Nanostructure

Abstract

The invention provides isolated polypeptides, fusion proteins, isolated nucleic acid molecules, nucleic acid vectors, host cells, nanostructures, composite nanostructures, and pharmaceutical compositions. The invention further provides methods of treating a disease or disorder, medical uses, and uses involving said products of the invention. The invention further provides methods of forming a nanostructure and methods of forming a composite nanostructure. The isolated polypeptides and fusion proteins have the ability to form a nanostructure in the presence of ATP.

Description

[0001]Our Reference: P605181PC00 Nanostructure Field of Invention The present invention relates to polypeptides having the ability to form a nanostructure in the presence of ATP. The present invention also relates to nucleic acid molecules, nucleic acid vectors, host cells, nanostructure, composite nanostructure, and pharmaceutical compositions. The present invention also relates to methods of treating a disease of disorder, and methods of forming a nanostructure. Background to the Invention Actin is a small, highly conserved protein that plays a crucial role in the structure and function of cells. Actin monomers (G-actin) polymerize to form filaments (F- actin) in the presence of ATP. Actin also possesses ATPase activity, and the filaments disassemble upon ATP hydrolysis and ADP release. This activity is modulated by numerous proteins, which forms the basis for most cellular processes in eukaryotic cells, including muscle contraction, cell migration, cell division, intracellular transport, and signal transduction. The bacterial cytoskeleton is a complex network of protein filaments that provides structural integrity and shape to the cell, similar to the cytoskeleton in eukaryotic cells. These filaments are often composed of proteins homologous to actin, although in bacteria each protein performs a specific function. Intriguingly, structural studies have revealed that even though the overall fold and ATPase activity is similar across actin-like proteins, they adopt very distinct filamentous architectures; nonetheless, all actin-like proteins characterized to date form two- stranded filaments. The targeted delivery of therapeutic molecules to target cells and tissues, is probably the biggest challenge in modern medicine. Most drugs are delivered through the systemic blood circulation, and consequently the vast majority of therapeutic agents never reach the affected organs. This often leads to very low efficacy, and / or side effects. For example, it is estimated that in chemotherapy treatments, 90% of anticancer molecules never reach the tumour site (1). In recent years, effort has been put towards the development of protein nanoparticles, to facilitate targeted drug delivery. These technologies are based on proteins that spontaneously assemble into nanocage structures, encapsulating the drug of choice. However, a major limitation of this, is that the assembly and disassembly of the nanocages cannot be modulated, i.e., the cargo drug cannot easily be incorporated, nor released at the desired site. There have been efforts to develop controllable assembly nanocages (2), but they are inefficient, lead to heterogeneous particles, and are not reversible. Summary of the Invention According to a first aspect, there is provided an isolated polypeptide having the ability to form a nanostructure in the presence of ATP, wherein the isolated polypeptide comprises: (a) an amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2; or (b) an amino acid sequence having at least 70% sequence identity to SEQ ID NO:1 or SEQ ID NO:2. The inventors have identified novel bacterial actin homologues and modified versions thereof that can form a nanostructure, controlled by the presence and subsequent hydrolysis of ATP (or an analogue thereof). The polypeptides can be functionalised and therefore be used for delivery of molecules to target sites, such as the delivery of therapeutic agents to tumours in a patient. This has the advantage of reducing side effects and increasing efficacy of therapeutic agents, and provides a novel targeting and / or delivery platform for drugs and other molecules. It has also been determined that the nanostructure has innate ATPase activity, meaning that the nanostructure will disassemble after a period of time as a result of ATP hydrolysis, releasing any cargo molecule encapsulated inside. This allows targeting delivery and release of cargo. This is the first example of a nanostructure-forming protein, with fully tuneable assembly and disassembly. The inventors have also determined the structure of the nanostructure of one of the bacterial actin homologues, providing molecular insights on its cavity, as well as on the exposed surface regions that can be employed for further modifications. The term “polypeptide”, as used herein, refers to any peptide-bond-linked polymer of amino acids, regardless of size, length, secondary and tertiary structure, number of subunits or post-translational modification. Thus, the term “polypeptide” is to be understood as covering the terms “peptide”, “protein”, “amino acid chain”, and “amino acid sequence”. Polypeptides of the invention may include at least one chemical modification, such as lipidation, glycosylation and phosphorylation. The term “isolated polypeptide”, as used herein, refers to a polypeptide that is in a form or environment that does not occur in nature, such as (1) any polypeptide that does not naturally occur, (2) any polypeptide that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any polypeptide that is manually modified by man with respect to that polypeptide as found in nature in admixture with other components, such as other polypeptides, secondary metabolites, salts, et alia, or (4) any polypeptide modified by increasing the amount of the polypeptide in relation to other components with which it is naturally associated. The term “amino acid”, as used herein, refers to organic compounds containing the functional groups amine (-NH2) and carboxylic acid (-COOH) and its zwitterions, typically and preferably, along with a side chain specific to each amino acid. The term “amino acid” typically and preferably includes amino acids that occur naturally, such as proteinogenic amino acids (produced by RNA- translation), non-proteinogenic amino acids (produced by other metabolic mechanisms, e.g., posttranslational modification), standard or canonical amino acids (that are directly encoded by the codons of the genetic code) and non- standard or non-canonical amino acids (not directly encoded by the genetic code). Naturally occurring amino acids include non-eukaryotic and