Caspase-2 variants and crystalline forms thereof

Abstract

Disclosed herein are conformationally stable variants of a single-chain circular permuted caspase, crystalline forms thereof, and methods of their preparation and use.

Description

[0001] 2024-337 09531.607W01

[0002] CASPASE- 2 VARIANTS AND CRYSTALLINE FORMS THEREOF

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. provisional patent application no. 63 / 748,735, filed January 23, 2025, the entirety of which is incorporated herein by reference.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0006] This invention was made with government support under AG062199 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] 1. BACKGROUND

[0008] Caspases are a family of cysteine aspartyl proteases known for their classical role in cell death and inflammation.1Improper functioning of caspases in these pathways has been linked to various diseases, including cancer and inflammatory diseases, ischemia / reperfusion-related conditions (e.g., stroke), and neurodegenerative diseases.2The broad range of diseases associated with caspases makes them an appealing target for drug discovery. Notably, the Caspl inhibitor VX-765 was recently approved by the FDA for human clinical trials for the treatment of HIV- 1.3There have been a handful of other caspase inhibitors in clinical trials which also demonstrate promising safety profiles.1' Despite such clinical relevance, most caspase inhibitors do not make it past clinical trials due to problems such as inadequate efficacy, poor target specificity, or adverse side effects.24These challenges largely stem from a limited understanding of the biological functions of caspases, as redundancy among them complicates the study of individual effects. Therefore, selective caspase probes are essential for advancing knowledge in this area.

[0009] Caspase-2 (Casp2) plays a role in Alzheimer’s Disease (AD).10' ’ Research has shown that Casp2 cleaves tau protein and forms a toxic partnership where the tau fragment generated migrates to the dendritic spine and blocks synaptic function.16 17In addition to AD, Casp2 has also been implicated in diet-induced obesity,18metabolic dysfunction-associated steatohepatitis (MASH),19retinal ganglion cell death,2021synaptic weakening,22and neurodegeneration caused by ischemia.23,24The development of a selective probe for Casp2 could significantly advance therapeutic research, as no such probes are currently available. Recent studies have shown that modifying peptide inhibitors by introducing a THIQ moiety or a 2,4-diaminobutyric acid (Dab) at the P2 position can greatly enhance selectivity over Caspase-3 (Casp3).25,26Additional improvements in selectivity' can be made by adding homoglutamic acid (hGlu) at P4 or indoline-2-carboxylic acid (Ide) at P5, or by replacing the more reactive aldehyde warhead with weaker electrophiles, like a nitrile.27-29Structure-activity' relationship (SAR) campaigns2024-337 09531.607W01

[0010] have made these developments possible but fall short of obtaining co-crystal structures with lead compounds. The protein data bank (PDB) contains only 11 crystal structures of Casp2, with no new structures deposited since 2011.2630No co-crystal structure of a non-peptide inhibitor has been published with Casp2. These crystallographic limitations stem from expression difficulties, with limited quantities of Casp2 creating a significant research bottleneck.2’31Strategies to improve expression and crystallization of Casp2 are needed to implement structure-based design into Casp2 drug design.

[0011] Drug design for caspases faces significant challenges due to their complex activation mechanisms. All caspases are synthesized as inactive zymogens (inactive proteins that are converted into an enzyme when activated) that are activated through dimerization by adaptor proteins, facilitating subsequent autocatalytic cleavage.’2This process requires cleavage of the zymogen at a specific aspartic acid residue between the large and small subunits, which are processed into a dimeric catalytic subunit, two of which associate with one another to form a heterotetramer, which can form either before cleavage, when it is referred to as a procaspase, or after the caspase has been activated.33

[0012] The determination of procaspase and active caspase structures revealed that the termini of the cleaved large and small subunits are in close proximity. This insight has facilitated the creation of constitutively active caspases by swapping the order of the large and small subunits in the caspase gene, leaving the N-terminal portion of the large subunit free and eliminating the need for cleavage.34See FIG.

[0013] 1. These enzymes are commonly called “reverse caspases” due to the swapped order of their subunits. It is also accurate to use the term “circular permutation” to describe this technique, as the term refers to mutations that alter the sequence of amino acids but maintain the same overall 3D structure.

[0014] There are examples of this strategy being used successfully with caspase-3 (Casp3), caspase-6 (Casp6), caspase-7 (Casp7), and the Drosophila caspase drICE.35-37Recently, circularly permuted caspase-2 (cpCasp2) was generated using the same methods; specifically, the large and small subunits were swapped, and a Gly-Ser linker was inserted betw een them.31A depiction of the cpCasp2 gene can be found in FIG. 1. This enzy me is constitutively active and more catalytically efficient than wild-type Casp2 (wtCasp2). Additionally, cpCasp2 exhibits higher quantitative expression yields than wtCasp2, which is significant given that wtCasp2 expression has proven difficult for researchers, with limited quantities creating a bottleneck for projects involving this enzyme.25’1

[0015] 2. SUMMARY

[0016] This invention is directed to structurally stable forms of cpCasp2, methods of stabilizing circularly permuted caspases, methods of facilitating the crystallization of stabilizing circularly permuted caspases, and methods of using cry stal structure information obtained from such crystallization for the screening of potential caspase inhibitors.2024-337 09531.607W01

[0017] One embodiment of the invention is directed to a polypeptide, which polypeptide is a variant of a single-chain circular permuted caspase-2 (cpCasp2) comprising the following structure from N- to C-tenninus: a small subunit, which is a small subunit of caspase-2 or a functionally active variant thereof; a linker consisting of n glycine, serine, proline and / or alanine residues wherein n is 2 to 10 (e.g.. 4 to 8); and a large subunit, which is a large subunit of caspase-2 or a functionally active variant thereof; which polypeptide comprises a modified loop region, which modified loop region is a contiguous amino acid sequence of the formula XssqLXLSr, wherein each Xssis independently an amino acid residue of the small subunit, q is 2-10, L is a chemical bond or the linker, each XLSis independently an amino acid residue of the large subunit, and r is 2-11. provided that the sum of q + r > 13.

[0018] In some embodiments, the polypeptide is functionally active. In some embodiments, the linker is GS, GG. GPPAG, GPAGGPG, GPSGG. GGSGG, SGAGSAAGSG. (GS)mwherein m is 1 to 4. GSG. or G4S. In some, the linker is GS, GG, GPPAG. GPAGGPG. or GPSGG.

[0019] In some embodiments, the polypeptide binds to AcVDVAD-CHO with an IC50 equal to or greater than about 30 nM (e.g.. greater than about 40, 50, 60, or 70 nM). In some, the polypeptide binds to AcIDVAD-CHO with an IC50 equal to or greater than about 70 nM (e.g., greater than about 80, 90, 100. or 110 nM).

[0020] The polypeptide may comprise an affinity tag (e.g., a hexahistidine tag) or a solubility enhancement tag.

[0021] This invention also encompasses crystalline forms of the polypeptides disclosed herein. In one case, the crystalline form is a co-crystal with AcVDVAD-CHO. In another, it is a co-crystal with MUR-65.

[0022] This invention also encompasses isolated nucleotide sequences encoding the polypeptides disclosed herein. It also encompasses vectors (e.g., bacterial expression vectors) comprising such nucleotide sequences. Also encompassed is an expression cassette comprising the nucleotide sequences operably linked to regulatory elements. This invention further encompasses host cells and host cell lines expressing the polypeptides disclosed herein.

[0023] One embodiment of the invention encompasses a method of facilitating crystallization of a circularly permuted caspase or a functional variant thereof comprising the following structure from N- to C-terminus; a small subunit, which is a small submit of the caspase or a functionally active variant thereof; a linker consisting of n glycine, serine, proline and / or alanine residues wherein 11 is 2 to 10 (e.g, 4 to 8); and a large subunit, which is a large submit of the caspase or a functionally active variant thereof;

[0024] which circularly permuted caspase or a functional variant thereof comprises a loop region, which loop region is a contiguous amino acid sequence that consists of 10 residues at the C-terminus of the small subunit, the linker, and 11 residues at the N-terminus of the large subunit; which method comprises2024-337 09531.607W01

[0025] removing amino acid residues from the loop region and / or replacing amino acid residues in the loop region to provide a modified loop region that is more conformationally stable than the loop region.

[0026] In particular methods, the caspase is caspase-2. In some methods, the linker is GS, GG, GPPAG, GPAGGPG, GPSGG, GGSGG, SGAGSAAGSG, (GS)mwherein m is 1 to 4, GSG, or G4S. In some, the linker is GS, GG, GPPAG, GPAGGPG, or GPSGG.

[0027] Preferred methods do not materially affect the secondary structure of the polypeptide. Preferred methods do not materially affect the binding site of the polypeptide. In some methods, the crystallization is a co-crystallization with a compound that inhibits wild-type caspase activity.

[0028] Another embodiment of the invention encompasses a method of screening compounds for their ability to bind to a caspase having a binding site, which method comprises: obtaining a crystal structure of a caspase variant, which variant is a circularly permuted variant of the caspase or a functional variant thereof; modeling in silico interactions between the binding site and a compound; and measuring binding of the compound to the caspase or caspase variant in vitro.

[0029] In one embodiment of this method, the crystal structure is a structure of the caspase variant cocrystallized with a caspase inhibitor. In a particular embodiment, the caspase is caspase-2. In certain embodiments, the caspase inhibitor is AcVDVAD-CHO, AcIDVKD-CHO, AcVDVRD-CHO, AcDVPD-CHO, AcDVAD-CHO, AcDEVD-CHO. AcDRTD-CHO, AcAKD-CHO, AcTRD-CHO, or MUR-65. In particular embodiments, the caspase variant has at least 80. 85, 90, or 95 sequence homology with cpCasp2.

[0030] 3. BRIEF DESCRIPTION OF THE FIGURES

[0031] Aspects of some embodiments of the invention may be understood from the attached figures. FIG. 1A provides a simplified depiction of the caspase genes and subunit arrangement into the active homodimer. FIG. IB provides a cartoon depiction of Casp2 bound to inhibitor AcVDVAD-CHO. The 12 stranded P-sheet drives dimerization. The active site loops are LI, L2, L2’. L3. and L4.

