I, L, N;

[0570] - I (at position 7 of helix a) may be exchanged for: V, L;

[0571] - A (at position 9 of helix a) may be exchanged for: S, C; loop a / b:

[0572] - G (of loop a / b) may be exchanged for: N, S, Q; helix b:

[0573] - A (at position 1 of helix b) may be exchanged for: G, V, I;

[0574] - L (at position 2 of helix b) may be exchanged for: I, V, P;

[0575] - L (at position 5 of helix b) may be exchanged for: I, F, M;

[0576] - V (at position 6 of helix b) may be exchanged for: L, I, C;

[0577] - L (at position 8 of helix b) may be exchanged for: I, F, C;

[0578] - L (at position 9 of helix b) may be exchanged for: I, M, S; loop b / c:

[0579] - SP (of loop b / c) may be exchanged for: HA, KN, PD, SN, YP, KD, SG, particularly exchanged for HA, KN, PD, SN, YP, KD; helix c:

[0580] - A (at position 8 of helix c) may be exchanged for: T, C, S;

[0581] - L (at position 9 of helix c) may be exchanged for: T, A, V, I; - A (at position 11 of helix c) may be exchanged for: T, V, G, C;

[0582] - L (at position 12 of helix c) may be exchanged for: I, V;

[0583] - I (at position 15 of helix c) may be exchanged for: L, V, A;

[0584] - A (at position 16 of helix c) may be exchanged for: T, S, V, L.

[0585] 16. The polypeptide sequence according to item 15, wherein the polypeptide sequence comprises one substitution according to the rules of item 15.

[0586] 17. The polypeptide sequence according to any one of the preceding items, wherein the polypeptide sequence is 100% identical to the sequence given in item 1 to 9.

[0587] 18. An armadillo repeat protein comprising the polypeptide sequence according to any one of items 1 to 17.

[0588] 19. A polypeptide domain comprising the polypeptide sequence according to any one of items 1 to 17.

[0589] 20. A binding motif comprising the polypeptide sequence according to any one of items 1 to 17.

[0590] Wherever alternatives for single separable features such as, for example, an X residue or a B residue or a loop sequence are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein. Thus, any of the alternative embodiments for an X residue may be combined with any of the alternative embodiments of a B residue and these combinations may be combined with any loop sequence mentioned herein.

[0591] The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

[0592] Description of the Figures

[0593] Fig. 1 dArmRP library and selection setup, a) Schematic drawing of a dArmRP as used for YSD. The N-cap and first 2 repeats harbor the lock mutation, recognizing the peptide's LSF motif at the C-terminal end, shown in olive. Repeats 4 and 5 contain the randomized library residues engaging target amino acid X shown in red. Ala residues are introduced to spatially flank the amino acid X, preventing a potential selection bias and reducing the basal peptide affinity to a range suitable to discriminate the impact of binding the target amino acid X (sequence shown corresponds to SEQ ID NO 346). b) Model of a consensus dArmRP with the 7 library positions highlighted in blue as used in the selection. In orange is depicted the Arg side chain engaging the pocket, c) YSD display setup with the dArmRP linked to the cell wall. A C- terminal myc tag is detected by standard antibodies as expression control (shown in green) in parallel to the target binding (shown as a peptide with an orange label).

[0594] Fig. 2 Characterization of Hits, a) Kd data measured by fluorescence anisotropy for the selected Tyr pocket with peptides of the sequence KAKXKAKRKLSF (SEQ ID NO 346), differing in one amino acid (X), shown as a bar graph. The y-axis is 1 / Kd, while the numbers above the bar represent the measured Kd in nM b) 1.4 A resolution structure of the selected Tyr pocket interacting with its target amino acid. The selected residues are labelled with residue type and residue number as by residue count per repeat. In brackets, the respective designation of the variable position X as used in the description is shown, c) Kd data measured by fluorescence anisotropy for the selected Topi His pocket, shown as a bar graph, d) Representation of the NGS binder specificity as a function of their enrichment in the last selection round. The specificity factor is defined as the affinity to the designated target divided by the affinity to the next best non-cognate amino acid. The average specificity shown here is the mean of the specificity factor for the two best off-targets. The enrichment factor is the ratio of the abundance fraction of a binder in the F4 pool, divided by its abundance fraction in pool F3. Binders enrichedF4-1 to enrichedF4-4 were selected from the top200 most abundant binders in the F4 pool, solely based on their enrichment factor. They would not have been analyzed based on their abundance, as it was not high. However, their specificities are at least as good as those from the 10 most abundant binders (Topi to Top10). This finding demonstrates the correlation between the enrichment factor and the binder specificity.

[0595] Fig. 3 Generation of double pockets, a) Schematic overview for combining two single pockets into a double pocket sequence using the Tyr single pocket as an example. Only the variable residues are shown, X12-X24 represent the library residues from the single pocket selection. Due to the same basic structure of each repeat, the residue X22 of the first single pocket sequence is the same as residue X12 of the adjacent pocket, likewise X24 of the first pocket and X14 of the adjacent pocket as indicated with the pink arrows. Therefore, a double pocket graft involves only twelve point mutations but in position X22 and X24 two different amino acids have to be considered. The double pocket sequence shows the respective designation of the variable positions X as used in the description, b) upper: Schematic drawing of a dArmRP with a grafted double Tyr pocket. The N-cap and the first two repeats still harbour the mutations required to bind the “LSF”-lock motive on the C- terminal end of the peptide (olive). The mutations introduced by the double pocket graft are located in repeat 3, 4 and 5 indicated by the dark blue colour. Repeat 4 harbours the two residues that are shared between the two Tyr pockets (pink). The peptide has been adapted to contain two spatially neighbouring Tyr as target amino acids, lower: Schematic drawing of the TyroM6. To generate a full (KY)X(SEQ ID NO 351) binder, repeat4, as in the double pocket construct, replaces the internal repeats rep2-5. The rep1 and rep6 in the full construct correspond to rep3 and rep5 in the double pocket construct, c) Model of the Tyr double pocket with the two shared residues X22 and X24 in pink, all others X12-X34 in blue. Shown here is the most successful variant with an lie in X22 and an Glu in X24.

[0596] Fig. 4 TyroTag Binder Analysis, a) Kd data measured by fluorescence anisotropy for the TyrM4 binder shown as a bar graph. The y-axis is 1 / Kd, while the numbers above the bar represent the measured Kd in nM. The Ala substitution of a Tyr or Lys in the target peptide reduces the affinity by a factor of 3.5 for Lys and a factor of 10 for the Tyr substitutions, regardless of the position, b) Detection of (KY)e-GFP (SEQ ID NO 352) fusion in an E. coli lysate in a WB assay. Left’. HA-tagged TyroM6 dArmRP was used as a primary agent to detect the (KY)e peptide (SEQ ID NO 351). The dArmRP was stained by its fused HA-tag, using a primary anti-HA antibody and secondary antibody combination. Right. Detection of GFP with standard antibodies. Sequences shown in this figure correspond to SEQ ID NOs 351-357.

[0597] Fig. 5 shows 1.6 A resolution structure of the selected Ile1-binding pocket interacting with its cognate target amino acid. The selected residues are labelled with residue type and residue number as by residue count per repeat. In brackets, the respective designation of the variable position X as used in the description is shown.

[0598] Fig. 6 shows 2.0 A resolution structure of the assembled lle-Z-lle motif binder (from the selected lie-binding pocket) interacting with its cognate target peptide motif l-Z-l. The crucial residues are labelled with residue type and residue number as by residue count per repeat. In brackets, the respective designation of the variable position X as used in the description is shown.