eukaryotic amino acids. The term “amino acid”, as used herein, also includes unnatural amino acids that are chemically synthesized. Moreover, the term covers alpha- (a-), beta- (b- ), gamma- (g-) and delta- (d-) etc. amino acids as well as mixtures thereof in any ratio, and, if applicable, any isomeric form of an amino acid, i.e., its D- and L- stereoisomers (alternatively addressed by the (R) and ( S) nomenclature) as well as mixtures thereof in any ratio, such as in a racemic ratio of 1:1. Amino acids in this invention are typically in L-configuration. The term “D-stereoisomer”, “L- stereoisomer”, “D-amino acid” or “L-amino acid” refers to the chiral alpha carbon of the amino acids. Amino acids can include modifications and / or attached compounds and residues, for example residues used for peptide synthesis, such as Boc, Fmoc or both. The isolated polypeptide has the ability to form a nanostructure in the presence of adenine triphosphate (ATP). This means when ATP is present (and complexed with a suitable divalent cation, such as Mg2+), the polypeptide multimerises with other polypeptides to assemble into a nanostructure. In certain embodiments, the isolated polypeptide has the ability to multimerise into a protein nanostructure when Mg2+-ATP is present. It will be understood by the skilled person that the polypeptide may also assemble into a nanostructure in the presence of other compounds, and is not limited to only multimerising in the presence of ATP. For example, the isolated polypeptide may also have the ability to assemble into a nanostructure in the presence of ATP analogues. The skilled person could easily determine a polypeptide’s ability to form a nanostructure using established methods known in the art. For example, a polypeptide may be mixed with ATP and Mg2+and assessed for nanostructure formation using dynamic light scattering. As a further example, nanocage formation may be verified using ultracentrifugation followed by SDS-PAGE analysis, dynamic light scattering and negative stain transmission electron microscopy. The term “nanostructure” as used herein refers to a protein structure with an internal cavity formed by the multimeric assembly of polypeptide subunits. In some embodiments, the nanostructure is a nanocage or nanotube. The term “nanostructure formation” refers to the assembly of the nanostructure. In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:1 or SEQ ID NO:2.In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 99.5% sequence identity to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 99.9% sequence identity to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the isolated polypeptide comprises an amino acid sequence having 100% sequence identity to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the isolated polypeptide consists of an amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2. In all cases, the skilled person will recognise that the isolated polypeptide must have the ability to form a nanostructure in the presence of ATP. In the description above, the term “sequence identity” is used to refer to the similarity of two sequences. For the purpose of this invention, it is defined here that in order to determine the percent identity of two sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence for optimal alignment with a second amino or nucleic acid sequence). The nucleotide / amino acid residues at each position are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (i.e. overlapping positions) x 100). Generally, the two sequences are the same length. A sequence comparison is typically carried out over the entire length of the two sequences being compared. The skilled person will be aware of the fact that several different computer programs are available to determine the identity between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two nucleic acid sequences is determined using the sequence alignment software Clone Manager 9 (Sci-Ed software - www.scied.com) using global DNA alignment; parameters: both strands; scoring matrix: linear (mismatch 2, OpenGap 4, ExtGap 1). Alternatively, the percent identity between two amino acid or nucleic acid sequences can be determined using the Needleman and Wunsch (1970) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. A further method to assess the percent identity between two amino acid or nucleic acid sequences can be to use the BLAST sequence comparison tool available on the National Center for Biotechnology Information (NCBI) website (www.blast.ncbi.nlm.nih.gov), for example using BLASTn for nucleotide sequences or BLASTp for amino acid sequences using the default parameters. In some embodiments, the isolated polypeptide has the ability to form a nanotube in the presence of ATP, wherein the isolated polypeptide comprises: (a) an amino acid sequence shown in SEQ ID NO:1; or (b) an amino acid sequence having at least 70% sequence identity to SEQ ID NO:1. In some embodiments, the isolated polypeptide has the ability to form a nanocage in the presence of ATP, wherein the isolated polypeptide comprises: (a) an amino acid sequence shown in SEQ ID NO:2; or (b) an amino acid sequence having at least 70% sequence identity to SEQ ID NO:2. According to a second aspect, there is provided a fusion protein having the ability to form a nanostructure in the presence of ATP, wherein the fusion protein comprises the isolated polypeptide as described above and at least one unrelated polypeptide. The term “fusion protein” as used herein refers to a chimeric protein created through the joining of two or more polynucleotides encoding separate polypeptides. In a more particular sense, the term “fusion protein” may also refer to a polypeptide comprising two or more heterologous polypeptides or peptides covalently linked, either directly or via an amino acid or peptide linker. The polypeptides forming the fusion polypeptide are typically linked C-terminus to N- terminus, although they can also be linked C-terminus to C- terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The term “fusion polypeptide” can be used interchangeably with the term “fusion protein”. Techniques for producing fusion polypeptides are known in the art and include linking the coding sequences encoding the polypeptides such that they are in structure and that expression of the fused polypeptide is under the control of the same promoter(s) and terminator. Fusion proteins can also be constructed using intein technology in which post-translational fusions are created (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779). The at least one unrelated polypeptide may be any suitable polypeptide that does not affect the nanostructure forming ability of