[0032] FIG. 2 provides an annotated cpCasp2 Sequence. An N-terminal 6-his tag was inserted into the sequence. The dot marks the beginning of the small (pl2) subunit which includes residues 334-452. The “linker” follows and is numbered as 1001 and 1002. Immediately following is the large (pl8) subunit from residues 1170 to 1333. Secondary’ structures are labeled as follows: -sheets (B), a-helix (H); loops (L): turns (T).

[0033] FIG. 3 show s ColabFold model of cpCasp2. Important structures like the engineered loop (L5) and helix 2 (H2) are labeled. The cartoon is shaded by confidence scores, from dark to light: 90 < x < 100; 70 < x < 90; 50 < x < 70; and x < 50.2024-337 09531.607W01

[0034] FIG. 4 shows the sequences of modified loop regions of particular embodiments of the invention, which may contain modified small and / or large cpCasp2 subunits with or without an engineered linker.

[0035] FIG. 5 provides a pET-30a(+) vector map. Gene sequences were inserted into the pET-30a(+) vector between the Ndel and EcoRV restriction sites.

[0036] FIGS. 6A-E provide purification information for some mutants of the invention. FIG.6A shows a zoomed-in IEX trace for A127-132cpCasp2. This mutant elutes with many overlapping peaks, making it difficult to obtain pure protein. These peaks were further investigated by SEC as shown in FIGS. 6C-E. This trace is representative of the IEX traces of most of the mutant enzymes, with the exception of JFlcpCasp2. FIG.6B provides a zoomed-in IEX trace of JFlcpCasp2. This mutant elutes as one main peak with a slight shoulder, a small second peak which mostly overlaps the main peak. FIGS. 6C-E contain the SEC traces of isolated peaks from A127-132cpCasp2 IEX.

[0037] FIGS. 7A-D provide results from molecular dynamics simulations. FIG. 7A shows the root mean square deviation (RMSD) over time plot for cpCasp2. The RMSD does not stabilize, suggesting the enzyme is relatively flexible. FIG. 7B. RMSD over time plot for JFlcpCasp2. The RMSD stabilizes quickly after the start of the simulation, suggesting a stable enzyme. FIG. 7C. The ColabFold model of cpCasp2 edited to show regions of stability (dark shading) or flexibility (light shading), based on average root mean square fluctuation (RMSF) calculations. FIG. 7D. Radius of gyration of L5 for each enzy me. cpCasp2 has the largest rg, with all the mutants having a significantly (p<0.05) shorter rgwhen analyzed in a one-way ANOVA test.

[0038] FIG. 8A shows a standard curve generated using free 7-amino-4-trifluoromethyl coumarin (AFC) to convert relative fluorescent units (RFU) to moles. FIG. 8B shows a comparison of Michaelis-Menten plots for wtCasp2 and cpCasp2.

[0039] FIGS. 9A-E provide a comparison of crystal structures of JFlcpCasp2 and wtCasp2 with bound AcVDVAD-CHO. FIG. 9A. Alignment of JFlcpCasp2 (PDBid 8vp4) and wtCasp2 (PDBid 3r6g). RMSD of alignment is 0.364. FIG.9B. Polder Omit map of AcVDVAD-CHO bound to JFlcpCasp2 (isomesh at 3a). FIG. 9C. H-bond interactions of AcVDVAD-CHO bound to wtCasp2. FIG. 9D. H-bond interactions of AcVDVAD-CHO bound to JFlcpCasp2. FIG. 9E. H-bond interactions of MUR-65 bound to JFlcpCasp2.

[0040] FIG. 10 provides data collection and processing statistics for two crystal structures of the invention, wherein JFlcpCasp2 was co-crystalized with AcVDVAD-CHO or MUR-65.

[0041] 4. DETAILED DESCRIPTION

[0042] While circularly permuted caspases have been available for over a decade, there have been no reports of their crystallization and our attempts to crystallize cpCasp2 were not successful. This invention2024-337 09531.607W01

[0043] is directed, in part, to novel mutant forms of cpCasp2 that may be cry stalized and used, for example, in discovering new inhibitors of the protein.

[0044] 4.1. DEFINITIONS

[0045] Unless otherw ise indicated, the term “about” means ± 10% of the indicated range.

[0046] Unless otherwise indicated, the term “caspase-2” refers to human wild-type caspase-2.

[0047] As used herein, the term “functionally active” w hen referring to a cpCasp2 variant means that, upon dimerization, the variant performs the same function (e.g., proteolytic cleavage) in vitro as activated wild-type caspase-2.

[0048] The terms “mutant” and “variant” are used interchangeably.

[0049] The term “wild-ty pe” generally refers to a phenoty pe, genoty pe, or gene that predominates in a natural population of organisms or strain of organisms in contrast to that of natural or recombinant mutant variants. In other words, “wild-ty pe” refers to the form or forms of a gene commonly occurring in nature in a given species. The term “wild-type” with respect to amino acid sequences or domains within caspase-2, cpCasp2, or mutants thereof, means the amino acid or domains thereof found in nature.

[0050] 4.2. CASPASE-2 MUTANTS

[0051] Caspase-2 mutants of this invention are derived from circularly permuted, or “reversed”, forms of the protein, referred to as “cpCasp2”. Examples of cpCasp2 proteins are described, for example, in U.S. patent no. 6,379,950 to Alnemri and international patent application publication WO 2021 / 028590 to Jungbauer et al. (“Jungbaurer”). Small subunits of capsase-2 include human wild-type subunit (SEQ ID. No. 2) and variations thereof, including those described in lungbaurer. Large subunits of capsase-2 include human wild-ty pe subunit (SEQ ID. No. 3) and variations thereof, including those described in Jungbaurer. Boundaries of die small and large subunits of human and other species’ capsase-2 are well documented. See, e.g., Jungbaurer at 47.

[0052] The mutants of this invention may contain a tag (e.g., at the N-terminus) to allow for purification, to increase solubility', or to achieve other purposes for which tags are commonly used.

[0053] Examples of affinity tags include polyhistidine tags, poly-arginine tags, peptide substrates for antibodies, chitin binding domains, RNAse S peptide, protein A, 11 -galactosidase, a FLAG tag, a Strep II tag. streptavidin-binding peptide (SBP). calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP). an S-tag, an HA-tag. a c-Myc tag, a SUMO tag. E.coli thioredoxin. NusA, chloramphenicol. 5-acetyl transferase CAT, LysRS. ubiquitin, calmodulin, and lambda gpV.

[0054] Examples of solubility enhancement tags include T7C, T7B, T7B1, T7B2, T7B3, T7B3, T7B4, T7B5, T7B6, T7B6, T7B7, T7B8, T7B9, T7B10, T7B11, T7B12, T7B13, T7A, T7A1, T7A2, T7A3,2024-337 09531.607W01

[0055] T7A4, T7A5, T3, Nl, N2, N3, N4, N5, N6, N7, T7AC, calmodulin-binding peptide (CBP), DsbA, DsbC, poly Arg, poly Lys, G Bl domain, protein D, Z domain of Staphylococcal protein A, and thioredoxin.

[0056] The variants described herein are more confonnationally stable — or exhibit less structural disorder at room temperature — than their corresponding wild-type caspase or circularly permuted variants thereof. This enhanced stability makes it easier to crystalize the proteins either alone or with compounds that bind to their active sites. Methods of assessing or estimating a protein’s stability, or structural order, are well known and may be quantified by, for example, the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values of residues within the polypeptide chain that connects the small and large caspase subunits (e.g., what is referred to herein as Loop 5 of cpCasp2). These values may be calculated by in silico molecular modeling. Other methods of assessing conformational stability include differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR) spectroscopy, and optical spectroscopy -based methods such as circular dichroism (CD), UV absorbance and fluorescence. See, e.g., Atsavapranee, B., et al., “Fundamentals to function: Quantitative and scalable approaches for measuring protein stability ” Cell Systems 2021; 12:547-560.

[0057] Preferred variants of this invention do not materially affect the secondary structure of the polypeptide. In particular, they do not disrupt the secondary' structures of the small and large caspase subunits they contain. Preferred variants also do not materially affect the proteins’ binding site. This may be determined by, for example, measuring the catalytic efficiency of a variant or its ability to bind to a compound to which the corresponding wild-type caspase binds (e.g., AcVDVAD-CHO or AcIDVAD-CHO).

[0058] 4.2.1. Mutant Design

[0059] Residue numbering for cpCasp2 follows wtCasp2 numbering as much as possible to simplify comparisons. Where the subunits are swapped, 1000 has been added to the wtCasp2 numbering to keep the sequence in ascending order. The entire sequence of cpCasp2 with relevant loops and secondary' structure elements labeled can be found in FIG.2. For ease of reference, the loops were numbered sequentially, with the engineered loop becoming Loop 5 (L5). The reengineering of cpCasp2 for crystallography was aided by the use of ColabFold39(open-source AlphaFold). The highest-ranking ColabFold model is depicted in FIG.3. This investigation focused on the structural integrity of the engineered loop, which links the N- and C-tennini of the large and small subunits. ColabFold failed to find an ordered structure for the engineered loop L5 (confidence scores <70), suggesting that this section of the protein is not well ordered.40This led to the hypothesis that the unstructured “floppy” loop could interfere with the enzyme’s ability to form the highly ordered lattice necessary for crystallization.2024-337 09531.607W01

[0060] Using this ColabFold model to explain the failed attempts at crystallization, a new series of enzymes with a shortened L5 was designed. These mutants can be categorized into two families based on how their sequence was designed. The first family includes mutants A126-129cpCasp2, A126-131cpCasp2, A126-132cpCasp2, A127-131cpCasp2. A127-132cpCasp2, and A125-132cpCasp2, and was designed by simply deleting amino acids from L5. earning them the title of "deletion mutants”. Six mutants in this family were named according to the amino acids of cpCasp2 that were deleted. Due to the significant gaps in the numbering of cpCasp2 (e.g., amino acid 452 precedes 1001 due to the rearrangement of the subunits), a purely sequential numbering system was adopted, starting from Metl instead of Met327. For instance, the mutant with amino acids Thrl26 through Glyl29 removed is designated as A126-129cpCasp2. This sequential numbering is implemented solely for the sake of simplifying mutant nomenclature.

[0061] The second family of mutants is more complex in design. Similar to the deletion mutants, portions of the L5 region surrounding the G-S linker were removed, but in this case, additional small, turn-promoting amino acids were incorporated to enhance loop stability. This group is referred to as “engineered mutants,” examples of which include what are referred to herein as JFlcpCasp2, JF2cpCasp2, JF3cpCasp2, and JF4cpCasp2.