[0599] Fig. 7 shows leucine binding pockets. A) 1.55 A resolution structure of the selected Leu_B5 pocket interacting with a non-cognate residue, alanine. B) 1.4 A resolution structure of the selected Leu_B5 pocket interacting with a noncognate residue, isoleucine. C) 1.6 A resolution structure of the selected Leu_B5 pocket interacting with its cognate target amino acid. D) 1.8 A resolution structure of the selected Leu_B5 pocket interacting with a noncognate residue, arginine. E) 1.55 A resolution structure of the selected Leu_B5 pocket interacting with a non-cognate residue, valine. F) 1.3 A resolution structure of the selected Leu_B5 pocket interacting with a noncognate residue, tyrosine. G) 1.5 A resolution structure of the selected Leu_C10 pocket interacting with a non-cognate residue, alanine. H) 1.5 A resolution structure of the selected Leu_C10 pocket interacting with a non- cognate residue, isoleucine. I) 1.45 A resolution structure of the selected Leu_C10 pocket interacting with its cognate target amino acid. J) 1.4 A resolution structure of the selected Leu_C10 pocket interacting with a non- cognate residue, arginine. K) 1.55 A resolution structure of the selected Leu_C10 pocket interacting with a non-cognate residue, valine. L) 1.5 A resolution structure of the selected Leu_C10 pocket interacting with a non- cognate residue, tyrosine.

[0600] The selected residues of Fig. 7 A-L are labelled with residue type and residue number as by residue count per repeat. In brackets, the respective designation of the variable position X as used in the description is shown.

[0601] Fig. 8 shows 1.6 A resolution structure of the selected Trp-binding pocket interacting with its cognate target amino acid. The selected residues are labelled with residue type and residue number as by residue count per repeat. In brackets, the respective designation of the variable position X as used in the description is shown.

[0602] Fig. 9 shows 1.75 A resolution structure of the assembled T rp-Z-T rp motif binder (from the selected Trp-binding pocket) interacting with its cognate target peptide motif W-Z-W. The crucial residues are labelled with residue type and residue number as by residue count per repeat. In brackets, the respective designation of the variable position X as used in the description is shown.

[0603] Examples

[0604] Example 1: Yeast Surface Display

[0605] Yeast surface display (YSD) was chosen as a selection technology to generate specific modules for different amino acids based on the dArmRP consensus scaffold. To ensure the correct binding register of the repetitive peptide target, a hydrophobic interaction was grafted onto the consensus scaffold to specifically recognizing an Leu-Ser-Phe (LSF) motif in the target peptide, thereby anchoring it in the desired position as shown in Figure 1.

[0606] A structure-based, rationally designed library with seven randomized positions was designed and integrated in a dArmRP with six internal repeats as shown in Figure 1. The N-terminal part of the dArmRP was adapted to accommodates the LSF lock motif to ensure the correct binding register of the target peptide. The peptides used for the YSD selection contain the variable target amino acid X positioned to engage with the randomized dArmRP module. A schematic drawing of the selection setup is shown in Figure 1. The residues spatially flanking the target amino acid X in the peptide were changed to Ala to minimize selection bias towards these flanking sites. The introduction of Ala additionally decreased the general affinity of the target peptide from the picomolar to the low nanomolar range, which had been found previously to be ideal to distinguish the binding energy contributed by individual target amino acid side chains. Nonetheless, for correct target binding, the complete peptide must be engaged by the dArmRP. Hence, during selection, the additional energetic gain resulting from favorable interactions with the target amino acid X has to be distinguished from the basal peptide binding energy.

[0607] Example 2: Selection with Single Side Chain Resolution

[0608] The main challenge of the dArmRP module selection is the need to select for modules that have a high selectivity for only one amino acid side chain in a peptide, but not necessarily the highest affinity. To this end, differently labelled off-target peptides were used as competitors during the selection process. Such off-target peptides are identical except containing any other residue type in the variable position X except the target amino acid. Since the target and off-target peptides differ in only this one amino acid, a large part of the respective affinities results from the constant part, which is identical in both the target and the off-target. We will refer to this affinity, attributed to the constant part of the peptide, as the basal (peptide) affinity. Hence, the selection requires to discriminate the affinity differences given by the target side chain in comparison to the off-target side chains, on top of the basal peptide affinity (single side chain resolution).

[0609] The selection complexity is further exacerbated by the relatively flat topography of the dArmRP module binding surface. Large amino acids cannot be excluded from binding by generating a small cavity, since they can potentially avoid repulsions or clashes by adapting a different rotamer conformation. Hence, the depletion of cross-reactive binders, which show a high affinity for the target, but also for other amino acids in this position, is the most critical step for a successful selection.

[0610] In order to achieve this depletion of cross- reactive binders, an excess of competitor peptides is typically added during the sorting experiments, decreasing the signal intensity in the target signal channel for yeast cells that simultaneously bind to target and competitor and hence display a cross-reactive dArmRP. In theory, variants that then still maintain a high target signal should be specific for the target and non-cross reactive for all competitors used during this selection. However, this assumption turned out to be oversimplified when distinguishing very similar epitopes, as the discrimination of high-affinity but cross- reactive binders from high-affinity specific binders has proven to be very challenging with this setup. We attribute this to the fact that the highest affinity binders are not necessarily the most specific ones, which renders it very difficult to set the right target-to-competitor ratio: too high a competitor concentration will eventually also affect target specific binders (due to the same basal affinity of the constant parts of the target peptide), whereas too low a concentration will not lead to a notable shift in target signal intensity for cross- reactive binders. Also, the definition of a too high or too low competitor concentration is highly dependent on the precise affinities for the target and off-target peptides and obviously differs for the individual binders contained in the selection pools. Using an excess of competitor peptides during the selection is therefore always a compromise to keep lower affinity but higher specificity binders versus depleting high-affinity but cross-reactive variants. The above described “standard” competition strategy was successfully applied to deplete binders with very low general affinity or those with high off-target specificity, but high amounts of cross-reactive binders were still obtained. Thus, this standard competitor selection only proved to be very useful to enrich a pool of binders all with a high affinity for the designated target but variable specificity.

[0611] To address the lack of specificity we developed here an additional competition setup which weighs the specificity of the binder over their affinity. This new strategy was termed “reverse competitor” selection because the target peptide is employed as competitor to decrease the binding signal of the desired clones. It follows the rationale that, compared to cross-reactive binders, specific binders are more susceptible to competition when using their designated target as competitor.

[0612] For a reverse competition, a mix of target peptide and off-target peptides, labelled with the same dye {e.g., mNeongreen) are incubated with an excess of differently labelled target peptides as competitor {e.g., labelled with mRuby3). The competition effect (fewer bound mNeongreen-peptides which results in a lower mNeongreen signal) is most prominent for specific binders because, out of the mNeongreen-labelled peptides present, they dominantly engage the target peptide, which can effectively be competed with the competitor, since target and competitor are the same peptide (with a different label) and hence have the same affinity to the binder. In contrast, cross-reactive binders engage the mNeongreen target and off-targets peptides with similar affinities and therefore the fraction of bound mNeongreen peptide that can be effectively competed with target-competitor is lower. This effect is potentiated for binders with slightly higher affinities for off-targets compared to the designated target, since the competition effect is obviously weaker when competing a higher affinity peptide with a lower affinity one.

[0613] Hence, in a reverse competitor setup, specific binders are singled out because they do preferentially engage the designated target (used in the function of a competitor). In contrast, in a normal competition setup, the variants selected are those that do not mainly engage the employed (off-target) competitors, which does not automatically mean that they are specific for the designated target. Additionally, by using the target peptide in the function of a competitor, the selection stringency is increasing with lower competitor concentration, since the competitor is the target itself. This significantly decreases the risk of false positives due to a high competitor concentration. As for any competition experiment, the degree of competition depends on the binder’s affinities for target and off-targets, respectively, and the target / competitor concentrations.