the fusion protein in the presence of ATP. Preferably, the at least one unrelated polypeptide provides an additional function to the nanostructure. In some embodiments, the at least one unrelated polypeptide is selected from the group consisting of: a label, a purification tag, a therapeutic moiety, an antigen, a targeting peptide, an antibody fragment, a peptide that enhances the biocompatibility, solubility, secretion, or half-life of the fusion protein or nanostructure, a conjugation domain, and any combination thereof. The label may be any suitable label for identifying the fusion protein or the cleaved products thereof, or the assembled nanostructure. For example, the label may be a fluorescent label, such as GFP. Various labels and fluorescent labels are known to the skilled person. The purification tag may be any suitable tag that aids in the purification of the fusion polypeptide or the cleaved products thereof, or the assembled nanostructure. Various purification tags are known to the skilled person, including polyhistidine tags (e.g., HisX6), GST peptide, FLAG peptide, streptavidin binding peptide, V5 epitope peptide, Myc peptide or HA peptide. The therapeutic moiety may be any suitable moiety that is intended to provide a therapeutic effect. In some embodiments, the therapeutic moiety is a therapeutic peptide, a cytokine (e.g., TRAIL) a receptor agonist peptide, a therapeutic enzymatic subunit, an antigen, or an antibody fragment. The antigen may be an exogenous antigen, an endogenous antigen or an autoantigen. The antigen may be a derived from a pathogenic organism (such as a bacterium, virus, fungus or parasite), a foreign substance (such as an allergen), from the subject (i.e., self-antigen) or from tumour cells (e.g., tumour-associated antigens and tumour-specific antigens). Examples include influenza virus hemagglutinin, Malaria circumsporozoite protein (CSP), AraR, HER2, and PSA. The targeting peptide may be any suitable peptide or polypeptide that targets the assembled nanostructure to a certain location, such as a cell type or tissue. For example, the targeting peptide may be a peptide that targets glioma cells (e.g., GKRK peptide), cancer cells (e.g., IL4 receptor-binding peptides, RGD4C peptide, α-melanocyte-stimulating hormone peptide, SIRPα, NRP1-binding peptides such as CendR peptides, GE11 peptide, folate receptor A binding peptides such as C7 peptide), clots (e.g., CNAGESSKNC), hepatocytes (preS1 peptide), dying cells (Scarf1) or transepithelial movement (e.g., transferrin). The peptide that enhances the biocompatibility, solubility, secretion, or half-life of the fusion protein or nanostructure may be any suitable peptide known to the skilled person, such as a PAS polypeptide (a polypeptide consisting of proline, alanine and serine residues) or an albumin binding domain. The conjugation domain may be any linker designed to conjugate a functional ligand to the nanostructure. Exemplary conjugation domains include SpyCatcher (of the SpyTag / SpyCatcher system) and related systems (e.g., SpyStapler- mediated SpyTag / BDTag, SpyTag / SpyDock), avidin or streptavidin (for binding to biotin) and a transpeptidase recognition sequence (e.g., sortase A recognition sequence). In some embodiments, the fusion protein further comprises a linker between the fused polypeptides. Any suitable linker can be used, provided the linker does not affect the nanostructure-forming ability of the fusion protein. In some embodiments, the linker is a peptide linker. The linker sequence may be chosen based on its ability to adopt a flexible extended confirmation and / or their inability to adopt a secondary structure that could interact with the fused polypeptides. The peptide linker may comprise glycine and serine residues (Gly / Ser linkers). The peptide linker may comprise glycine, serine, histidine and / or asparagine residues. Specific suitable linkers include, but are not limited to, poly-Gly, poly- His, poly-Asn or poly-Ser, those described in Klein et al., Design and characterization of structured protein linkers with differing flexibilities, Protein Engineering, Design and Selection, Volume 27, Issue 10, October 2014, Pages 325–330, or Briers et al., mBio, 2014, 5:e0l379-l4, those described in Maratea et al., Gene 40:39 to 46, 1985, and those described in Murphy et al., PNAS, 83:8258 to 8262 (1986). The linker sequence may be from 1 to about 20 amino acid residues in length. In some embodiments, the linker sequence may be between about 8 and about 12 amino acids in length. In some embodiments, the fusion protein further comprises a cleavage site between the fused polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378- 381; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48. The isolated polypeptide and the at least one unrelated polypeptide may be in any orientation. For example, the isolated polypeptide may be N-terminal to the at least one unrelated polypeptide, or the isolated polypeptide may be C-terminal to the at least one unrelated polypeptide. In some embodiments, the isolated polypeptide is between at least two unrelated polypeptides. Typically, the C- terminal end of the at least one unrelated polypeptide is connected to the N- terminal end of the isolated polypeptide. In some embodiments, the at least one unrelated polypeptide comprises at least two unrelated polypeptides. In some embodiments, the at least one unrelated polypeptide comprises at least three unrelated polypeptides. In some embodiments, the at least one unrelated polypeptide comprises at least four unrelated polypeptides. According to a third aspect, there is provided an isolated nucleic acid molecule encoding the isolated polypeptide as described herein or the fusion protein as described herein. The term "nucleic acid molecule" as used herein refers to any linear or sequential array of nucleotides and nucleosides, for example cDNA, genomic DNA, mRNA, tRNA, oligonucleotides, oligonucleotides and derivatives thereof. Nucleic acids may include derivatized or modified nucleotides and nucleosides such as, without limitation, halogenated nucleotides such as, but not limited to, 5-bromouracil and derivatized nucleotides such as nucleotides identified by biotin. The term "isolated nucleic acid molecule", as used herein, covers, for example, (a) a DNA that has the sequence of part of a naturally occurring genomic molecule, but not flanked by at least one of the sequences flanking that part of the molecule in the genome of the species in which it occurs naturally and; (b) an isolated nucleic acid that has been incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote such that the resulting genomic DNA or vector is not identical to the