[0062] Deletion mutants have betw een 2 and 12 (e.g., 4 to 8) amino acid deletions on either side of the original cpCasp2 linker, while the engineered mutants started with removal of a larger portion (e.g., 9 or 10) of amino acids. Using Coot,41the loop was then built back out with, for example, 2 to 7 smaller, turnpromoting amino acids ( / .e., glycine, proline). This model was then exported to Maestro42in Schrodinger and energy' minimized to predict if L5 was long enough to preserve the enzy me’s secondary structure leading up to the loop. Preferred embodiments of this invention comprise an L5 portion that does not distort or completely disorder Helix 2 (H2), which borders L5. See FIG. 3.

[0063] FIG. 4 shows the sequences of modified loop regions of particular embodiments of the invention, which may contain modified small and / or large cpCasp2 subunits with or without an engineered linker. Sequence numbering for all the mutants follows that of cpCasp2. The amino acids added to L5 of the engineered mutants are numbered starting from 1001, consistent with numbering for the cpCasp2 G-S linker.

[0064] 4.2.2. Expression and Purification

[0065] The cpCasp2 gene without the solubility' tag, described by Cserjan-Puschmann et al., was inserted into apET30a (+) vector (GenScript; Piscataway, NJ) atNdel-EcoRV restriction sites.31A depiction of the cpCasp2 plasmid is shown in FIG. 5. Expression conditions were rapidly optimized, yielding an average cell pellet of 6 g per liter of culture. Purification of the pellet through immobilized2024-337 09531.607W01

[0066] metal affinity chromatography (IMAC) followed by ion exchange chromatography (IEX) resulted in a final yield of 9 mg of protein.

[0067] Mutants were cloned in the same maimer as cpCasp2, varying only in sequence. Complete sequences of particular embodiments of the invention are provided below. None of the mutations made were detrimental to the expression of the protein in E. coli. The average cell pellet size per liter of expression culture was 5g for all of the mutants that were made, comparable to that of cpCasp2. Protein yields after crude purification IMAC (nickel affinity) varied among the mutants, ranging from 10 to 25 mg per liter of expression culture. The most notable difference in purification occurred during IEX; the UV traces for all mutants, except JFlcpCasp2, displayed overlapping peaks near the expected elution point, which could indicate differing ionization states or truncated mutant forms. To further investigate this elution pattern, SEC and sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were conducted on each IEX peak. The first IEX peak elutes off the SEC as one species, while the following peaks all elute as two or more, although they all run the same size on the SDS-PAGE. Based on these observations, the peaks may represent different oligomeric states of the mutants. See FIG.6.

[0068] Among the mutants made, JFlcpCasp2 retained purification that was most similar to that of cpCasp2, both in quantity and quality. Unlike the other mutants, which displayed overlapping peaks after IEX, cpCasp2 and JFlcpCasp2 have elution patterns with one prominent peak each. See FIG. 6. This is particularly important as crystallography experiments necessitate a homogenous protein sample; thus, maintaining a single peak was crucial for subsequent experiments. While most mutants experienced a loss of more than 50% of the sample during IEX due to the formation of different oligomeric states, nearly 60% of JFlcpCasp2 was successfully retained (Table 1). This retention partially accounts for the higher protein yields observed in JFlcpCasp2. compared to the other mutants.

[0069] Table 1. Ion Exchange Chromatography Statistics

[0070]

[0071] 2024-337 09531.607W01

[0072] 4.2.3. Molecular Dynamics of cpCasp and Mutants

[0073] Molecular Dynamics simulations were run through Desmond, as implemented in Schrddinger / 2022.2,43on all the mutants to further predict their relative stability. The root mean square deviation (RMSD) and root mean square fluctuation (RMSF) were calculated for whole proteins, and the radius of gyration (rg) for L5. The RMSD is the average distance between the atom’s position at any given timepoint in the simulation, superimposed with the atoms in the first frame.44Frequently, this measurement is used to determine the stability or e lastici ty of a structure over the course of the simulation.44Stable structures typically exhibit a steady RMSD, while flexible structures tend to display more variation in RMSD. As such, the average RMSD and standard deviation (o) were calculated for each simulation, disregarding the first 25 nanoseconds (ns) for initial equilibration of the systems, as a quantitative measure for stability. Under this analysis, the most stable mutant was JFlcpCasp2 (o = 0.147 A), followed by A127-131 cpCasp2 and A126-132 cpCasp2 (o = 0.189 and 0.194 A respectively). The RMSD plots of cpCasp2 and JFlcpCasp2 over time indicate a clear increase in stability of JFlcpCasp2 over cpCasp2, which never reaches equilibrium. See FIG. 7A and 7B. Preferred mutants of the invention have an RMSD < 2 A.

[0074] The RMSF describes portions of the structure (e.g., residues) that deviate from their average position the most.45Gratifyingly, there were no added regions of flexibility in any of the mutants, suggesting that the secondary' structure was not significantly altered. FIG. 7C presents the AlphaFold model of cpCasp2. highlighting regions of high flexibility (RMSF > 15 A) in lighter shading, while depicting more stable regions (RMSF < 15 A) arc darker. The low confidence residues at the termini have been cut off for clarity in the model.

[0075] Focusing on die flexibility of L5, the radius of gy ration of this loop was calculated for each mutant to determine how compact the designed loops might be. Each mutant exhibited a significantly smaller average radius of gyration for L5 compared to cpCasp2 (p < 0.05), as depicted in FIG. 7D. This suggests that the shorter loops sustain a more compact conformation throughout the simulation. Overall, the molecular dynamics studies support the hypothesis that shortening L5 of cpCasp2 increases enzyme stability.

[0076] 4.2.4. Enzyme Kinetics and Stability

[0077] Saturation Binding Experiment. Michaelis-Menten kinetics were measured through a fluorescence-based binding saturation assay with a known fluorescent substrate, Z-VDVAD-AFC (Cayman Chemicals). Using free 7-amino-4-trifluoromethyl coumarin (AFC), a standard was generated to convert relative fluorescent units (RFU) to moles (FIG. 8B). This conversion allowed calculation of the catalytic constant ( / vcat) and determination of enzyme efficiency

[0078]

[0079] Table 2 lists the enzyme constants and rates measured for wtCasp2, cpCasp2. and embodiments of the invention. The Michaelis2024-337 09531.607W01

[0080] constant (KM) of cpCasp2 was calculated to be 128.1 pM + 5.66 pM, which is statistically different from wtCasp2 with a KM of 61.85 pM ± 7.88 pM (Table 2). FIG. 8A shows a comparison of the two Michaelis-Menten plots. While the catalytic constants for these two enzymes were not different, due to the substrates higher binding affinity for wtCasp2. cpCasp2 had a significantly lower catalytic efficiency (Table 2).

[0081] When conducting the analysis of the mutant enzymes, enzyme constants and rates were compared with cpCasp2. rather than wtCasp2. to evaluate what affect the mutations had on the enzyme. Notably, none of die mutants had a significantly different binding affinity (K ) with the substrate (Z-VDVAD-CHO), indicating that the mutations made have not disrupted the active site. A few differences could be observed, however, in turnover rates (feat) and enzyme efficiency. Mutants A127-13fcpCasp2 and A127-132cpCasp2 had significantly higher featvalues (p < 0.05) when analyzed by one-way ANOVA. These two mutants additionally had significantly higher enzy me efficiency than cpCasp2, as did A126-129cpCasp2. ft is plausible that the reduction in L5 length has mitigated the interference between two enzyme molecules in solution, thereby increasing the accessibility of the active site for the substrate. However, although this hypothesis holds merit, not all mutations resulted in improved turnover rates, suggesting that this may not fully account for the observed effects. Additionally, JFfcpCasp2 exhibits a significantly lower catalytic efficiency than cpCasp2, further implying there are other variables affecting the function of these enzymes. One potential contributing factor is the stability or flexibility of the enzyme. The predicted additional stability in JFlcpCasp2 from the MD studies indicates a reduction in flexibility, possibly affecting the accessibility of the active site and reducing the turnover rate.46This same pattern can be seen in other engineered mutants, with lower catalytic constants and enzyme efficiency than cpCasp2 and the deletion mutants.

[0082] Table 2. Enzyme Efficiency of wtCasp2, cpCasp2, and cpCasp2 Mutants

[0083]

[0084] 2024-337 09531.607W01

[0085]

[0086] Fluorometric Enzyme Assay. After confirming the functionality of the mutant enzymes, the next step was to verify their sensitivity to inhibition, a requirement for their potential utilization in cocrystallization studies. The inhibition profile of each mutant was evaluated using previously characterized inhibitors that showed a range of activity.25,38Tables 3 and 4 provide IC50 values for selected inhibitors with wtCasp2, cpCasp2, and various embodiments of the invention, the N of which for each is 3. AcVDVAD-CHO was purchased from Cayman Chemicals (Ann Arbor, MI); the synthesis and characterization of AcIDVKD-CHO, AcIDVKD-CHO, AcVDVRD-CHO and AcDEVD-CHO are described by Bresinsky et al.25; the synthesis and characterization of AcDVPD-CHO, AcDVAD-CHO, AcDRTD-CHO, AcAKD-CHO, AcTRD-CHO are described by Bresinsky et al.382024-337 09531.607W01

[0087] Table 3. ICso Values for Selected Inhibitors

[0088]

[0089]

[0090] 2024-337 09531.607W01

[0091] Table 4. ICso Values for Selected Inhibitors

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[0093]

[0094]

[0095] For this assay, statistical significance was calculated with respect to wtCasp2 to assess reliability in using these enzymes as tools to evaluate inhibitors designed for wtCasp2. None of the compounds exhibit a statistically significant variance in IC50 values among the mutants when compared to the wild-fipe enzyme. This is crucial as it indicates that not only can we effectively inhibit the function of the mutants, but also that the inhibitors interact similarly within the active sites. This instills confidence that any co-crystal structures obtained with our compounds and mutant enzymes would yield information reflective of compound interactions with the wild-type enzyme.