[0614] The parallel sorts of a standard and reverse competition selection clearly demonstrate the ability of the reverse competition setup to deplete cross-reactive variants. As will be discussed below, the deep sequencing of selection pools at different stages of selection allowed us to visualize the effect of the reverse competition strategy, again indicating its efficacy to separate cross- reactive from specific binders.

[0615] In our understanding, the success of the reverse competitor over the standard competitor selection depends on the constitution of the epitopes that have to be distinguished and the conformational space the binder can sample to engage the target epitope. We expect that the reverse competitor strategy outperforms a standard competition approach whenever the discrimination of highly similar epitopes is required and / or if the conformational space that the binder can sample is limited. As an additional advantage, the reverse competition setup does not require the designated target to be labelled as long as the off-targets can be detected (although it provides an additional layer of control and is therefore recommended if possible). This feature renders the reverse competitor strategy amenable for targets that are difficult to produce.

[0616] Example 3: Selection of Tyr and His pockets

[0617] Based on the described strategies, selections against His and Tyr were performed as the amino acid x, in the context of an otherwise identical peptide. Hits were identified from the last selection pool by a single-clone screen of yeast using flow cytometry. The sequence- verified hits were then expressed in E. coli and characterized by measuring the Kd for different peptides by fluorescence anisotropy to determine the affinity and specificity of the selected modules. The selectivity and affinity trends observed in the yeast single clone assay were confirmed. The obtained affinities for the best Tyr- and His-module are shown in Figure 2. With a Kd of 5 nM in the context of the chosen peptide, the pocket selected for Tyr shows a high affinity for its target and at the same time discriminates to the next best target (Trp) by a factor of more than 10. All other amino acids are lower in affinity by at least a factor 20. A high-resolution crystal structure (Figure 2) was obtained, elucidating the interaction of the Tyr side chain and the selected Tyr-pocket. An H-bond network between the selected residues K29, E33 and R30 orientates the E33 in an ideal conformation to form a C-H - n stacking interaction with its Cp hydrogen to the aromatic moiety of the Tyr side chain. Additionally, the Tyr hydroxyl head group of the incoming Tyr side chain is engaged in an H-bond to residue Q33. Upon grafting a Trp as the target amino acid into the peptide structure (Pymol), a clash with R30 is observed, whereas the spatially fitting Phe lacks the hydroxyl group to undergo the H-bond with Q33. Hence, the observed binding mode supports the experimental Kd data.

[0618] The characterization data for the topi His pocket (named after the next-generation sequencing (NGS) ranking discussed below) reveal a similar performance in terms of specificity and affinity as the Tyr pocket, as shown in Figure 2. The Topi His pocket was measured to have an affinity of 8 nM for its designated target in this peptide context, discriminates against Arg as the next best binding amino acid by a factor of 13 and all other measured amino acids by more than a factor of 30. Hence, also the topi His pocket fulfils the criteria with regards to specificity and selectivity required for a modular binder assembly approach and validates the above-described selection strategy.

[0619] Example 4: Next Generation Sequencing

[0620] The selection rounds for the His modules were analyzed by llumina Miseq next generation sequencing (NGS). The 7 library positions were extracted from the processed data for each single round. A comparison of the amino acid distribution over different selection rounds, as well as the visualization of the selection progress by projecting the sequence differences as distances using UMAP, impressively visualizes the effect of the different selection strategies. The data confirm the desired distribution of amino acids for the randomized library positions in the native pool, which is very close to the designed one. The first round (FACS1) was sorted with low stringency, aiming mainly to separate binding vs. non-binding variants and to decrease the library size. Hence, the FACS1 pool still maintains a high diversity. Enrichment of individual amino acids in different library positions is observed after FACS2 and remains similar for FACS3, both of which employ a standard competitor selection strategy to deplete binders with predominant off-target specificity and enrich binders with high affinity for the target. The most significant change in amino acid distribution is observed for the reverse competition sort in FACS4, which aims at the separation of the high-affinity but cross-reactive binders from high-affinity specific binders, as described above. Interestingly, during that step, in some positions the earlier amino acids that were enriched are significantly reduced again. The shift in sequence space upon reverse competitor selection is also clearly observed in the LIMAP plots. The sequence space explored by the FACS4 pool comprises only a small portion of that of the FACS3 pool, underlining the importance of this selection strategy to retrieve modules with the desired selectivity.

[0621] The 10 most frequent clones as identified by NGS were additionally characterized as shown in Table 1. All of them show high affinity for the His target but differ in their specificity. Comparing the relative abundance of the top10 binders in pools FACS3 and FACS4, it can be observed that over-proportionally enriched binders in the step from FACS3 to FACS4 tend to have the better specificity. Hence, the selectivity of individual binders can be estimated based on their enrichment factor that is calculated as a ratio of the respective sequence abundance fraction in FACS3 and FACS4. The correlation of this enrichment with measured selectivity mirrors the success of the reverse competition. It can also be seen that the selection for affinity (FACS3, standard competition), in comparison, has not enriched specific clones, while the direct selection for selectivity (FACS4, reverse competition) did achieve the intended purpose.

[0622] We next wanted to test the hypothesis that a notable enrichment from FACS3 to FACS4 would conversely help to identify highly selective clones. Therefore, binders with a high enrichment factor were picked out of the top200 NGS hits and characterized for their selectivity. Three out of four tested binders indeed show a specificity factor average above 5, which makes them more specific than 50 % of the top 10 binders (Figure 2). Hence, the enrichment factor allows to harvest highly sequence-diverse binders from the NGS data alone, which would be very unlikely to have been identified otherwise.

[0623] Example 5: Binder Generation Based on Assembly of Selected Modules

[0624] Based on the selected Tyr pocket, a binder with two adjacent Tyr pockets was designed. The assembly of two adjacent pockets presents some challenges because the central H3 helix is contributing to both pockets (Figure 3). A screening was performed investigating 4 variants that are obtained from rationally combining the selected amino acids in the critical positions as shown in Figure 3, as either of the shared positions could prefer the identity of the residue from the "left" or the "right" pocket. Retaining a Glu in the shared library position X7 proved to be important to obtain the best affinity towards the designated target.

[0625] The most promising combination for the double Tyr motif was then used to create a (KY)4 (SEQ ID NO 351) and (KY)e (SEQ ID NO 351) - binder, respectively, by propagating these double pockets. The binders and their corresponding targets were expressed in E. coli and characterized by FA measurements. The TyrM4 and TyrM6 dArmRPs show a very high affinity to their respective targets. For comparison, the consensus M4 dArmRP binds with 250 nM to its cognate (KR)4 (SEQ ID NO 358) target peptide, whereas the TyrM4 binds its cognate target (KY)4 (SEQ ID NO 351) with 3 nM (cf. Figure 4). The TyrM6 affinity to its cognate target was estimated to be below picomolar, as the Kd could not be exactly determined, because in a FRET assay to measure the off-rate, no disassociation of the (KY)e (SEQ ID NO 351) target was detected over the maximal measuring time of 16 h.

[0626] However, assuming a 10% dissociation of the complex over the 16 h measuring time would have been observed, the dissociation rate can be estimated to be smaller than 1.7 x 10-6s’1. Together with the association rate that was determine to be 1.3 x 107M'1s’1, it follows that the equilibrium dissociation constant for the TyrM6 and its cognate target (KY)e (SEQ ID NO

[0627] 351) is below 1.5 10’13M.