naturally occurring DNA from which the nucleic acid isolate was obtained; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), ligase chain reaction (LCR) or chemical synthesis, or a restriction fragment; (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein; and (e) a recombinant nucleotide sequence that is part of a hybrid sequence that is not naturally occurring. Isolated nucleic acid molecules of the present invention can include, for example, naturally occurring allelic variants as well as isolated nucleic acid molecules modified by nucleotide deletions, insertions, inversions or substitutions. Isolated nucleic acid molecules of the invention can be DNA, RNA, or nucleic acid analogues. Nucleic acid analogues can be modified at the base moiety, sugar moiety, or phosphate backbone. Such modification can improve, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety can include deoxyuridine for deoxythymidine, and 5-methyl-2′-deoxycytidine or 5-bromo-2′-deoxycytidine for deoxycytidine. Modifications of the sugar moiety can include modification of the 2′ hydroxyl of the ribose sugar to form 2′-O-methyl or 2′-O-allyl sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, for example, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7:187-195; and Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5-23. In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone. Isolated nucleic acid molecules of the invention can be produced by standard techniques, including, without limitation, common molecular cloning and chemical nucleic acid synthesis techniques. Isolated nucleic acid molecules of the invention may be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides (e.g., using phosphoramidite technology for automated DNA synthesis in the 3′ to 5′ direction). For example, one or more pairs of long oligonucleotides (e.g., >100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase can be used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector. Isolated nucleic acid molecules of the invention may be obtained by mutagenesis. For example, a reference sequence (e.g., the nucleotide sequence set forth in SEQ ID NO:3 or SEQ ID NO:4) can be mutated using standard techniques, including oligonucleotide-directed mutagenesis and / or site-directed mutagenesis through PCR. See, Short Protocols in Molecular Biology, Chapter 8, Green Publishing Associates and John Wiley & Sons, edited by Ausubel et al., 1992. According to a fourth aspect, there is provided an isolated nucleic acid molecule encoding a polypeptide or a fusion protein having the ability to form a nanostructure in the presence of ATP, wherein the isolated nucleic acid molecule comprises a nucleotide sequence shown in SEQ ID NO:3 or SEQ ID NO:4, or a codon-optimised version thereof. The isolated nucleotide sequences may be codon optimised to enhance expression in particular host cells. Codon optimisation methods are known in the art and refer to modifying the nucleotide sequence in order to enhance protein expression in a host cell of interest by replacing one or more codons of the native sequence with codons that are more frequently used in the genes of that host cell or in the genes of the host the cell was derived from. Various species exhibit particular bias for certain codons of a particular amino acid. In certain embodiments, the isolated nucleotide sequences are codon optimised to enhance expression in a host cell selected from bacteria, yeast, fungi, plant, mammalian and / or insect cells. In some embodiments, the isolated nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO:3 or SEQ ID NO:4. According to a fifth aspect, there is provided a nucleic acid vector comprising the isolated nucleic acid molecule as described herein. A vector may be any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA, although RNA vectors are also available. Vectors include, but are not limited to, plasmids and phagemids. A cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase. In some embodiments, the nucleic acid vector comprises a promoter sequence. A promoter may include an untranslated nucleic acid usually located upstream of the coding region that contains the site for initiating transcription of the nucleic acid. The promoter region may also include other elements that act as regulators of gene expression. In further embodiments of the invention, the expression vector contains an additional region to aid in selection of cells that have the expression vector incorporated. The promoter sequence is often bounded (inclusively) at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. In some embodiments, the nucleic acid vector comprises one or more marker sequences suitable for use in the identification and selection of cells which have been transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., β- galactosidase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques. Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined. An expression vector is one into which a desired nucleic acid may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Expression refers to the transcription and / or translation of an endogenous gene, transgene or coding region in a cell. A coding sequence and regulatory sequences are operably joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide. The nucleic acid vector may be any suitable vector, such as a prokaryotic vector, a eukaryotic vector or a viral vector. The prokaryotic vector may be a plasmid vector, a phage vector, a phagemid vector, a cosmid vector or a bacterial artificial chromosome vector. The eukaryotic vector may be a yeast vector, an insect cell vector, a mammalian vector, or a plant cell vector. The viral vector may be an adenovirus vector, a lentivirus vector, or a retrovirus vector. According to a sixth aspect, there is provided a host cell comprising the nucleic acid vector as described herein. The host cell may be any suitable host, such as a prokaryotic cell or a eukaryotic cell. As used herein, the term "host cell" refers to a cell which harbours a nucleic acid vector of the invention, as well as a cell that is suitable for use in expressing a recombinant gene or protein. It is not intended that the present invention be limited to any particular type of cell. Indeed, it is contemplated that any suitable cell will find use in the present invention as a host cell. A host cell according to the invention may permit the expression of a nucleic acid molecule of the invention. Thus, the host cell may be, for example, a bacterial, a yeast, a fungal, a plant, an insect or a mammalian cell. In some embodiment, the host cell is a bacterial, a yeast, or a mammalian cell. According to a seventh aspect, there is