[0096] 4.2.5. Crystallography

[0097] While some of the other mutants were also screened for crystal conditions, multiple conditions were quickly found that resulted in crystallization of the mutant JFlcpCasp2 with the canonical inhibitor AcVDVAD-CHO. Following this, crystallography experiments were focused on this one mutant to streamline workflow. Buffer conditions which lead to crystals were repurchased from Molecular Dimensions (the ECO-PACT crystallization screen - individual reagents) for future experiments. Crystals chosen for data collection grew in 0.1 M tris, pH 8.0. 0.2 M sodium chloride, and 20% w / v PEG 6000, appeared in 24 hours, and grew to full size in 2-4 days. JFlcpCasp2 - AcVDVAD-CHO co-crystals form a new orthorhombic crystal system (space group P212121) with unit cell parameters of a = 55.867 A. b = 101.548 A, and c = 112.555 A, and two molecules (homodimer) in the asymmetric unit.

[0098] Diffraction to 1.51 A confirms the ColabFold predicted tertiary structure of the JFlcpCasp2 monomer (RMSD of overlay = 0.397). This structure also confinns that the circular pennutation of wtCasp2 does not affect its affinity to dimerization, as JFlcpCasp2 with AcVDVAD-CHO generates a homodimer nearly identical to that of wtCasp2 with the same inhibitor (RMSD of overlay = 0.364) (FIG.

[0099] 9 A).47A majority of the variability between JFlcpCasp2 and wtCasp2 can be accounted for by differences in active site loop LI conformation, with alignment of these loops (JFlcpCasp2Asnl207-Glyl222 and wtCasp2Asn207-Gly222) generating an RMSD of 1.189, significantly higher than that of the overall structures.47

[0100] Covalent binding of the C-tenninal aspartic acid of the inhibitor to the catalytic cysteine (Cysl320) is supported by well-defined electron density for the entire peptide, with placement bias minimized using a Polder omit map. See FIG.9B. When compared to the published structure of wtCasp2 with AcVDVAD-CHO (PDBid 3r6g)47, the binding motifs of the peptide inhibitor are identical. See FIGS.

[0101] 9C-D. The backbone of the peptide inhibitor makes hydrogen bonding interactions with residues Thr380. Tyr(l)240, Arg378, Ala376, and His(l)277. The nitrogen at P2 also makes hydrogen bonding interactions with a water molecule in both structures. Similar binding interactions are also seen in both structures in the side chains of Pl, P4, and P5. See FIGS. 9C-D.

[0102] The co-crystal structure of JFlcpCasp2 with AcVDVAD-CHO not only established the atomic structure of JFlcpCasp2, but also confirmed that the active site and binding mode of peptide inhibitorsremained unchanged compared to wtCasp2. This provides reasonable evidence that JFlcpCasp2 can be used as a surrogate for wtCasp2 in structure-based design for further inhibitor development. In fact, to further demonstrate the utility of this Casp2 construct, crystals of JFlcpCasp2 were prepared and characterized using the potent and selective Casp2 inhibitor MUR-65.29

[0103]

[0104] MUR-65

[0105] Co-crystals of JFlcpCasp2 and MUR-65 diffracted to 1.959 A and form a new monoclinic crystal system (space group P21) not seen in any previously published Casp2 structures. Cell parameters for this complex are a = 63.288 A, b = 130.246 A, and c = 78.300 A. with angles a and y = 90 ° and = 104.56 °. The asymmetric unit is comprised of four molecules, or two homodimers, in which one homodimer is related to the other through a mirrored image with a 104.56° rotation about a single symmetry axis.

[0106] Despite the higher order symmetry, the homodimer of the co-crystal structure with MUR-65 is not significantly different than that of the structure with AcVDVAD-CHO (RMSD of overlay = 0.214).

[0107] The active site interactions between JFlcpCasp2 and AcVDVAD-CHO are conserved in the structure with MUR-65. Interestingly, the P4 hGlu makes two new hydrogen bonding interactions with Arg417 and Asn379. See FIG. 9E. Interactions with these residues have never been seen before, and although MUR-65 does not have increased affinity at Casp2 compared to AcVDVAD-CHO, MUR-65 is 794-fold selective for Casp2 over Casp3, while AcVDVAD-CHO has no Casp2 selectivity29The equivalent position to Arg417 in Casp3 is Phc247, which docs not have the ability to form H-bonds. Thus, targeting the Arg417 (cpCasp2 numbering) may be an effective strategy for gaining Casp2 selectivity.

[0108] 4.3. MATERIALS AND METHODS

[0109] 4.3.1. Protein Expression, Isolation, and Purification

[0110] cpCasp2. A circularly permuted caspase-2 (cpCasp2) based on modifications to wtCasp2 (UniProt P42575) suggested by Cserjan-Puschmann et al. (2020)31was expressed. The gene encoding 6H-Casp2334-452-GS-Casp2170-333 with a D347 — > A347 mutation was inserted into a pET30a(+) vector (GenScript, Piscataway, NJ, USA) using Ndel and EcoRV restriction sites. The transformation into Lemo21 cells was performed as recommended by the vendor (New England Biolabs, Ipswich, MA, USA), and the plates were incubated overnight at 37 °C. A single colony was picked for primary culture using asterile wooden stick, added to 5 mL sterile Luria broth (LB) media containing kanamycin and chloramphenicol to final concentrations of 50 pg / mL and 30 pl / inL respectively (Kan50. Cm30), and incubated for 6-8 hours at 37 °C and 270 rpm. The primary culture was then diluted 1:10 into a 200mL shake flask containing sterile LB and antibiotics. This secondary culture was incubated overnight at 37 °C and 270 rpm and then diluted 1:50 into 1 L shake flasks the following morning. The cultures were incubated at 37 °C and 270 rpm, and the optical density (OD) was monitored until it reached betw een 0.6 and 0.8. Once the desired OD was reached, the cells were induced overnight with 0.1 mM isopropyl- b-D-thiogalactosidase (IPTG) at 18 °C and 270 rpm. After induction, the cells were harvested through centrifugation at 5000 g for 20 min. Typical yield was about a 3.5 g cell pellet per liter. The pellets were stored at -20 °C until purification.

[0111] Pellets were resuspended in lysis buffer (lOOmM Tris, pH 8.0, lOOmM NaCl, and 20mM Imidazole with 2.5 pM Leupeptin, 5 pM Pepstatin, 0.5 pM DNase, 1 mM PMSF, and 10 mM MgCL) and lysed through Bonification (30 seconds on followed by 30 seconds off for 8 min). The lysate was clarified through centrifugation at 20,000 g for 40 min. The supernatant was filtered through a 0.45 pm filter before it was purified on a Cytiva AKTA Pure system (Marlborough, MA). Purification was achieved through nickel-affmity chromatography on a HisTrap FF crude column (Cytiva) followed by anion exchange chromatography on a HiTrap Q anion exchange column (Cytiva). Protein for enzymatic assays was concentrated to 0.5 mg / mL in buffer (25mM Tris, pH 8.0 and 150mM NaCl), cryoprotected with 5% glycerol, flash frozen in liquid nitrogen (LN2) and stored at -80°C.

[0112] Further purification was necessary for crystallographic experiments. Following ion exchange chromatography. SEC was run using a HiPrep Sephacryl S-200 HR preparative SEC column (Cytiva) on a Cytiva AKTA Pure system. Protein was concentrated to 0.5 mg / mL in SEC buffer (25mM Tris pH 8.0, 150mM NaCl, and 5mM DTT) with 5% glycerol, aliquoted to 500 pL and stored at -80°C.

[0113] Deletion Mutants. All six cpCasp2 mutant genes were purchased as mutations of our cpCasp2 gene described above (GenScript. Piscataway, NJ, USA). Expression and purification of each mutant was performed as described for cpCasp2 with no further optimization. Typical pellet size for all deletion mutants for 1 L of expression culture was about 6 g. Protein yield for each mutant can be found in the Table 1.

[0114] Re-Engineered Mutants. All four re-engineered mutants were purchased as new plasmids from GenScript. Sequences were inserted into a pET30a(+) vector using Ndel and EcoRV restriction sites by GenScript. The transformation and further expression and purification were carried out as described above for cpCasp2. Typical pellet size for all mutants for 1 L of expression culture was about 6 g. Protein yield after IEX for each mutant can be found in Table 1. wherein the values are nonnalized to volume of expression culture.4.3.2. Caspase Enzyme Assays

[0115] Saturation Binding Experiments. Michalis-Menten kinetics (KM values) were determined experimentally through a fluorometric assay. All Casp2 mutants were diluted to 21 nM in assay buffer (100 mM MES, pH 6.5, 150 mM NaCl. 0.1% CHAPS, 1.5% sucrose, and 10 rnM added on the day of the assay). Enzymes were then added (19 pL) to appropriate wells in a black 384-well Corning 4514 assay plate (to reach a final assay concentration of 20 nM). Ten serial dilutions (2-fold) of an AFC substrate (Z-VDVAD-AFC) were prepared in DMSO beginning at 10 mM. The dilutions were plated in duplicate into a Coming 3656 transfer plate and then added to the assay plate in 1 pL aliquots per well (final assay concentrations of 0-500 pM). mixing 10 times, using a BiomekFX (Beckman Coulter). After addition of the AFC substrate the assay was immediately run. Fluorescence from free AFC was read at 37 °C every 5 min over an hour using a CLARIOstar (BMG Labtech) plate reader (Xex= 400 nm, XCm = 505 nm). The 40 min time point was reported, consistent with reported literature.

[0116] Fluorometric Enzyme Assay (96 well). Compound affinity for caspases was measured in fluorometric assays. wtCasp2, cpCasp2, and Casp3 were produced in house as described previously.25,38AFC fluorogcnic substrates Z-VDVAD-AFC and AcDEVD-AFC and control peptides AcVDVAD-CHO and AcDEVD-CHO were purchased from Bachem (Torrance / CA, USA). The enzyme was diluted in buffer: 100 mM MES (pH 6.5) for wtCasp2 and cpCasp2 or 100 mM HEPES (pH 7.0) for Casp3, plus 150 mM NaCl, 0.1% CHAPS, 1.5% sucrose, and 10 mM DTT. Enzyme concentrations were 5 nM / well for wtCasp2 and cpCasp2 and 2 nM / well for Casp3. Enzyme in buffer (96.5 pL) was added per well in a black Coming 335696-well assay plate. Test compounds were serially diluted in DMSO and plated in triplicate in a Coming 3357 transfer plate. The test compound was added to assay plates in 1 pL aliquots per well and mixed 10 times using a BiomekFX (Beckman Coulter). The compound and enzyme mixture was incubated at 37 °C for 5 min. The BiomekFX was then used to add and mix 2.5 pL of the AFC substrate in DMSO from a transfer plate (final assay concentrations: 25 pM Z-VDVAD-AFC for wtCasp2 and cpCasp2, 10 pM AcDEVD-AFC for Casp3) to the assay plate for a total assay volume of 100 pL in the assay plate. Fluorescence from free AFC was read at 37 °C every 5 min over an hour using a CLARIOstar (BMG Labtech) plate reader (Zex= 400 nm, z.Cm = 505 nm).