[0628] The impact of individual amino acids of the (KY)n(SEQ ID NO 351) peptide to the binding affinity was also investigated. Similar as for the original (KR)n(SEQ ID NO 351) binder, a very regular contribution to the free energy of binding was observed for the (KY)n(SEQ ID NO 351) binder TyrM4, depending on the nature of the respective amino acid, but regardless of its position in the target peptide. As shown in Figure 4, the substitution of a Tyr by an Ala reduces the affinity by a factor of 10, whereas the exchange from a Lys to an Ala results in a 2.5 times diminished affinity. This validates the modularity of the TyroMX binders and the success of introducing the new Tyr pockets.

[0629] The TyrMX binders thus serve as proof of principle that binders can be assembled based on modules selected by YSD. The characteristics of a modular binder are maintained, for instance the ability to tune the affinity by adjusting the length of the binder or the regular binding energy contribution of individual amino acids in the target peptide, as shown in Figure 4.

[0630] A rather obvious application for modular peptide binders is their use as detection agents in assays such as western blots (WB). To investigate the performance of the dArmRP as staining reagents, an HA-tagged TyrM6 was used to detect blotted (KY)e-GFP (SEQ ID NO

[0631] 352) in a WB assay. The TyrM6 itself was stained via a standard anti-HA antibody sandwich. As a control, the GFP was also detected via an anti-GFP antibody sandwich. As shown in Figure 4, the dArmRP specifically detects the expressed (KY)e-GFP (SEQ ID NO 352) in the E. coli cell lysate without background. This result clearly demonstrates the potential for the use of custom made dArmRPs as sequence-specific protein detection agents. Example 6: Crystal Structures

[0632] In the crystal structures (Fig. 5-9), it can be seen that the non-cognate side chains do not enter the pocket at all, or very little, or in the case of Alanine, make no interactions. This fact explains the strong discrimination in binding constants, even though only a single amino acid in the peptide differs.

[0633] Example 7: Varying parameters of the pocket

[0634] To proof the versatility of the pocket structures, the inventors varied the following parameters:

[0635] - Sequence of the peptide anchor (LSF and RQR): Table 3A and 3B

[0636] - Location of the pocket relative to the N-terminus of the Armadillo repeat protein: Table 3C

[0637] - Flanking residues of the specific peptide residue: Table 3D

[0638] - Different B residues: known sequence variations from Table 2, SEQ ID NOs 85-184, and sequences of BYM6 (SEQ ID NO. 394) and LKDVV (SEQ ID NO. 395).

[0639] SEQ ID NOs. 381-391 were tested against the wildtype peptide target. All of these proteins show strong binding with a Kd value of <10nm, thus, they fold correctly.

[0640] Materials and Methods

[0641] Target generation

[0642] Initially, chemically synthesized, biotinylated target peptides were preincubated with labelled Streptavidin (SA) to detect target binding in yeast flow cytometry experiments. To avoid the avidity effect, the total concentration of peptide-SA was lowered, and more stringent gates were set. However, the effect between affinity and cytometry signal was obscured, suggesting that an avidity effect within the tetrameric peptide-SA complex is the cause.

[0643] To overcome this issue, monovalent targets were produced as peptide fusions to fluorescent proteins, restoring the correlation between peptide binding signal in flow cytometry experiments and fluorescence anisotropy measurements. To avoid selection of binders against the fluorescent proteins, different versions (mNeonGreen, mRuby) were used either in conjunction or in alternation.

[0644] Yeast Surface Display and FACS

[0645] The tryptophan-auxotrophic S. cerevisiae strain EY100 was used in all experiments. Yeast transformants were grown at 30°C in SD-CAA media (6.9 g / l yeast nitrogen base without amino acids, 5 g / l casamino acids, 20 g / l D-glucose, 35.3 mM tribasic sodium citrate, 35.3 mM citric acid). Expression of the display construct was induced at an ODeoo of 1 and cultivated for 24 h at 20 °C in SG-CAA media (6.9 g / l yeast nitrogen base without amino acids, 5 g / l casamino acids, 2 g / l anhydrous D-glucose, 18 g / l anhydrous D-galactose, 38 mM dibasic sodium phosphate, 55 mM monobasic sodium phosphate).

[0646] Yeast Library Preparation

[0647] The yeast display vector pCTCON2 (G. Chao et al., Nat. Protoc. 1, 755-768 (2006)) was modified to contain restriction sites directly at the N- and C- terminus of the dArmRP Gene to facilitate the downstream processing of the selected variants. The initial randomized library inserts used for the His selections were ordered at Geneart Synthesis, using trinucleotide technology (B. Virnekas et al., Nucleic Acids Res 22: 5600-5607. Nucleic acids research 22, 5600-5607 (1995)). In the randomized positions, all amino acids were allowed except Pro, Gly and Cys, with the exception of position X2 and X7 (Fig. 1), where also Gly was allowed, since Gly was observed in some natural ArmRP at these positions.

[0648] For the Tyr selection, a second design was used, in which the position X3 was limited to the amino acids Vai, Thr, Ala and Ser. This library was created by the MAX technology (M. D. Hughes et al., Removing the redundancy from randomised gene libraries) and amplified by PCR using HR primers (HR-fwd: AAT GAA CAA ATC TTG CAA GAG GCC TTG TGG GCC CTC (SEQ ID NO 359), HR-rev: ATC GAT TAC TGC TTG GAT TTG CTC ATT ACC CCC GCT AG (SEQ ID NO 360)). The pCTCON2 library backbone was linearized by restriction digest with Nhel (NEB) and BbvCI (NEB), located within the dArmRP gene, and purified via a PCR Purification Kit (Qiagen). The yeast libraries were created by homologous recombination of the library insert and the linearized library backbone, and were used to cotransform EBY100 cells by electroporation. The cells were made competent using the lithium-acetate method (J. A. Van Deventer, K. D. Wittrup, Methods Mol. Biol. 1131, 151-181 (2014)). For the transformation, the cell suspension was mixed with 3 pg linearized backbone and 6 pg library insert, transferred to a chilled cuvette (0.2 cm Gene Pulse, BioRad), and pulsed at 1.5 kV, 25 mF, 200 V using Gene Pulser Xcell (Bio-Rad). The recovered cells were cultivated in SD-CAA until ODeoo of 5 was reached. After transformation, dilution series were plated to estimate the actual library size.

[0649] Flow Cytometry

[0650] Flow cytometry experiments were performed in PBSAE buffer (137 mM sodium chloride, 3 mM potassium chloride, 8 mM dibasic sodium phosphate, 1.5 mM monobasic potassium phosphate, 1 g / l bovine serum albumin, 2 mM EDTA). Cell staining was performed on ice using 5x107cells / 50 pl PBSAE buffer. The required expression culture volume was collected and the pellet resuspended in 50 pl mouse anti-c-myc antibody (Sigma, M4439), 1 / 1000 diluted in PBSAE. After incubating for 30 min while rotating at 4 °C, the yeast cells were centrifuged (4000 g, 5 min) and the pellet resuspended in 50 pl PBSAE with goat anti- mouse-AF647 (Invitrogen, A21235, 1 / 1000 diluted) and the desired concentration of peptide- mNeongreen or -mRuby3 fusions. The mixture was incubated for 60 min while rotating at 4 °C. Subsequently, the cells were centrifuged (4000 g, 5 min) and the pellet resuspended in 200 pl PBSAE directly before subjecting the cells to flow cytometry analysis or FACS.