provided a nanostructure formed by the multimerisation of the isolated polypeptide as described herein or the fusion protein as described herein in the presence of ATP or an analogue thereof. The nanostructure is formed in the presence of ATP or an analogue thereof. As discussed above, the skilled person would understand that the ATP or analogue would be in any necessary complex for function, such as in complex with Mg2+. In some embodiments, the nanostructure is formed in the presence of ATP. In some embodiments, the nanostructure is formed in the presence of ATP, AMPPNP (adenylyl imidodiphosphate), AMPPCP (adenylyl methylenediphosphate), AMPCPP (alpha,beta-methylene-triphosphonate) or ATP-γ-S (adenosine 5’-(gamma- thiotriphosphate)). In some embodiments, the nanostructure further comprises at least one chemically-conjugated functional ligand. The at least one chemically-conjugated functional ligand may be present on the exterior, the interior and / or at an interface of the nanostructure. In some embodiments, the at least one chemically-conjugated functional ligand is present on the exterior (i.e., the outer surface) of the nanostructure. The functional ligand may be any ligand that functionalises the nanostructure in a suitable manner. The term “functional ligand” may be used interchangeably with the term “functionalising moiety”. Functionalising ligands for attachment to nanostructures are known to the skilled person, for example in Sengonul et al. (“Surface Modification of Protein Nanocontainers and Their Self-Directing Character in Polymer Blends. Polymer (Guildf). 2007 Jun 15;48(13):3632-3640). In some embodiments, the functional ligand is selected from the group consisting of: biotin, polymers (e.g., PEG), diagnostic agents, therapeutic agents, masking peptides (e.g., PASE masking peptide), targeting peptides (e.g., RGD polypeptide), antigens, aptamers, hormones, vitamins (e.g., folic acid), nanobodies, and antibodies or fragments thereof (e.g., EP1). The functional ligand(s) may be chemically conjugated to the nanostructure by any suitable bioconjugation technique. In some embodiments, chemical conjugation is achieved by exploiting reactive groups of the amino acids of the nanostructure (e.g., maleimides for cysteine thiols; N-hydroxy-succinimides (NHS) for lysine amines; and carbodiimides for glutamate or aspartate carboxylates), by click chemistry (e.g., copper-catalyzed azide–alkyne cycloaddition (CuAAC) reaction), or by transpeptidation (e.g., sortase mediated transpeptidation). In some embodiments, amino acids of the underlying structure are modified to provide suitable reactive groups. The functional ligand(s) may be conjugated, either directly or indirectly through the use of a linker. Linkers are well known in the art; for example, homo- or hetero- bifunctional linkers are well known (See 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, as well as the 2003 catalog). Preferred linkers include, but are not limited to, alkyl groups (including substituted alkyl groups and alkyl groups containing heteroatom moieties), short alkyl groups, esters, amide, amine, epoxy groups, ethylene glycol and derivatives. In some embodiments, the functional ligand(s) are attached to one or more outward facing residues (e.g., residues 53-61, 130-135, 180-193, 225-271 of SEQ ID NO:1). In some embodiments, the functional ligand(s) are attached to one or more inward facing residues (e.g., residues 105-114; 143-148; 216-222; 244-250 of SEQ ID NO:1). The linker may be photocleavable (i.e., the linker includes a photolabile group that is cleavable by a specific wavelength of light), pH-responsive (i.e., the linker is a compound that can degrade at a certain pH or pH range) or enzyme-cleavable (i.e., the linker contains a cleavage site for an enzyme). According to an eighth aspect, there is provided a composite nanostructure comprising the nanostructure as described herein and at least one cargo molecule encapsulated therein. The term “composite nanostructure” as used herein refers to the nanostructure in which a specific substance is loaded in the internal cavity of the nanostructure. For example, when doxorubicin, an anticancer drug, is loaded inside a protein nanocage, it becomes a doxorubicin composite nanocage. “Composite nanostructure” can be used interchangeably with “loaded nanostructure” or “complex nanostructure”. The term “encapsulated” as used herein refers to the confinement, containment or association of a cargo molecule within the nanostructure. “Encapsulated” may be used interchangeably with “entrapped”. The cargo molecule may be any molecule that is suitable for encapsulation inside the nanostructure. Typically, the cargo molecule is a water-soluble molecule. In some embodiments, the cargo molecule is a single domain water soluble protein. In some embodiments, the cargo molecule is a diagnostic agent or a therapeutic agent. In some embodiments, the at least one cargo molecule is a diagnostic agent. The term "diagnostic agent" as used herein, refers to any agent that can produce a diagnostic signal detectable by any means in a subject. The diagnostic agent may be a radioisotope, paramagnetic label (e.g., gadolinium or iron oxide), fluorophore, dye (such as Cy3, Cy5.5, Alexa680, Dylight680, or Dylight800), or any other molecule with the desired properties suited for the practice of the present invention. Typically, the diagnostic agent is an imaging agent, such as a dye, a contrast agent, or a metal nanoparticle. The imaging agent may be any agent known to one skilled in the art to be useful for imaging a cell, tissue or a biofilm, and in certain embodiments being a medical imaging agent. Examples of medical imaging agent include, but are not limited to, magnetic resonance imaging (MRI) agents, nuclear magnetic resonance imaging (NMR) agents, positron emission tomography (PET) agents, x-ray agents, optical agents, ultrasound agents and neutron capture therapy agents. In some embodiments, the at least one cargo molecule is a therapeutic agent. The term “therapeutic agent” as used herein refers to any biologically, physiologically or pharmacologically active substance that acts locally or systemically in and / or on a subject and is administered to a subject for purposes of treatment, mitigation, cure or prevention of a medical condition or enhancement of a desired physical or mental development or condition. The therapeutic agent may be a drug, a protein, a protein fragment, a peptide, an antigen, an antibody, an antibody fragment, an enzyme, a nucleic acid, an oligonucleotide, or an extract of biological material. The term "antibody" includes antibody fragments, as are known in the art, including Fab, Fab2, single