[0117] GraphPad Prism version 9 was used to calculate the IC50 by fitting the dose-response data with four parameter variable slope nonlinear regression.48The 40-minute time point was reported, consistent with reported literature.25,28,50,31

[0118] 4.3.3. Computational Methods

[0119] AlphaFold. Proteins were all modeled using the open source version of AlphaFold (ColabFold).39Each mutant sequence was copied into the "query sequence” and then the program was run with the default settings. The output model with the highest rank (rank 1) was chosen for each mutant.Desmond Molecular Dynamics. Prior to running molecular dynamics on all mutants, the 6-his tag was removed as well as the low confidence regions from AlphaFold (residues 1-30). The models were uploaded to Maestro and a protein minimization was run through the protein prep menu. Salt (0.15 M) was added to the system through Desmond System Builder. Desmond Molecular Dynamics program was opened, and the system input was defined as the last trajectory frame. Time was set to 100 ns and run interaction analysis was set to on. All other default settings were used, and the simulation was run to completion (4-6 hours).

[0120] Root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values over time were recorded in Maestro and exported to Prism version 10 for analysis. Radius of gy ration (rg) for L5 (residues 443-1180) were calculated in Maestro and exported to Prism version 10 for data analysis.

[0121] 4.3.4. Crystallography

[0122] Co-crystallization. Initial crystallography experiments with JFlcpCasp2 utilized nanoliter crystallization screens to screen for crystallization conditions. The three screens run were Hampton Research PEG / ion and PEGRx screens and Molecular Dimensions PACT Screen. Enzyme (2.5 mg / mL) was incubated with 50LLM inhibitor for 30 minutes before setting up cry stallization trays. Trays were set up on the Phoenix liquid handler (Art Robbins Inc.) at the nanoliter crystallization facility at the University' of Minnesota. Initial co-crystal hits of JFlcpCasp2 and the covalent inhibitor Ac-VDVAD-CHO were found in multiple conditions within the PACT screen, with the best diffraction resulting from 20% w / v PEG 6000, 0.2M sodium chloride, and 0.1M Tris, pH 8.0. These conditions were carried forward for crystallization of other peptidic inhibitors (MUR compounds). Plate-like crystals grew at room temperature within 24 hours and grew to full size in about 4 days. Cry stals were flash frozen in LN2 and shipped to NSLSII for data collection.

[0123] Data Collection. Diffraction data was collected at the AMX beamline at the National Synchrotron Light Source II (NSLSII) in Upton, New York, USA in both December 2023, and February' 2024.

[0124] Collection was completed at 100 K using a wavelength of 1.00 Angstroms (A) and a Eiger 9M detector. Data was processed using XDS52and re-scaled with ap scale using CCi / 2> 0.5 as criteria for resolution cutoff.

[0125] Molecular Replacement and Structure Refinement. A dimer was generated out of the AlphaFold model of JFlcpCasp2 (monomer) by aligning two monomer units to each chain of the co-crystal structure of wtCasp2 with bound AcVDVAD-CHO (PDBid 3r6g). The two JFlcpCasp2 monomers were saved as one PDB file and this was used as the search model for the molecular replacement of the first JFlcpCasp2 structure (JFlcpCasp2 bound to AcVDVAD-CHO). The deposited PDB file of this first structure (PDBid 8vp4) was used as the search model for all following JFlcpCasp2 co-crystal structures. All molecular replacement was carried out in Phaser53Crystals were either monoclinic or orthorhombic form with a homodimer or two homodimers in the asymmetric unit. Iterative rounds of refinement and model building were carried out in Phenix and Coot.41,54The pentapeptide inhibitor (AcVDVAD-CHO) was built directly into the electron density using Coot. Alternatively, a covalent inhibitor docking protocol employing the ‘"covalent docking” (CovDock) module of the Schrodinger Softw are Suite (Schrodinger, LLC, New York, NY. Version 2021.1) was implemented to predict the pose of MUR-65 in the JFlcpCasp2 active site before it was built into the electron density.35

[0126] The protein chain with the docked compound was then aligned with each chain of the MR solution in PyMOL so the docked compounds could be uploaded directly into the electron density in Coot. The covalent bond between Cys320 and the Pl aspartic acid was added through the restraints tab in Python, with 1.5 A length restriction and a sigma of 0.5. All refined structures were validated with MolProbilyC

[0127] Molecular Modeling. The crystal structure of JFlcpCasp2 (PDBid 8vp4) was imported into Maestro42in the Schrodinger Software Suite (Schrodinger, LLC, New York, NY, Version 2021.1). The protein was then prepared using the module “protein preparation wizard” with the default protein parameters. Hydrogen atoms were added, and water molecules that were beyond 5 A from heterocyclic groups were deleted. Hydrogen bonds were optimized, the partial charges were assigned, and the protein structure was energy -minimized using the OPLS3e force field. Following this preparation, the covalent bond connecting the ligand to the protein was broken, and the now-separated aldehyde (reactive functional group) and cysteine (nucleophilic reaction group) were reconstituted by adjusting bond orders, adding hydrogens, and minimizing these groups in place. This free ligand (the “workspace ligand”) was employed to create the covalent docking grid used in the covalent docking and scoring (vide infra). The individual target receptor was set up using the reactive cysteine residue (Cys320). The “reaction type” SMARTS string {[H]C=O} was built as a customized nucleophilic addition to a double bond. The “box center” for the docking grid was set using the “centroid of (the) workspace ligand”. Docking w as performed in the “pose prediction (thorough)” mode. A “minimization radius” of 3.0 A was used, “perform MM-GBSA scoring” w as selected, and three (3) “output poses per ligand reaction site” were selected (only the lowest energy pose is reported).

[0128] 4.3.5. Accession Codes

[0129] Atomic coordinates and reflection data for crystallographic complexes of JFlcpCasp2 with peptides AcVDVAD-CHO and MUR-65 (FIG. 10) have been deposited into the Protein Data Bank with accession codes 8vp4. and 9c2y.4.4. REFERENCES

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[0197] All publications (e g., patents and patent applications) cited above are incorporated herein by reference in their entireties.

[0198] 5. SEQUENCES

[0199] SEQ ID. No. 1 - Human caspase-2 MAAPSAGSWSTFQHKELMAADRGRRILGVCGMHPHHQETLKKNRVVLAKQLLLSELLEHLLEKDIITLEMRELIQA KVGSFSQNVELLNLLPKRGPQAFDAFCEALRETKQGHLEDMLLTTLSGLQHVLPPLSCDYDLSLPFPVCESCPLYKKLR LSTDTVEHSLDNKDGPVCLQVKPCTPEFYQTHFQLAYRLQSRPRGLALVLSNVHFTGEKELEFRSGGDVDHSTLVTLF KLLGYDVHVLCDQTAQEMQEKLQNFAQLPAHRVTDSCIVALLSHGVEGAIYGVDGKLLQLQEVFQLFDNANCPSLQ NKPKMFFIQACRGDETDRGVDQQDGKNHAGSPGCEESDAGKEKLPKMRLPTRSDMICGYACLKGTAAMRNTKR GSWYIEALAQVFSERACDMHVADMLVKVNAUKDREGYAPGTEFHRCKEMSEYCSTLCRHLYLFPGHPPT SEQ ID. No. 2 - Caspase-2 small subunit AGKEKLPKMRLPTRSDMICGYACLKGTAAMRNTKRGSWYIEALAQVFSERACDMHVADMLVKVNAUKDREGYAP GTEFHRCKEMSEYCSTLCRHLYLFPGHPPT SEQ ID. No. 3 - Caspase-2 large subunit GPVCLQVKPCTPEFYQTHFQLAYRLQSRPRGLALVLSNVHFTGEKELEFRSGGDVDHSTLVTLFKLLGYDVHVLCDQT AQEMQEKLQNFAQLPAHRVTDSCIVALLSHGVEGAIYGVDGKLLQLQEVFQLFDNANCPSLQNKPKMFFIQACRG DETDSEQ ID. No. 4 - cpCasp2 with N-terminal His tag and GS linker MHHHHHHGKNHAGSPGCEESAAGKEKLPKMRLPTRSDMICGYACLKGTAAMRNTKRGSWYIEALAQVFSERAC DMHVADMLVKVNALIKDREGYAPGTEFHRCKEMSEYCSTLCRHLYLFPGHPPTGSGPVCLQVKPCTPEFYQTHFQL AYRLQSRPRGLALVLSNVHFTGEKELEFRSGGDVDHSTLVTLFKLLGYDVHVLCDQTAQEMQEKLQNFAQLPAHRVT DSCIVALLSHGVEGAIYGVDGKLLQLQEVFQLFDNANCPSLQNKPKMFFIQACRGDETDRGVDQQD SEQ ID. No. 5 - Nucleotide insert sequence of plasmid for cpCasp2 with N-terminal His tag and GS linker catatgcaccatcatcaccatcatggcaaaaatcatgcaggtagtccgggttgtgaagaaagcgcagcaggtaaagaaaaactgccgaaaatgc gtctgccgacccgtagcgatatgatttgtggttatgcatgtctgaaaggcaccgcagcaatgcgtaataccaaacgtggtagctggtatattgaagc actggcacaggtttttagcgaacgtgcatgtgatatgcatgttgcagatatgctggttaaagtgaacgccctgattaaagatcgtgaaggttatgca ccgggtacagaatttcatcgttgtaaagaaatgagcgagtattgtagcaccctgtgtcgtcatctgtacctgtttccgggtcatcctccgaccggatc cggtccggtttgtctgcaggttaaaccgtgtacaccggaattttatcagacccattttcagctggcatatcgtctgcagagccgtccgcgtggtctggc actggttctgagcaatgttcattttaccggtgaaaaagaactggaatttcgtagcggtggtgatgttgatcatagtaccctggttaccctgtttaaact gctgggttatgacgttcatgttctgtgtgatcagaccgcacaagaaatgcaagagaaactgcagaattttgcacagctgcctgcacatcgtgttacc gatagctgtattgttgcactgctgagccatggtgttgaaggtgcaattatggtgtggatggcaaactgctgcaactgcaagaagtgtttcagctgtt tgataatgcaaattgtccgagcctgcagaataaaccgaaaatgttttttatccaggcctgccgtggtgatgaaaccgatcgtggtgttgatcagcag gattaataagatatc