[0651] Plasmid construction forArmRP expression

[0652] For protein expression, a pQE30LIC_3C-based vector, which contains a SC-protease- cleavable MRGSHis6-tag, was used (S. Hansen et al., J. Am. Chem. Soc. 138, 3526-3532 (2016)). Selected dArmRPs variants were subcloned from the display vector pCTCON2 using restriction enzymes BamHI and Hindi 11. DNA fragments coding for rationally designed dArmRP pockets or assemblies were ordered from IDT or Twist Bioscience and subcloned using restriction enzymes Nhel and BbvCI. The target-peptide fusions were obtained by ligation of the BamHI-, Kasl-digested expression vector variant with the sticky-end dsDNA insert encoding the target peptide that was obtained by heat-induced annealing of the respective ssDNA sequences. To obtain mCherry-dArmRP fusions, the dArmRP were subcloned using restriction enzymes BamHI and Hindi 11 in an expression vector coding for an MRGSHis6-tag-3C-mCherry fusion N-terminal to the BamHI site. All construct sequences were confirmed by DNA sequencing (Microsynth).

[0653] Expression and Purification

[0654] Protein expression was done in autoinduction medium for 15 h at 25 °C. The expression cultures were centrifuged (5,000 g, 5 min), resuspended in resuspension buffer (RB) (50 mM Tris pH 8.0, 500 mM NaCI, 20 mM imidazole). Protease inhibitors Petabloc SC (50 pg / ml), Leupeptin (5 pg / ml), Pepstatin-A (1 pg / ml) were added. The cells were lysed by sonication and the insoluble contents removed by centrifugation (25,000 g, 20 min). The crude extracts were loaded onto Ni-NTA superflow resin columns (3 ml, Qiagen), 30 column volumes (CV) of RB were applied for washing. Elution was achieved using 2.5 CV of EB (RB with 300 mM imidazole). Human rhinovirus 3C-protease (2% w / w) was mixed with eluted protein fractions and the reaction mixtures were dialyzed against PBS (137 mM sodium chloride, 3 mM potassium chloride, 8 mM dibasic sodium phosphate, 1.5 mM monobasic potassium phosphate) to cleave off the His-tags. Proteins destined for crystallization were dialyzed against crystallization buffer (CB) (50 mM Tris pH 7.4 and 300 mM NaCI). Protease and uncleaved proteins were removed by reverse IMAC. The monomeric protein fractions were isolated by SEC on an AKTA Explorer chromatography system using a HiLoad 16 / 60 Superdex 200 pg column and the same buffers as used for dialysis. Affinity Characterization

[0655] For Kd determination, fluorescence anisotropy measurements were performed as described in (S. Hansen et al., J. Am. Chem. Soc. 138, 3526-3532 (2016)). As reporter, target peptide- sfGFP or -mNeongreen fusions were used. The assay was performed in black non-binding 96-well plates (Greiner bio-One). All measurements were performed in PBS supplied with 0.05 % (v / v) Tween 20. Binding kinetics were determined using a FRET binding assay as described in (S. Hansen et al., J. Am. Chem. Soc. 138, 3526-3532 (2016); P. Ernst et al., ACS Chem. Biol. 15, 457-468 (2020)). The dArmRP variants were produced as mCherry fusions for the FRET assay.

[0656] Western blot

[0657] The newly generated dArmRP binders were HA-tagged for the use as primary detection agents. The C-terminal HA tag was detected with commercial antibodies, primary mouse anti-HA (Sigma), secondary goat anti-mouse Daylight790 conjugate (Biotium). As positive control, GFP was detected with primary mouse anti-GFP (Cell Signaling), secondary goat anti-mouse Daylight790 conjugate (Biotium). The assay was performed in TBS (137 mM NaCI, 50 mM Tris). The samples were separated by SDS / PAGE Mini-PROTEAN TGX™ 4- 20% gels and blotted onto a nitrocellulose membrane using a Trans-Blot® Turbo™ transfer system (BioRad) according to the manufacturer's protocol (mixed MW, 25 V, 7 min). The membranes were air-dried at RT for 1 h and blocked for 20 min, RT, with TBSC (TBS containing 1x Casein Blocking buffer (Thermo Scientific)). The primary incubation with the HA-tagged dArmRP (5 pg / ml) or anti-GFP mAb (1 :1000 diluted) was performed ON at 4 °C in 5 ml TBSC. The membranes were washed with TBST (TBS supplied with 0.1 % Tween20) and incubated at RT for 1 h with the anti-HA antibody (1:2000) in 5 ml TBST. The incubation step was also performed for the GFP membrane with 5 ml TBST. The membranes were washed with TBST and incubated at RT for 1 h with the secondary mAb in 5 ml TBST. After a final wash, the fluorescence intensity was detected on an Odyssey imaging system (Ll- COR Biosciences).

[0658] Crystallization

[0659] The crystallization chaperone DARPin D12 (22) and a GGSGG-linked (SEQ ID NO 361) target peptide were N- terminally fused to the selected dArmRP variants. The proteins were concentrated to 40 - 70 mg / ml in 50 mM Tris / HCI at pH 8 and 300 mM NaCI using Amicon centrifugal concentrators (Amicon Ultra Centrifugal Filters, Merck Millipore). Sitting drop vapor diffusion crystallization was performed in I ntelli-Plates R96-3 LV (Art Robbins, California) with a mother-liquor to protein ratio of 1 :1, 2:1 and 3:1 per condition. The plates were set up and kept at 20 °C. Prior to data collection, the crystals were incubated for 10 s in the mother liquor containing 20 % (v / v) ethylene glycol and flash-frozen in liquid nitrogen. Data were collected at the Paul Scherer Institute with the Synchrotron light source, (PSI, Villigen, Switzerland). The data were processed using XDS, XSCALE and XDSCONV for analysis. Molecular replacement using CCP4 with Phaser was employed to solve the structure. The refinement was done in CCP4 using refmac5 and in Phenix with PhenixRefine. The Tyr module structure was obtained in 2 M (NH^SOt, 0.1 M Bis-Tris, pH 5.5.

[0660] NGS

[0661] Yeast cells were lysed by mixing an approximately 1-mm diameter globule of freshly grown cells with 10 pL 0.02 M NaOH and incubated for 10 min at 95 °C. 5 pL of lysate were used as PCR template to amplify the NGS amplicon using Taq Polymerase and the provided Q- solution (HotStarTaq DNA Polymerase, Cat No. / ID: 20320). The adaptor ligation, the Illumina Miseq run and pre-processing of the data was performed by the Functional Genomics Center Zurich. From the obtained FASTQ files, approximately 99% of the reads were paired, these were combined into single reads by FLASH (T. Magoc, S. L. Salzberg, Bioinformatics 27, 2957-2963 (2011)) with a minimum / maximum overlap of 230 / 300 base pairs. Single reads were then mapped back to the wild-type dArmRP sequence using Bowtie2 (B. Langmead, S. L. Salzberg, Nature Methods 9, 357-359 (2012)) with triple penalties for gap opening and extension and the non-coding sequences excluded. The remaining sequences were clustered at 100% identity by a custom Python script. To visualize the highly dimensional sequence space, sequences were one-hot encoded and mapped to 2 dimensions by UMAP (L. H. Mclnnes, J.; Melville, J. (UMAP: Uniform Manifold Approximation and Projection for Dimension Reduction. (htps: / / arxiv.orQ / abs / 1802.03426)).

[0662] Experimental Kp Measurements:

[0663] Table 1 : Kd measurements of NGS hits

[0664] Kd data of the ten most abundant hits (topi to top1O) of the His selection present in the FACS4 pool and the variants selected based on their enrichment factor (enrichedF4-1 to enrichedF4- 4). The Kd was determined by a fluorescence anisotropy assay. For the calculation of the specificity factor the Kd of the off-target R- and Y- peptides is divided by the Kd of the designated H-peptide target. The enrichment factor is the ratio of the abundance fraction of the respective clone in FACS3 and FACS4 (numbers not shown). The rank in FACS4 is based on the absolute abundance ranking of the variants in FACS4.