chain antibodies (Fv for example), chimeric antibodies, single-chain antibodies, and fusion proteins including an antigen-binding portion of an antibody and a non-antibody protein, etc. The antibodies may be humanized antibodies or human antibodies. The term "antibody" includes antibodies of any isotype, fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, fusion proteins including an antigen-binding portion of an antibody and a non-antibody protein, and non- immunoglobulin-based protein scaffolds as known in the art. The antibodies may be detectably labelled, e.g., with a radioisotope, an enzyme which generates a detectable product, a fluorescent protein, and the like. The antibodies may be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin-avidin specific binding pair), and the like. In some embodiments, the at least one cargo molecule comprises a plurality of cargo molecules. In some embodiments, the at least one cargo molecule comprises at least two different cargo molecules. In some embodiments, the at least one cargo molecule comprises at least three different cargo molecules. In some embodiments, the at least one cargo molecule comprises at least four different cargo molecules. In some embodiments, the at least one cargo molecule comprises at least five different cargo molecules. In some embodiments, the at least one cargo molecules comprises no more than five different cargo molecules. In some embodiments, the at least one cargo molecules comprises no more than four different cargo molecules. In some embodiments, the at least one cargo molecules comprises no more than three different cargo molecules. In some embodiments, the at least one cargo molecules comprises no more than two different cargo molecules. In some embodiments, the cargo molecule is a therapeutic protein, suicide protein, tumour suppressor protein, transcription factor, kinase inhibitor, kinase, regulatory protein, apoptotic protein, anti-apoptotic protein, microbial antigen, viral antigen, bacterial antigen, parasite antigen, cell antigen, differentiation factor, immortalizing factor, toxin, enzyme, antisense construct, diagnostic imaging agent or contrast agent, isotope, dye, antibacterial agent, antifungal agent, antiviral agent, antiproliferation agent, cytostatic, immunosuppressant, histamine receptor antagonist, vitamin, analgesic, anti-neoplastic agent, hormone, anti-inflammatory agent, adhesion molecule, receptor molecule, therapeutic organic molecule, organic inhibitor, peptide. In some embodiments, the at least one cargo molecule is an anti-neoplastic agent. The term “anti-neoplastic agent” as used herein refers to any compound that inhibits and combats tumour or neoplasm development. In some embodiments, the at least one cargo molecule is an antineoplastic agent selected from the group consisting of: alkylating agents, antimetabolites, topoisomerase inhibitors, antibiotics (e.g., Doxorubicin), mitotic inhibitors, protein kinase inhibitors, enzymes (e.g., L-asparaginase), proteasome inhibitors, PARP inhibitors, monoclonal antibodies, or a combination thereof. In some embodiments, the composite nanostructure further comprises at least one chemically-conjugated functional ligand. The description in relation to the chemically-conjugated functional ligand of the seventh aspect is as defined above. According to a ninth aspect, there is provided a pharmaceutical composition comprising the nanostructure as described herein or the composite nanostructure as described herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" is employed herein to refer to those materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. The term "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject extract from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminium hydroxide; alginic acid; sterile distilled water; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and / or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations. See Remington: The Science and Practice of Pharmacy, 19th Ed. (Easton, Pa.: Mack Publishing Co., 1995), which discloses typical carriers and conventional methods of preparing pharmaceutical formulations. Diluents, wetting agents, fillers, extenders, binders, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as colouring agents, release agents, coating agents, perfuming agents, sweeteners, preservatives and antioxidants can also be present in the compositions. The pharmaceutical composition may be a liquid, gel, or gel-like formulation (e.g., suspensions, solutions, emulsions, aqueous solutions). The pharmaceutical composition may be administered parenterally. The pharmaceutical composition may be administered parenterally via various routes, including intravenous injection, intranasal inhalation, intramuscular administration, intraperitoneal administration, transdermal absorption, etc. According to a tenth aspect, there is provided a method of treating a disease or disorder comprising administering a therapeutically effective amount of the nanostructure as described herein, the composite nanostructure as described herein, or the pharmaceutical composition as described herein to a subject in need thereof, wherein the nanostructure, the composite nanostructure or the pharmaceutical composition comprises a therapeutic agent. The term “treating a disease or disorder” as used herein refers to alleviating, ameliorating or reducing the severity or frequency of, inhibiting the progress of, reversing or abrogating a medical condition or one or more symptoms or complications associated with the condition, and alleviating, ameliorating or eradicating one or more causes of the condition. Treating the disease or disorder includes ameliorating at least one symptom of the particular disease or disorder, even if the underlying pathophysiology is not affected, such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain. Reference to “treatment” of a disease or disorder includes prevention of the disease or disorder. The terms “prevent”, “preventing” and “prevention” include precluding, reducing the risk of developing and delaying the onset of a medical condition or one or more symptoms or complications associated with the condition. Administration may be oral or parenteral administration. When administered parenterally, the nanostructure, composite nanostructure or pharmaceutical composition may be administered via various routes, including intravenous injection, intranasal inhalation, intramuscular administration, intraperitoneal administration, transdermal absorption, etc. The nanostructure, composite nanostructure or pharmaceutical composition may be administered as a single dose or in multiple doses. The nanostructure, composite