[0200] SEQ ID. No. 6 - Sequence of A126-129cpCasp2 with N-terminal His tag and no linker MHHHHHHGKNHAGSPGCEESAAGKEKLPKMRLPTRSDMICGYACLKGTAAMRNTKRGSWYIEALAQVFSERAC DMHVADMLVKVNALIKDREGYAPGTEFHRCKEMSEYCSTLCRHLYLFPGHPPPVCLQVKPCTPEFYQTHFQLAYRL QSRPRGLALVLSNVHFTGEKELEFRSGGDVDHSTLVTLFKLLGYDVHVLCDQTAQEMQEKLQNFAQLPAHRVTDSCI VALLSHGVEGAIYGVDGKLLQLQEVFQLFDNANCPSLQNKPKMFFIQACRGDETDRGVDQQD SEQ ID. No. 7 - Nucleotide insert sequence of plasmid for A126-129cpCasp2 with N-terminal His tag and no linker catatgcaccatcatcaccatcatggcaaaaatcatgcaggtagtccgggttgtgaagaaagcgcagcaggtaaagaaaaactgccgaaaatgc gtctgccgacccgtagcgatatgatttgtggttatgcatgtctgaaaggcaccgcagcaatgcgtaataccaaacgtggtagctggtatattgaagc actggcacaggtttttagcgaacgtgcatgtgatatgcatgttgcagatatgctggttaaagtgaacgccctgattaaagatcgtgaaggttatgca ccgggtacagaatttcatcgttgtaaagaaatgagcgagtattgtagcaccctgtgtcgtcatctgtacctgtttccgggtcatcctccgaccggatc cggtccggtttgtctgcaggttaaaccgtgtacaccggaatttatcagacccattttcagctggcatatcgtctgcagagccgtccgcgtggtctggc actggttctgagcaatgttcattttaccggtgaaaaagaactggaatttcgtagcggtggtgatgttgatcatagtaccctggttaccctgtttaaact gctgggttatgacgttcatgttctgtgtgatcagaccgcacaagaaatgcaagagaaactgcagaattttgcacagctgcctgcacatcgtgttacc gatagctgtattgttgcactgctgagccatggtgttgaaggtgcaatttatggtgtggatggcaaactgctgcaactgcaagaagtgtttcagctgtt tgataatgcaaattgtccgagcctgcagaataaaccgaaaatgttttttatccaggcctgccgtggtgatgaaaccgatcgtggtgtgatcagcag gattaataagatatc

[0201] SEQ ID. No. 8 - Sequence of A126-131cpCasp2 with N-terminal His tag and no linker MHHHHHHGKNHAGSPGCEESAAGKEKLPKMRLPTRSDMICGYACLKGTAAMRNTKRGSWYIEALAQVFSERAC DMHVADMLVKVNALIKDREGYAPGTEFHRCKEMSEYCSTLCRHLYLFPGHPPCLQVKPCTPEFYQTHFQLAYRLQS RPRGLALVLSNVHFTGEKELEFRSGGDVDHSTLVTLFKLLGYDVHVLCDQTAQEMQEKLQNFAQLPAHRVTDSCIVA LLSHGVEGAIYGVDGKLLQLQEVFQLFDNANCPSLQNKPKMFFIQACRGDETDRGVDQQDSEQ ID. No. 9 - Nucleotide insert sequence of plasmid for A126-131cpCasp2 with N-terminal His tag and no linker catatgcaccatcatcaccatcatggcaaaaatcatgcaggtagtccgggttgtgaagaaagcgcagcaggtaaagaaaaactgccgaaaatgc gtctgccgacccgtagcgatatgatttgtggttatgcatgtctgaaaggcaccgcagcaatgcgtaataccaaacgtggtagctggtatattgaagc actggcacaggtttttagcgaacgtgcatgtgatatgcatgttgcagatatgctggttaaagtgaacgccctgattaaagatcgtgaaggttatgca ccgggtacagaatttcatcgttgtaaagaaatgagcgagtattgtagcaccctgtgtcgtcatctgtacctgtttccgggtcatcctccgaccggatc cggtccggtttgtctgcaggttaaaccgtgtacaccggaattttatcagacccattttcagctggcatatcgtctgcagagccgtccgcgtggtctggc actggttctgagcaatgttcattttaccggtgaaaaagaactggaatttcgtagcggtggtgatgttgatcatagtaccctggttaccctgtttaaact gctgggttatgacgttcatgttctgtgtgatcagaccgcacaagaaatgcaagagaaactgcagaattttgcacagctgcctgcacatcgtgttacc gatagctgtattgttgcactgctgagccatggtgttgaaggtgcaatttatggtgtggatggcaaactgctgcaactgcaagaagtgtttcagctgtt tgataatgcaaattgtccgagcctgcagaataaaccgaaaatgttttttatccaggcctgccgtggtgatgaaaccgatcgtggtgttgatcagcag gattaataagatatc

[0202] SEQ ID. No. 10 - Sequence of A126-132cpCasp2 with N-tcrminal His tag and no linker MHHHHHHGKNHAGSPGCEESAAGKEKLPKMRLPTRSDMICGYACLKGTAAMRNTKRGSWYIEALAQVFSERAC DMHVADMLVKVNALIKDREGYAPGTEFHRCKEMSEYCSTLCRHLYLFPGHPPLQVKPCTPEFYQTHFQLAYRLQSR PRGLALVLSNVHFTGEKELEFRSGGDVDHSTLVTLFKLLGYDVHVLCDQTAQEMQEKLQNFAQLPAHRVTDSCIVAL LSHGVEGAIYGVDGKLLQLQEVFQLFDNANCPSLQNKPKMFFIQACRGDETDRGVDQQD SEQ ID. No. 11 - Nucleotide insert sequence of plasmid for A126-132cpCasp2 with N-tcrminal His tag and no linker catatgcaccatcatcaccatcatggcaaaaatcatgcaggtagtccgggttgtgaagaaagcgcagcaggtaaagaaaaactgccgaaaatgc gtctgccgacccgtagcgatatgatttgtggttatgcatgtctgaaaggcaccgcagcaatgcgtaataccaaacgtggtagctggtatattgaagc actggcacaggtttttagcgaacgtgcatgtgatatgcatgttgcagatatgctggttaaagtgaacgccctgattaaagatcgtgaaggttatgca ccgggtacagaatttcatcgttgtaaagaaatgagcgagtattgtagcaccctgtgtcgtcatctgtacctgtttccgggtcatcctccgaccggatc cggtccggtttgtctgcaggttaaaccgtgtacaccggaattttatcagacccattttcagctggcatatcgtctgcagagccgtccgcgtggtctggc actggttctgagcaatgttcattttaccggtgaaaaagaactggaatttcgtagcggtggtgatgttgatcatagtaccctggttaccctgtttaaact gctgggttatgacgttcatgtctgtgtgatcagaccgcacaagaaatgcaagagaaactgcagaattttgcacagctgcctgcacatcgtgttacc gatagctgtattgttgcactgctgagccatggtgttgaaggtgcaatttatggtgtggatggcaaactgctgcaactgcaagaagtgtttcagctgtt tgataatgcaaattgtccgagcctgcagaataaaccgaaaatgttttttatccaggcctgccgtggtgatgaaaccgatcgtggtgttgatcagcag gattaataagatatc

[0203] SEQ ID. No. 12 - Sequence of A127-131cpCasp2 with N-terminal His tag and no linker MHHHHHHGKNHAGSPGCEESAAGKEKLPKMRLPTRSDMICGYACLKGTAAMRNTKRGSWYIEALAQVFSERAC DMHVADMLVKVNALIKDREGYAPGTEFHRCKEMSEYCSTLCRHLYLFPGHPPTCLQVKPCTPEFYQTHFQLAYRLQS RPRGLALVLSNVHFTGEKELEFRSGGDVDHSTLVTLFKLLGYDVHVLCDQTAQEMQEKLQNFAQLPAHRVTDSCIVA LLSHGVEGAIYGVDGKLLQLQEVFQLFDNANCPSLQNKPKMFFIQACRGDETDRGVDQQD

[0204] SEQ ID. No. 13 - Nucleotide insert sequence of plasmid for A127-131cpCasp2 with N-terminal His tag and no linker catatgcaccatcatcaccatcatggcaaaaatcatgcaggtagtccgggttgtgaagaaagcgcagcaggtaaagaaaaactgccgaaaatgc gtctgccgacccgtagcgatatgatttgtggttatgcatgtctgaaaggcaccgcagcaatgcgtaataccaaacgtggtagctggtatattgaagc actggcacaggtttttagcgaacgtgcatgtgatatgcatgttgcagatatgctggttaaagtgaacgccctgattaaagatcgtgaaggttatgca ccgggtacagaatttcatcgttgtaaagaaatgagcgagtattgtagcaccctgtgtcgtcatctgtacctgtttccgggtcatcctccgaccggatc cggtccggtttgtctgcaggttaaaccgtgtacaccggaattttatcagacccattttcagctggcatatcgtctgcagagccgtccgcgtggtctggc actggttctgagcaatgttcattttaccggtgaaaaagaactggaatttcgtagcggtggtgatgttgatcatagtaccctggttaccctgtttaaactgctgggttatgacgttcatgttctgtgtgatcagaccgcacaagaaatgcaagagaaactgcagaattttgcacagctgcctgcacatcgtgttacc gatagctgtattgttgcactgctgagccatggtgttgaaggtgcaatttatggtgtggatggcaaactgctgcaactgcaagaagtgtttcagctgtt tgataatgcaaattgtccgagcctgcagaataaaccgaaaatgttttttatccaggcctgccgtggtgatgaaaccgatcgtggtgttgatcagcag gattaataagatatc