[0665] Kd (nM) Specificity Factor NGS H- R- Y- Enrichme Rank eptid peptide eptid Average factor FACS4 topi | 7.8 104.0 292.0 37.6 25.5 118 1 top2 14.9 109.7 81.7 5.5 6.4 30 top3 | 11.6 72.7 65.0 5.6 6.0 68 3

[0666] Table 2: shows armadillo structures available via PDB accession numbers.

[0667] Further Armadillo Repeat Protein sequences are given in SEQ ID NO 85-184.

[0668] Table 3A: shows Kd values for targets. Single-pocket binders were tested against the peptide KAKXKAKRKLSF (SEQ ID NO 346), wherein X is the target amino acid. Motif binders (Tyr-X-Tyr and Trp-X-Trp) were tested against the peptide KAKXKXKAKLSF (SEQ ID NO 362). (c) stands for “cognate”, (n.d.) stands for “not determined” Table 3B: shows Kd values for targets. Motif binders (lle-Z-lle, Tyr-Z-Tyr and Trp-Z-Trp) were tested against the peptide KAKXKXARQR (SEQ ID NO 396) (c) stands for “cognate”, (n.d.) stands for “not determined”

[0669] Table 3C: shows Kd data with different locations of the Y pocket. The Tyrosine-binding pocket was placed between different internal modules (M3 / M4, M4 / M5, M5 / M6) and was tested against three target peptides containing the tyrosine residue facing either pocket M3 / M4, M4 / M5 or M5 / M6. Table 3D: shows Kd values for targets with other flanking residues. The tyrosine-binding pocket placed between internal modules M4 / M5 was tested against two peptides (KRKXKRKAALSF) confronting the pocket with either tyrosine or phenylalanine (X being either Y or F). The peptide residues facing the flanking binding pockets contain two arginines instead of alanine residues.

[0670] Sequences

[0671] Table 4: shows sequences of binders for respective targets.

[0672] Further polypeptide sequences are disclosed by the sequence listing.

[0673] Table 7: Full length sequences of the target peptides (=Peptide (underlined) fused to GFP).

[0674] Cited prior art documents:

[0675] All scientific publications and patent documents cited in the present specification are incorporated by reference herein. Madhurantakam, C., Varadamsetty, G., Griitter, M. G., Pluckthun, A. and Mittl, P. R. (2012) Structure-based optimization of designed armadillo-repeat proteins. Protein Sci. 21, 1015-1028.

[0676] Reichen, C., Hansen, S., Forzani, C., Honegger, A., Fleishman, S. J., Zhou, T., Parmeggiani, F., Ernst, P., Madhurantakam, C., Ewald, C., Mittl, P. R., Zerbe, O., Baker, D., Caflisch, A. and Pluckthun, A. (2016) Computationally designed armadillo repeat proteins for modular peptide recognition. J. Mol. Biol. 428, 4467-4489.

[0677] Reichen, C., Madhurantakam, C., Pluckthun, A. and Mittl, P. R. (2014) Crystal structures of designed armadillo repeat proteins: Implications of construct design and crystallization conditions on overall structure. Protein Sci. 23, 1572-1583. Hansen, S., Tremmel, D., Madhurantakam, C., Reichen, C., Mittl, P. R. and Pluckthun, A. (2016) Structure and energetic contributions of a designed modular peptide-binding protein with picomolar affinity. J. Am. Chem. Soc. 138, 3526-3532. Hansen, S., Kiefer, J. D., Madhurantakam, C., Mittl, P. R. E. and Pluckthun, A.

[0678] (2017) Structures of designed armadillo repeat proteins binding to peptides fused to globular domains. Protein Sci. 26, 1942-1952.

Claims

Claims1. A polypeptide sequence providing Tyr-residue-binding specificity to an armadillo repeat protein sequence, said polypeptide sequence comprising a sequence consisting of two repeat units 1 and 2, wherein each repeat unit comprises three helices a, b, and c, wherein the helices a and b are connected via a loop a / b, and the helices b and c are connected via a loop b / c, and wherein two armadillo repeats are connected via a loop c / a; wherein the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below, with X not included in calculating identity:• helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 001);• loop a1 / b1consists of a single glycine G;• helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 002);• loop b1 / c1consists of a sequence serine-proline SP;• helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 003); and• loop c1 / a2consists of 1 to 20 amino acids• helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 004);• loop a2 / b2consists of a single glycine G;• helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 005);• loop b2 / c2consists of a sequence serine-proline SP;• helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ ID NO 006);• loop c2 / a3consists of 1 to 20 amino acids; wherein X11-X25have the following definition:wherein optionally, 1 or 2 amino acids of X12, X14, X15, X21, X22, X23, X24may be exchanged for a different amino acid, wherein X11, X13, and X25each independently is any amino acid; wherein B1-B13have the following definition:- B1: is selected from the group of D, E, N, P;- B2: is selected from the group of Q, N, E, A;- B3: is selected from the group of Q, L, A;- B4: is selected from the group of S, T, A, V;- B5: is selected from the group of N, D;- B6: is selected from the group of P, E, Q;- B7: is selected from the group of N, A, R, K;- B8: is selected from the group of K, R, Q, E;- B9: is selected from the group of S, T, G;- B10: is selected from the group of N, D, E, S;- B11: is selected from the group of E, N, P;- B12: is selected from the group of I, V, L;- B13: is selected from the group of L, I, V.

2. A polypeptide sequence providing Trp-residue-binding specificity to an armadillo repeat protein sequence, said polypeptide sequence comprising a sequence consisting of two repeat units 1 and 2, wherein each repeat unit comprises three helices a, b, and c, wherein the helices a and b are connected via a loop a / b, and the helices b and c are connected via a loop b / c, and wherein two armadillo repeats are connected via a loop c / a; wherein the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below, with X not included in calculating identity:• helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 007);• loop a1 / b1consists of a single glycine G;• helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 008);• loop b1 / c1consists of a sequence serine-proline SP;• helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA(SEQ ID NO 009); and loop c1 / a2consists of 1 to 20 amino acids• helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 010);• loop a2 / b2consists of a single glycine G;• helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 011);• loop b2 / c2consists of a sequence serine-proline SP;• helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ ID NO 012);• loop c2 / a3consists of 1 to 20 amino acids; wherein X11-X25have the following definition:wherein optionally, 1 or 2 amino acids of X12, X14, X15, X21, X22, X23, X24may be exchanged for a different amino acid, wherein X11, X13, and X25each independently is any amino acid; wherein B1-B13have the following definition:- B1: is selected from the group of D, E, N, P;- B2: is selected from the group of Q, N, E, A;- B3: is selected from the group of Q, L, A;- B4: is selected from the group of S, T, A, V;- B5: is selected from the group of N, D;- B6: is selected from the group of P, E, Q;- B7: is selected from the group of N, A, R, K;- B8: is selected from the group of K, R, Q, E;- B9: is selected from the group of S, T, G;- B10: is selected from the group of N, D, E, S;- B11: is selected from the group of E, N, P;- B12: is selected from the group of I, V, L;- B13: is selected from the group of L, I, V.