nanostructure or pharmaceutical composition may be administered at a dose of 0.1 mg / kg to 1 g / kg, more preferably at a dose of 1 mg / kg to 500 mg / kg. On the other hand, the dose can be appropriately adjusted according to the age, sex and condition of the subject. The term “therapeutically effective amount” as used herein refers to an amount of the nanostructure, composite nanostructure or pharmaceutical composition that, when administered to a subject, is sufficient to prevent, reduce the risk of developing, delay the onset of, slow the progression of or cause regression of the medical condition being treated, or to alleviate to some extent the medical condition or one or more symptoms or complications of that condition, at least in some fraction of the subjects taking that agent. The term “therapeutically effective amount” also refers to an amount of the nanostructure, composite nanostructure or pharmaceutical composition that is sufficient to elicit the biological or medical response of a cell, tissue, organ, system, animal or human which is sought by a researcher, veterinarian, medical doctor or clinician. The therapeutically effective amount may vary depending on such factors as the disease or condition being treated, the particular agent being administered, the size of the subject, or the severity of the disease or condition. One skilled in the art may empirically determine the effective amount of a particular agent without necessitating undue experimentation. The subject may be any living multi-cellular vertebrate organism, including human and non-human mammals, such as non-human primates (e.g., chimpanzees and other apes and monkey species), pigs, camels, bats, sheep, cows, dogs, cats, rodents (e.g., mice, rats and guinea pigs), birds and the like. In some embodiments, the subject is a mammal. In some embodiments, the subject is human. In some embodiments, the disease or disorder is selected from the group consisting of infectious diseases, acute and chronic inflammatory diseases, cancer, and transplantation and autoimmune diseases. In some embodiments, the disease is cancer. The cancer may be any cancer, including haematological cancers, leukaemias, lymphomas and multiple myeloma, as well as solid tumours and other non-blood and non-haematological cancers. In some embodiments, the disease is an infectious disease. The infectious disease may be any infectious disease caused by a pathogen, including infections caused by viruses, bacteria, fungi, parasites and prions. In some embodiments, the disease is an autoimmune disease. The autoimmune disease may be any autoimmune disease, including type 1 diabetes mellitus, rheumatoid arthritis, psoriasis, psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, Addison’s disease, Graves’ disease, Sjögren’s syndrome, Hashimoto’s thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, pemphigus vulgaris and coeliac disease. In some embodiments, the method further comprises co-administering an additional therapeutic agent to the subject. The additional therapeutic agent may be administered by any route, such as oral or parenteral administration. The additional therapeutic agent may be administered sequentially or simultaneously with the nanostructure, composite nanostructure or pharmaceutical composition. According to an eleventh aspect, there is provided the nanostructure as described herein, the composite nanostructure as described herein, or the pharmaceutical composition as described herein for use in treating a disease or disorder, wherein the nanostructure, the composite nanostructure or the pharmaceutical composition comprises a therapeutic agent. The embodiments defined with respect to the method of treatment of the tenth aspect are also applicable to the eleventh aspect. There is also provided a method of treating a cancer comprising administering a therapeutically effective amount of the nanostructure as described herein, the composite nanostructure as described herein, or the pharmaceutical composition as described herein to a subject in need thereof, wherein the nanostructure, the composite nanostructure or the pharmaceutical composition comprises a neoplastic agent, optionally wherein the neoplastic agent is doxorubicin. According to a twelfth aspect, there is provided the use of the nanostructure as described herein, the composite nanostructure as described herein, or the pharmaceutical composition as described herein for the manufacture of a medicament for treating a disease or disorder, wherein the nanostructure, the composite nanostructure or the pharmaceutical composition comprises a therapeutic agent. The embodiments defined with respect to the method of treatment of the tenth aspect are also applicable to the twelfth aspect. According to a thirteenth aspect, there is provided a method of forming a nanostructure, wherein the method comprises combining isolated polypeptides as described herein or fusion proteins as described herein with ATP or an analogue thereof, such that the isolated polypeptides or fusion proteins multimerise into a nanostructure. The description of “ATP or an analogue thereof” is as defined above with respect to the seventh aspect. The terms “multimerisation” and “multimerise” refer to the assembly of the monomeric polypeptides or fusion proteins into a multimeric nanostructure, forming a nanostructure. In some embodiments, the method further comprises conjugating at least one functional ligand to the assembled nanostructure. The at least one functional ligand may be conjugated to the exterior and / or the interior of the nanostructure. In some embodiments, the at least one functional ligand is conjugated to the exterior (i.e., the outer surface) of the nanostructure. The functional ligand may be any ligand that functionalises the nanostructure in a suitable manner. The description of the functional ligand according to the seventh aspect is equally applicable to this aspect. In some embodiments, the method further comprises preventing or delaying the disassembly of the nanostructure by: (i) freezing the assembled nanostructure; (ii) utilising a poorly hydrolysable ATP analogue. The term “preventing or delaying” as used herein refers to restricting the disassembly of the nanostructure, either entirely. (i.e., the nanostructure cannot disassemble) or over a certain time period. In other words, the innate ATPase activity of the polypeptide is slowed or deferred, such that the nanostructure is assembled for a greater length of time prior to disassembly. The term “disassembly” as used herein refers to the breakdown of the multimeric nanostructure into the individual subunits as a result of ATP (or analogue thereof) hydrolysis. As discussed