[0205] SEQ ID. No. 14 - Sequence of A127-132cpCasp2 with N-terminal His tag and no linker MHHHHHHGKNHAGSPGCEESAAGKEKLPKMRLPTRSDMICGYACLKGTAAMRNTKRGSWYIEALAQVFSERAC DMHVADMLVKVNALIKDREGYAPGTEFHRCKEMSEYCSTLCRHLYLFPGHPPTLQVKPCTPEFYQTHFQLAYRLQSR PRGLALVLSNVHFTGEKELEFRSGGDVDHSTLVTLFKLLGYDVHVLCDQTAQEMQEKLQNFAQLPAHRVTDSCIVAL LSHGVEGAIYGVDGKLLQLQEVFQLFDNANCPSLQNKPKMFFIQACRGDETDRGVDQQD SEQ ID. No. 15 - Nucleotide insert sequence of plasmid for A127-132cpCasp2 with N-terminal His tag and no linker catatgcaccatcatcaccatcatggcaaaaatcatgcaggtagtccgggttgtgaagaaagcgcagcaggtaaagaaaaactgccgaaaatgc gtctgccgacccgtagcgatatgatttgtggttatgcatgtctgaaaggcaccgcagcaatgcgtaataccaaacgtggtagctggtatattgaagc actggcacaggtttttagcgaacgtgcatgtgatatgcatgttgcagatatgctggttaaagtgaacgccctgattaaagatcgtgaaggttatgca ccgggtacagaatttcatcgttgtaaagaaatgagcgagtattgtagcaccctgtgtcgtcatctgtacctgtttccgggtcatcctccgaccggatc cggtccggttgtctgcaggttaaaccgtgtacaccggaattttatcagacccattttcagctggcatatcgtctgcagagccgtccgcgtggtctggc actggttctgagcaatgttcattttaccggtgaaaaagaactggaatttcgtagcggtggtgatgttgatcatagtaccctggttaccctgtttaaact gctgggttatgacgttcatgttctgtgtgatcagaccgcacaagaaatgcaagagaaactgcagaattttgcacagctgcctgcacatcgtgttacc gatagctgtattgttgcactgctgagccatggtgttgaaggtgcaatttatggtgtggatggcaaactgctgcaactgcaagaagtgtttcagctgtt tgataatgcaaattgtccgagcctgcagaataaaccgaaaatgttttttatccaggcctgccgtggtgatgaaaccgatcgtggtgttgatcagcag gattaataagatatc

[0206] SEQ ID. No. 16 - Sequence of A125-132cpCasp2 with N-terminal His tag and no linker MHHHHHHGKNHAGSPGCEESAAGKEKLPKMRLPTRSDMICGYACLKGTAAMRNTKRGSWYIEALAQVFSERAC DMHVADMLVKVNALIKDREGYAPGTEFHRCKEMSEYCSTLCRHLYLFPGHPLQVKPCTPEFYQTHFQLAYRLQSRP RGLALVLSNVHFTGEKELEFRSGGDVDHSTLVTLFKLLGYDVHVLCDQTAQEMQEKLQNFAQLPAHRVTDSCIVALL SHGVEGAIYGVDGKLLQLQEVFQLFDNANCPSLQNKPKMFFIQACRGDETDRGVDQQD SEQ ID. No. 17 - Nucleotide insert sequence of plasmid for A125-1232cpCasp2 with N-terminal His tag and no linker catatgcaccatcatcaccatcatggcaaaaatcatgcaggtagtccgggttgtgaagaaagcgcagcaggtaaagaaaaactgccgaaaatgc gtctgccgacccgtagcgatatgatttgtggttatgcatgtctgaaaggcaccgcagcaatgcgtaataccaaacgtggtagctggtatattgaagc actggcacaggtttttagcgaacgtgcatgtgatatgcatgttgcagatatgctggttaaagtgaacgccctgattaaagatcgtgaaggttatgca ccgggtacagaatttcatcgttgtaaagaaatgagcgagtattgtagcaccctgtgtcgtcatctgtacctgtttccgggtcatcctccgaccggatc cggtccggtttgtctgcaggttaaaccgtgtacaccggaattttatcagacccattttcagctggcatatcgtctgcagagccgtccgcgtggtctggc actggttctgagcaatgttcattttaccggtgaaaaagaactggaatttcgtagcggtggtgatgttgatcatagtaccctggttaccctgtttaaact gctgggttatgacgttcatgttctgtgtgatcagaccgcacaagaaatgcaagagaaactgcagaattttgcacagctgcctgcacatcgtgttacc gatagctgtattgttgcactgctgagccatggtgttgaaggtgcaatttatggtgtggatggcaaactgctgcaactgcaagaagtgtttcagctgtt tgataatgcaaattgtccgagcctgcagaataaaccgaaaatgttttttatccaggcctgccgtggtgatgaaaccgatcgtggtgttgatcagcag gattaataagatatc

[0207] SEQ ID. No. 18 - Sequence of JFlcpCasp2 with N-terminal His tag and GG linker MHHHHHHGKNHAGSPGCEESAAGKEKLPKMRLPTRSDMICGYACLKGTAAMRNTKRGSWYIEALAQVFSERAC DMHVADMLVKVNALIKDREGYAPGTEFHRCKEMSEYCSTLCRHLYLFPGGGVKPCTPEFYQTHFQLAYRLQSRPRGLALVLSNVHFTGEKELEFRSGGDVDHSTLVTLFKLLGYDVHVLCDQTAQEMQEKLQNFAQLPAHRVTDSCIVALLSH GVEGAIYGVDGKLLQLQEVFQLFDNANCPSLQNKPKMFFIQACRGDETDRGVDQQD SEQ ID. No. 19 - Nucleotide insert sequence of plasmid for JFlcpCasp2 with N-tenninal His tag and GG linker catatgcaccatcatcaccatcatggcaaaaatcatgcaggtagtccgggttgtgaagaaagcgcagcaggtaaagaaaaactgccgaaaatgc gtctgccgacccgtagcgatatgatttgtggttatgcatgtctgaaaggcaccgcagcaatgcgtaataccaaacgtggtagctggtatattgaagc actggcacaggtttttagcgaacgtgcatgtgatatgcatgttgcagatatgctggttaaagtgaacgccctgattaaagatcgtgaaggttatgca ccgggtacagaatttcatcgttgtaaagaaatgagcgagtattgtagcaccctgtgtcgtcatctgtacctgtttccgggtcatcctccgaccggatc cggtccggtttgtctgcaggttaaaccgtgtacaccggaattttatcagacccattttcagctggcatatcgtctgcagagccgtccgcgtggtctggc actggttctgagcaatgttcattttaccggtgaaaaagaactggaatttcgtagcggtggtgatgttgatcatagtaccctggttaccctgtttaaact gctgggttatgacgttcatgttctgtgtgatcagaccgcacaagaaatgcaagagaaactgcagaattttgcacagctgcctgcacatcgtgttacc gatagctgtattgttgcactgctgagccatggtgttgaaggtgcaatttatggtgtggatggcaaactgctgcaactgcaagaagtgtttcagctgtt tgataatgcaaattgtccgagcctgcagaataaaccgaaaatgttttttatccaggcctgccgtggtgatgaaaccgatcgtggtgttgatcagcag gattaataagatatc

[0208] SEQ ID. No. 20 - Sequence of JF2cpCasp2 with N-tenninal His tag and GPPAG linker MHHHHHHGKNHAGSPGCEESAAGKEKLPKMRLPTRSDMICGYACLKGTAAMRNTKRGSWYIEALAQVFSERAC DMHVADMLVKVNALIKDREGYAPGTEFHRCKEMSEYCSTLCRHLYLFPGGPPAGCTPEFYQTHFQLAYRLQSRPRG LALVLSNVHFTGEKELEFRSGGDVDHSTLVTLFKLLGYDVHVLCDQTAQEMQEKLQNFAQLPAHRVTDSCIVALLSH GVEGAIYGVDGKLLQLQEVFQLFDNANCPSLQNKPKMFFIQACRGDETDRGVDQQD SEQ ID. No. 21 - Nucleotide insert sequence of plasmid for JF2cpCasp2 with N-tenninal His tag and GPPAG linker catatgcaccatcatcaccatcatggcaaaaatcatgcaggtagtccgggttgtgaagaaagcgcagcaggtaaagaaaaactgccgaaaatgc gtctgccgacccgtagcgatatgatttgtggttatgcatgtctgaaaggcaccgcagcaatgcgtaataccaaacgtggtagctggtatattgaagc actggcacaggtttttagcgaacgtgcatgtgatatgcatgttgcagatatgctggttaaagtgaacgccctgattaaagatcgtgaaggttatgca ccgggtacagaatttcatcgttgtaaagaaatgagcgagtattgtagcaccctgtgtcgtcatctgtacctgtttccgggtcatcctccgaccggatc cggtccggtttgtctgcaggttaaaccgtgtacaccggaattttatcagacccattttcagctggcatatcgtctgcagagccgtccgcgtggtctggc actggttctgagcaatgttcattttaccggtgaaaaagaactggaatttcgtagcggtggtgatgttgatcatagtaccctggttaccctgtttaaact gctgggttatgacgttcatgttctgtgtgatcagaccgcacaagaaatgcaagagaaactgcagaattttgcacagctgcctgcacatcgtgttacc gatagctgtattgttgcactgctgagccatggtgttgaaggtgcaattatggtgtggatggcaaactgctgcaactgcaagaagtgtttcagctgtt tgataatgcaaattgtccgagcctgcagaataaaccgaaaatgttttttatccaggcctgccgtggtgatgaaaccgatcgtggtgttgatcagcag gattaataagatatc