3. A polypeptide sequence providing His-residue-binding specificity to an armadillo repeat protein sequence, said polypeptide sequence comprising a sequence consisting of two repeat units 1 and 2,wherein each repeat unit comprises three helices a, b, and c, wherein the helices a and b are connected via a loop a / b, and the helices b and c are connected via a loop b / c, and wherein two armadillo repeats are connected via a loop c / a; wherein the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below, with X not included in calculating identity:• helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 013);• loop a1 / b1consists of a single glycine G;• helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 014);• loop b1 / c1consists of a sequence serine-proline SP;• helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 015); and• loop c1 / a2consists of 1 to 20 amino acids• helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 016);• loop a2 / b2consists of a single glycine G;• helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 017);• loop b2 / c2consists of a sequence serine-proline SP;• helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ ID NO 018);• loop c2 / a3consists of 1 to 20 amino acids; wherein X11-X25have the following definition:wherein optionally, 1 or 2 amino acids of X12, X14, X15, X21, X22, X23, X24may be exchanged for a different amino acid,wherein X11, X13, and X25each independently is any amino acid; wherein B1-B13have the following definition:- B1: is selected from the group of D, E, N, P;- B2: is selected from the group of Q, N, E, A;- B3: is selected from the group of Q, L, A;- B4: is selected from the group of S, T, A, V;- B5: is selected from the group of N, D;- B6: is selected from the group of P, E, Q;- B7: is selected from the group of N, A, R, K;- B8: is selected from the group of K, R, Q, E;- B9: is selected from the group of S, T, G;- B10: is selected from the group of N, D, E, S;- B11: is selected from the group of E, N, P;- B12: is selected from the group of I, V, L;- B13: is selected from the group of L, I, V.

4. A polypeptide sequence providing lle-residue-binding specificity to an armadillo repeat protein sequence, said polypeptide sequence comprising a sequence consisting of two repeat units 1 and 2, wherein each repeat unit comprises three helices a, b, and c, wherein the helices a and b are connected via a loop a / b, and the helices b and c are connected via a loop b / c, and wherein two armadillo repeats are connected via a loop c / a; wherein the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below, with X not included in calculating identity:• helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 019);• loop a1 / b1consists of a single glycine G;• helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 020);• loop b1 / c1consists of a sequence serine-proline SP;• helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 021); and• loop c1 / a2consists of 1 to 20 amino acids• helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 022);• loop a2 / b2consists of a single glycine G;• helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 023);• loop b2 / c2consists of a sequence serine-proline SP;• helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ ID NO 024);• loop c2 / a3consists of 1 to 20 amino acids; wherein X11-X25have the following definition:wherein optionally, 1 or 2 amino acids of X12, X14, X15, X21, X22, X23, X24may be exchanged for a different amino acid, wherein X11, X13, and X25each independently is any amino acid; wherein B1-B13have the following definition:- B1: is selected from the group of D, E, N, P;- B2: is selected from the group of Q, N, E, A;- B3: is selected from the group of Q, L, A;- B4: is selected from the group of S, T, A, V;- B5: is selected from the group of N, D;- B6: is selected from the group of P, E, Q;- B7: is selected from the group of N, A, R, K;- B8: is selected from the group of K, R, Q, E;- B9: is selected from the group of S, T, G;- B10: is selected from the group of N, D, E, S;- B11: is selected from the group of E, N, P;- B12: is selected from the group of I, V, L;- B13: is selected from the group of L, I, V.

5. A polypeptide sequence providing Glu-residue-binding specificity to an armadillo repeat protein sequence, said polypeptide sequence comprising a sequence consisting of two repeat units 1 and 2,wherein each repeat unit comprises three helices a, b, and c, wherein the helices a and b are connected via a loop a / b, and the helices b and c are connected via a loop b / c, and wherein two armadillo repeats are connected via a loop c / a; wherein the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below, with X not included in calculating identity:• helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 025);• loop a1 / b1consists of a single glycine G;• helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 026);• loop b1 / c1consists of a sequence serine-proline SP;• helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 027); and• loop c1 / a2consists of 1 to 20 amino acids• helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 028);• loop a2 / b2consists of a single glycine G;• helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 029);• loop b2 / c2consists of a sequence serine-proline SP;• helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA(SEQ ID NO 030); loop c2 / a3consists of 1 to 20 amino acids; wherein X11-X25have the following definition:wherein optionally, 1 or 2 amino acids of X12, X14, X15, X21, X22, X23, X24may be exchanged for a different amino acid, wherein X11, X13, and X25each independently is any amino acid; wherein B1-B13have the following definition:- B1: is selected from the group of D, E, N, P;- B2: is selected from the group of Q, N, E, A;- B3: is selected from the group of Q, L, A;- B4: is selected from the group of S, T, A, V;- B5: is selected from the group of N, D;- B6: is selected from the group of P, E, Q;- B7: is selected from the group of N, A, R, K;- B8: is selected from the group of K, R, Q, E;- B9: is selected from the group of S, T, G;- B10: is selected from the group of N, D, E, S;- B11: is selected from the group of E, N, P;- B12: is selected from the group of I, V, L;- B13: is selected from the group of L, I, V.

6. A polypeptide sequence providing Leu-residue-binding specificity to an armadillo repeat protein sequence, said polypeptide sequence comprising a sequence consisting of two repeat units 1 and 2, wherein each repeat unit comprises three helices a, b, and c, wherein the helices a and b are connected via a loop a / b, and the helices b and c are connected via a loop b / c, and wherein two armadillo repeats are connected via a loop c / a; wherein the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below, with X not included in calculating identity:• helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 025);• loop a1 / b1consists of a single glycine G;• helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 026);• loop b1 / c1consists of a sequence serine-proline SP;• helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 027); and• loop c1 / a2consists of 1 to 20 amino acids• helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 028);• loop a2 / b2consists of a single glycine G;• helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 029);• loop b2 / c2consists of a sequence serine-proline SP;helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA(SEQ ID NO 030); loop c2 / a3consists of 1 to 20 amino acids; wherein X11-X25have the following definition:wherein optionally, 1 or 2 amino acids of X12, X14, X15, X21, X22, X23, X24may be exchanged for a different amino acid, wherein X11, X13, and X25each independently is any amino acid; wherein B1-B13have the following definition:- B1: is selected from the group of D, E, N, P;- B2: is selected from the group of Q, N, E, A;- B3: is selected from the group of Q, L, A;- B4: is selected from the group of S, T, A, V;- B5: is selected from the group of N, D;- B6: is selected from the group of P, E, Q;- B7: is selected from the group of N, A, R, K;- B8: is selected from the group of K, R, Q, E;- B9: is selected from the group of S, T, G;- B10: is selected from the group of N, D, E, S;- B11: is selected from the group of E, N, P;- B12: is selected from the group of I, V, L;- B13: is selected from the group of L, I, V.

7. A polypeptide sequence providing Tyr-Z-Tyr-motif-binding specificity to an armadillo repeat protein sequence, said polypeptide sequence comprising a sequence consisting of three repeat units 1 , 2, and 3,wherein each repeat unit comprises three helices a, b, and c, wherein the helices a and b are connected via a loop a / b, and the helices b and c are connected via a loop b / c, and wherein two armadillo repeats are connected via a loop c / a; wherein the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below with X not included in calculating identity:• helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 031);• loop a1 / b1consists of a single glycine G;• helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 032);• loop b1 / c1consists of a sequence serine proline SP;• helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 033); and• loop c1 / a2consists of 1 to 20 amino acids• helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 034);• loop a2 / b2consists of a single glycine G;• helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 035);• loop b2 / c2consists of a sequence serine proline SP;• helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ ID NO 036);• loop c2 / a3consists of 1 to 20 amino acids;• helix a3consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 037);• loop a3 / b3consists of a single glycine G;• helix b3consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 038);• loop b3 / c3consists of a sequence serine proline SP;• helix c3consists of a sequence B10B11X31B12B13X32X33ALX34ALX35NIA (SEQ ID NO 039);• loop c3 / a4consists of 1 to 20 amino acids;• wherein X11-X35have the following definition:wherein optionally, 1 , 2, 3 or 4 amino acids of X12, X14, X15, X21, X22, X23, X24, X25, X31, X32, X33, X34may be exchanged for a different amino acid, wherein X11, X13, and X35each independently is any amino acid; wherein B1-B13have the following definition:- B1: is selected from the group of D, E, N, P;- B2: is selected from the group of Q, N, E, A;- B3: is selected from the group of Q, L, A;- B4: is selected from the group of S, T, A, V;- B5: is selected from the group of N, D;- B6: is selected from the group of P, E, Q;- B7: is selected from the group of N, A, R, K;- B8: is selected from the group of K, R, Q, E;- B9: is selected from the group of S, T, G;- B10: is selected from the group of N, D, E, S;- B11: is selected from the group of E, N, P;- B12: is selected from the group of I, V, L;- B13: is selected from the group of L, I, V.