[0209] SEQ ID. No. 22 - Sequence of JF3cpCasp2 with N-terminal His tag and GPAGGPG linker MHHHHHHGKNHAGSPGCEESAAGKEKLPKMRLPTRSDMICGYACLKGTAAMRNTKRGSWYIEALAQVFSERAC DMHVADMLVKVNALIKDREGYAPGTEFHRCKEMSEYCSTLCRHLYLFPGGPAGGPGPCTPEFYQTHFQLAYRLQSR PRGLALVLSNVHFTGEKELEFRSGGDVDHSTLVTLFKLLGYDVHVLCDQTAQEMQEKLQNFAQLPAHRVTDSCIVAL LSHGVEGAIYGVDGKLLQLQEVFQLFDNANCPSLQNKPKMFFIQACRGDETDRGVDQQD SEQ ID. No. 23 - Nucleotide insert sequence of plasmid for JF3cpCasp2 with N-tenninal His tag and GPAGGPG linker catatgcaccatcatcaccatcatggcaaaaatcatgcaggtagtccgggttgtgaagaaagcgcagcaggtaaagaaaaactgccgaaaatgc gtctgccgacccgtagcgatatgatttgtggttatgcatgtctgaaaggcaccgcagcaatgcgtaataccaaacgtggtagctggtatattgaagc actggcacaggtttttagcgaacgtgcatgtgatatgcatgttgcagatatgctggttaaagtgaacgccctgattaaagatcgtgaaggttatgcaccgggtacagaatttcatcgttgtaaagaaatgagcgagtattgtagcaccctgtgtcgtcatctgtacctgtttccgggtcatcctccgaccggatc cggtccggttgtctgcaggttaaaccgtgtacaccggaattttatcagacccattttcagctggcatatcgtctgcagagccgtccgcgtggtctggc actggttctgagcaatgttcattttaccggtgaaaaagaactggaatttcgtagcggtggtgatgttgatcatagtaccctggttaccctgtttaaact gctgggttatgacgttcatgttctgtgtgatcagaccgcacaagaaatgcaagagaaactgcagaattttgcacagctgcctgcacatcgtgttacc gatagctgtattgttgcactgctgagccatggtgttgaaggtgcaattatggtgtggatggcaaactgctgcaactgcaagaagtgtttcagctgtt tgataatgcaaattgtccgagcctgcagaataaaccgaaaatgttttttatccaggcctgccgtggtgatgaaaccgatcgtggtgttgatcagcag gattaataagatatc

[0210] SEQ ID. No. 24 - Sequence of JF4cpCasp2 with N-terminal His tag and GPSGG linker MHHHHHHGKNHAGSPGCEESAAGKEKLPKMRLPTRSDMICGYACLKGTAAMRNTKRGSWYIEALAQVFSERAC DMHVADMLVKVNALIKDREGYAPGTEFHRCKEMSEYCSTLCRHLYLFPGGPSGGPCTPEFYQTHFQLAYRLQSRPR GLALVLSNVHFTGEKELEFRSGGDVDHSTLVTLFKLLGYDVHVLCDQTAQEMQEKLQNFAQLPAHRVTDSCIVALLS HGVEGAIYGVDGKLLQLQEVFQLFDNANCPSLQNKPKMFFIQACRGDETDRGVDQQD SEQ ID. No. 25 - Nucleotide insert sequence of plasmid for JF4cpCasp2 with N-terminal His tag and GPSGG linker catatgcaccatcatcaccatcatggcaaaaatcatgcaggtagtccgggttgtgaagaaagcgcagcaggtaaagaaaaactgccgaaaatgc gtctgccgacccgtagcgatatgatttgtggttatgcatgtctgaaaggcaccgcagcaatgcgtaataccaaacgtggtagctggtatattgaagc actggcacaggtttttagcgaacgtgcatgtgatatgcatgttgcagatatgctggttaaagtgaacgccctgattaaagatcgtgaaggttatgca ccgggtacagaatttcatcgttgtaaagaaatgagcgagtattgtagcaccctgtgtcgtcatctgtacctgtttccgggtcatcctccgaccggatc cggtccggtttgtctgcaggttaaaccgtgtacaccggaattttatcagacccattttcagctggcatatcgtctgcagagccgtccgcgtggtctggc actggttctgagcaatgttcattttaccggtgaaaaagaactggaatttcgtagcggtggtgatgttgatcatagtaccctggttaccctgtttaaact gctgggttatgacgttcatgttctgtgtgatcagaccgcacaagaaatgcaagagaaactgcagaattttgcacagctgcctgcacatcgtgttacc gatagctgtattgttgcactgctgagccatggtgttgaaggtgcaatttatggtgtggatggcaaactgctgcaactgcaagaagtgtttcagctgtt tgataatgcaaattgtccgagcctgcagaataaaccgaaaatgttttttatccaggcctgccgtggtgatgaaaccgatcgtggtgttgatcagcag gattaataagatatc

Claims

CLAIMSWhat is claimed is:

1. A polypeptide, which polypeptide is a variant of a single-chain circular permuted caspase-2 (cpCasp2) comprising the following structure from N- to C -terminus:a small subunit, which is a small subunit of caspase-2 or a functionally active variant thereof a linker consisting of n glycine, serine, proline and / or alanine residues wherein n is 2 to 10 (e.g..4 to 8); anda large subunit, which is a large subunit of caspase-2 or a functionally active variant thereof; which polypeptide comprises a modified loop region, which modified loop region is a contiguous amino acid sequence of the formula XssqLXLSr, wherein each Xssis independently an amino acid residue of the small subunit, q is 2-10, L is a chemical bond or the linker, each XLSis independently an amino acid residue of the large subunit, and r is 2-11, provided drat the sum of q + r > 13.

2. The polypeptide of claim 1, which is functionally active.

3. The polypeptide of claim 1, wherein the linker is GS, GG, GPPAG, GPAGGPG, GPSGG, GGSGG, SGAGSAAGSG, (GS)mwherein in is 1 to 4, GSG, or G4S.

4. The polypeptide of claim 3, wherein the linker is GS, GG, GPPAG, GPAGGPG, or GPSGG.

5. The polypeptide of any of claims 1-4, which binds to AcVDVAD-CHO with an IC50 equal to or greater than about 30 nM (e.g., greater than about 40, 50, 60, or 7011M).

6. The polypeptide of any of claims 1-4, which binds to AcIDVAD-CHO with an IC50 equal to or greater than about 70 nM (e.g., greater than about 80, 90, 100. or 110 nM).

7. The polypeptide of any of the preceding claims, which comprises an affinity tag or a solubility enhancement tag.

8. The polypeptide of claim 7. wherein the affinity tag is a hexahistidine tag.

9. The polypeptide of claim 8, which has a sequence selected from the group consisting of SEQ ID. Nos. 4, 6. 8, 10. 12, 14, 16, or 18.

10. The polypeptide of claim 9, which has SEQ ID. No. 18 (i.e., JFlcpCasp2).

11. A crystalline form of the polypeptide of any of the preceding claims.

12. The crystalline form of claim 11, which is a co-cry stal with AcVDVAD-CHO.

13. The crystalline form of claim 12, wherein the polypeptide has SEQ ID. No. 18 (i.e., JFlcpCasp2).

14. The crystalline form of claim 12, has at least one of the characteristics set forth in FIG.10.

15. The crystalline form of claim 14, which has space group P2i2i2i (e.g., as described in FIG. 10).

16. The crystalline form of claim 11, which is a co-crystal with MUR-65.

17. The crystalline form of claim 16, wherein the polypeptide has SEQ ID. No. 18 (i.e., JFlcpCasp2).

18. The crystalline form of claim 16, has at least one of the characteristics set forth in FIG.10.

19. The crystalline form of claim 18, which has space group P2i (e.g., as described in FIG.10).

20. An isolated nucleotide sequence encoding a poly peptide of any of claims 1-10.

21. A vector (e.g., a bacterial expression vector) comprising the nucleotide sequence of claim 20.

22. An expression cassette comprising the nucleotide sequence of claim 20 operably linked to regulatory elements.

23. A host cell or host cell line expressing the polypeptide of any of claims 1-10.

24. A method of facilitating crystallization of a circularly permuted caspase or a functional variant thereof comprising the following structure from N- to C-terminus:a small subunit, which is a small subunit of the caspase or a functionally active variant thereof; a linker consisting of n glycine, serine, proline and / or alanine residues wherein n is 2 to 10 (e.g., 4 to 8); anda large subunit, which is a large subunit of the caspase or a functionally active variant thereof; which circularly permuted caspase or a functional variant thereof comprises a loop region, which loop region is a contiguous amino acid sequence that consists of 10 residues at tire C-terminus of the small subunit, the linker, and 11 residues at the N-terminus of the large subunit;which method comprises removing amino acid residues from the loop region and / or replacing amino acid residues in the loop region to provide a modified loop region that is more conformationally stable than the loop region.

25. The method of claim 24, wherein the caspase is caspase-2.

26. The method of claim 24 or 25. wherein the linker is GS, GG, GPPAG. GPAGGPG. GPSGG. GGSGG, SGAGSAAGSG, (GS)mwherein m is 1 to 4, GSG, or G4S.

1. The method of claim 26, wherein the linker is GS, GG, GPPAG. GPAGGPG. or GPSGG.

28. The method of any of claims 24-27, which method does not materially affect the secondary structure of the polypeptide.

29. The method of any of claims 24-28, which method does not materially affect the binding site of the polypeptide.

30. The method of any of claims 24-29, wherein the crystallization is a co-crvstallization with a compound that inhibits wild-type caspase activity.

31. A method of screening compounds for their ability to bind to a caspase having a binding site, which method comprises:obtaining a cry stal structure of a caspase variant, which variant is a circularly permuted variant of the caspase or a functional variant thereof;modeling in silico interactions between the binding site and a compound; andmeasuring binding of the compound to the caspase or caspase variant in vitro.

32. The method of claim 31, wherein the crystal structure is a structure of the caspase variant co-crystallized with a caspase inhibitor.

33. The method of claim 31 or 32, wherein the caspase is caspase-2.

34. The method of claim 33, w herein the caspase inhibitor is AcVDVAD-CHO, AcIDVKD-CHO, AcVDVRD-CHO, AcDVPD-CHO, AcDVAD-CHO, AcDEVD-CHO. AcDRTD-CHO, AcAKD-CHO, AcTRD-CHO. or MUR-65.

35. The method of any of claims 31-34, w herein the caspase variant has at least 80, 85, 90, or 95 sequence homology with cpCasp2.

36. The method of claim 35, wherein the caspase variant has a sequence selected from the group consisting of SEQ ID. Nos. 4, 6, 8, 10, 12, 14, 16, or 18.

37. The method of claim 36, wherein the caspase variant has SEQ ID. No. 18 (i.e., JFlcpCasp2).

38. The method of claim 37, wherein the crystal structure of the caspase variant has at least one of the characteristics set forth in FIG. 10.

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