8. A polypeptide sequence providing Trp-Z-Trp-motif-binding specificity to an armadillo repeat protein sequence, said polypeptide sequence comprising a sequence consisting of three repeat units 1 , 2, and 3, wherein each repeat unit comprises three helices a, b, and c, wherein the helices a and b are connected via a loop a / b, and the helices b and c are connected via a loop b / c, and wherein two armadillo repeats are connected via a loop c / a; wherein the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below with X not included in calculating identity:• helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 040);• loop a1 / b1consists of a single glycine G;• helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 041);• loop b1 / c1consists of a sequence serine proline SP;• helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 042); and• loop c1 / a2consists of 1 to 20 amino acids• helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 043);• loop a2 / b2consists of a single glycine G;• helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 044);• loop b2 / c2consists of a sequence serine proline SP;• helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ ID NO 045);• loop c2 / a3consists of 1 to 20 amino acids;• helix a3consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 046);• loop a3 / b3consists of a single glycine G;• helix b3consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 047);• loop b3 / c3consists of a sequence serine proline SP;• helix c3consists of a sequence B10B11X31B12B13X32X33ALX34ALX35NIA (SEQ ID NO 048);loop c3 / a4consists of 1 to 20 amino acids;• wherein X11-X35have the following definition:wherein optionally, 1 , 2, 3 or 4 amino acids of X12, X14, X15, X21, X22, X23, X24, X25, X31, X32, X33, X34may be exchanged for a different amino acid, wherein X11, X13, and X35each independently is any amino acid; wherein B1-B13have the following definition:- B1: is selected from the group of D, E, N, P;- B2: is selected from the group of Q, N, E, A;- B3: is selected from the group of Q, L, A;- B4: is selected from the group of S, T, A, V;- B5: is selected from the group of N, D;- B6: is selected from the group of P, E, Q;- B7: is selected from the group of N, A, R, K;- B8: is selected from the group of K, R, Q, E;- B9: is selected from the group of S, T, G;- B10: is selected from the group of N, D, E, S;- B11: is selected from the group of E, N, P;- B12: is selected from the group of I, V, L;- B13: is selected from the group of L, I, V.

9. A polypeptide sequence providing I le-Z-l le-motif-binding specificity to an armadillo repeat protein sequence, said polypeptide sequence comprising a sequence consisting of three repeat units 1 , 2, and 3, wherein each repeat unit comprises three helices a, b, and c, wherein the helices a and b are connected via a loop a / b, and the helices b and c are connected via a loop b / c, and wherein two armadillo repeats are connected via a loop c / a; wherein the polypeptide sequence is >90%, >95%, or 100% identical to the sequence given below with X not included in calculating identity:• helix a1consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 040);• loop a1 / b1consists of a single glycine G;• helix b1consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 041);• loop b1 / c1consists of a sequence serine proline SP;• helix c1consists of a sequence B10B11X11B12B13X12X13ALX14ALX15NIA (SEQ ID NO 042); and• loop c1 / a2consists of 1 to 20 amino acids• helix a2consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 043);• loop a2 / b2consists of a single glycine G;• helix b2consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 044);• loop b2 / c2consists of a sequence serine proline SP;• helix c2consists of a sequence B10B11X21B12B13X22X23ALX24ALX25NIA (SEQ ID NO 045);• loop c2 / a3consists of 1 to 20 amino acids;• helix a3consists of the sequence B1B2IB3B4VIB5A (SEQ ID NO 046);• loop a3 / b3consists of a single glycine G;• helix b3consists of a sequence ALB6B7LVB8LLB9(SEQ ID NO 047);• loop b3 / c3consists of a sequence serine proline SP;• helix c3consists of a sequence B10B11X31B12B13X32X33ALX34ALX35NIA(SEQ ID NO 048); loop c3 / a4consists of 1 to 20 amino acids;• wherein X11-X35have the following definition:wherein optionally, 1 , 2, 3 or 4 amino acids of X12, X14, X15, X21, X22, X23, X24, X25, X31, X32, X33, X34may be exchanged for a different amino acid, wherein X11, X13, and X35each independently is any amino acid; wherein B1-B13have the following definition:- B1: is selected from the group of D, E, N, P;- B2: is selected from the group of Q, N, E, A;- B3: is selected from the group of Q, L, A;- B4: is selected from the group of S, T, A, V;- B5: is selected from the group of N, D;- B6: is selected from the group of P, E, Q;- B7: is selected from the group of N, A, R, K;- B8: is selected from the group of K, R, Q, E;- B9: is selected from the group of S, T, G;- B10: is selected from the group of N, D, E, S;- B11: is selected from the group of E, N, P;- B12: is selected from the group of I, V, L;- B13: is selected from the group of L, I, V.

10. The polypeptide sequence according to any one of the preceding claims, wherein B1- B13have the following definition:

11. The polypeptide sequence according to any one of the preceding claims, wherein the polypeptide sequence comprises one or two substitutions according to the rules below: helix a:- I (at position 3 of helix a) may be exchanged for: T, P, L;- V (at position 6 of helix a) may be exchanged for: I, L, N;- I (at position 7 of helix a) may be exchanged for: V, L;- A (at position 9 of helix a) may be exchanged for: S, C; loop a / b:- G (of loop a / b) may be exchanged for: N, S, Q; helix b:- A (at position 1 of helix b) may be exchanged for: G, V, I;- L (at position 2 of helix b) may be exchanged for: I, V, P;- L (at position 5 of helix b) may be exchanged for: I, F, M;- V (at position 6 of helix b) may be exchanged for: L, I, C;- L (at position 8 of helix b) may be exchanged for: I, F, C;- L (at position 9 of helix b) may be exchanged for: I, M, S; loop b / c:- SP (of loop b / c) may be exchanged for: HA, KN, PD, SN, YP, KD; helix c:- A (at position 8 of helix c) may be exchanged for: T, C, S;- L (at position 9 of helix c) may be exchanged for: T, A, V, I;- A (at position 11 of helix c) may be exchanged for: T, V, G, 0;- L (at position 12 of helix c) may be exchanged for: I, V;- I (at position 15 of helix c) may be exchanged for: L, V, A;- A (at position 16 of helix c) may be exchanged for: T, S, V, L.

12. The polypeptide sequence according to claim 10, wherein the polypeptide sequence comprises one substitution according to the rules of claim 10.

13. The polypeptide sequence according to any one of the preceding claims, wherein the polypeptide sequence is 100% identical to the sequence given in claim 1.

14. An armadillo repeat protein comprising the polypeptide sequence according to any one of claims 1 to 13.

15. An armadillo repeat protein comprising a plurality of polypeptide sequences as described in any one of claims 1 to 13.

16. A polypeptide domain comprising the polypeptide sequence according to any one of claims 1 to 13.

17. A binding motif comprising the polypeptide sequence according to any one of claims 1 to 13.

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