above, the nanostructure has innate ATPase activity and therefore the nanostructure will disassemble over time in the absence of intervention. For example, in an in vitro system at 25°C the nanostructure may disassemble within about 24 h. However, the skilled person will appreciate that the time to disassembly may depend on a number of factors, such as ATP / ATP analogue used, the environment the nanocage is in (in vitro, ex vivo, in vivo, etc), the temperature, the pH, etc. In some embodiments, the time to disassembly is 12-48 h. In some embodiments, the time to disassembly is 16-36 h. In some embodiments, the time to disassembly is 20-30 h. In some embodiments, the assembled nanostructure is frozen and maintained at a temperature between -100°C and -1°C. In some embodiments, the assembled nanostructure is frozen and maintained at a temperature between -80°C and - 20°C. In some embodiments, the poorly-hydrolysable ATP analogue is ATPγS or AMP- PNP. The term “poorly hydrolysable” as used herein refers to any ATP analogue that displays some level of ATP hydrolysis and at a lower level than ATP. The term “slowly hydrolysable” may be used interchangeably with “poorly hydrolysable” and “hydrolysis-resistant”. These terms are known to the skilled person, and the skilled person can easily determine the hydrolysis rate of ATP analogues. Exemplary ATP analogues are detailed in, for example, Lacabanne et al., ATP Analogues for Structural Investigations: Case Studies of a DnaB Helicase and an ABC Transporter. Molecules. 2020 Nov 12;25(22):5268. In some embodiments, the ATP is photocaged ATP, and wherein the method further comprises inducing the disassembly of the nanostructure by a laser pulse. In some embodiments, the photocaged ATP is NPE-caged ATP. The term “photocaged ATP” as used herein refers to ATP or an analogue thereof that includes a protecting group which can be removed by photolytic cleavage. Photocaged ATP is known to the skilled person, see for example, Jeschik et al., Photocaged and Mixed Photocaged Bioreversible-Protected ATP Derivatives as Tools for the Controlled Release of ATP, Eur. J. Org. Chem., 2020: 6776-6789. The skilled person is able to select the wavelength and duration of the laser pulse that is appropriate for the protecting group. In certain embodiments, the laser pulse has a wavelength of 340-380 nm. In certain embodiments, the laser pulse has a wavelength of about 160 nm. In some embodiments, the method further comprises inducing the disassembly of the nanostructure by exposing the nanostructure to a lower pH. The term “exposing the nanostructure to a lower pH” refers to placing the nanostructure in or delivering the nanostructure to a more acidic environment (i.e., more acidic than the pH at which the nanostructure was assembled), such that disassembly by ATP hydrolysis is induced. As an example, the tumour microenvironment is relatively more acidic (see Zhang et al., Tumor pH and its measurement. J Nucl Med. 2010 Aug;51(8):1167-70), and therefore delivery of the nanocage to the tumour microenvironment may induce the disassembly of the nanostructure. In some embodiments, the pH is lowered to pH 5.5-6.5. In some embodiments, the nanostructure is assembled at pH 7.5-8.5 and disassembly is induced at pH 5.5- 6.5. According to a fourteenth aspect, there is provided a method of forming a composite nanostructure, wherein the method comprises combining isolated polypeptides as described herein or fusion proteins as described herein with a cargo molecule and ATP or an analogue thereof, such that the isolated polypeptides or fusion proteins multimerise into a nanostructure with the cargo molecule encapsulated therein. The description above in relation to the method of forming a nanostructure according to the thirteenth aspect is also applicable to this aspect. The isolated polypeptides or fusion proteins may be combined with the cargo molecule and ATP or analogue thereof in any suitable manner which allows formation of the nanostructure around the cargo molecule, thus entrapping the cargo molecule inside the nanostructure. A skilled person will appreciate that all aspects of the invention, whether they relate to, for example, the polypeptide, the nanostructure, the medical uses, or a method of treatment etc., are equally applicable to all other aspects of the invention. In particular, aspects of the nanostructure, for example, may have been described in greater detail than in other aspects of the invention, for example, the medical use. However, the skilled person will appreciate where more detailed information has been given for a particular aspect of the invention, this information is generally equally applicable to other aspects of the invention. All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. Brief Description of the Drawings The invention will now be described in detail, by way of example only, with reference to the figures. Figure 1. A novel Bacterial actin homologue family forms a three-stranded filament. a) Unrooted phylogenic tree of the main families of actin orthologue. BeeR, which is most closely related to MamK, is in red. b) Pelleting assay for BeeR, Monomeric protein is present in the supernatant (S), whereas polymers are found in the Pellet fraction (P). BeeR is primarily monomeric in the absence of nucleotide, and polymerizes in the presence of ATP, as well as ADP. c) Negative- stain TEM micrograph of BeeR filaments in the presence of ATP. d) Cryo-EM structure of the BeeR filament, to 3.1 Å resolution. The three strands are coloured in dark blue, khaki, and magenta, respectively. e) Surface representation of the BeeR filament atomic model, coloured as in d), from the side and top. The diameter of the filament, and internal cavity, are indicated. f) Longitudinal view of the BeeR filament, illustrating the presence of a central cavity with a diameter between 2.5 and 3 nm wide. Figure 2. Structural basis for BeeR polymerization. a) Four adjacent BeeR subunits as shown, illustrating the various helical symmetry parameters that can be used to describe this helical structure. b) Lateral contact across the strands is shown, with close-up views of the main interacting residues on the right. Figure 3. Multiple Sequence Alignment of selected BeeR orthologues. BeeR sequences from various bacterial strains, as well as the sequences for MamK and MreB, are indicated. Conserved residues are in red boxes, similar residues are in red character. The secondary sequence of BeeR and MamK are shown on top (blue) and bottom (green), respectively. The * symbol indicates residues located at the cross-strand interface. Figure 4. Cryo-EM analysis of the BeeR filaments. a) representative electron micrograph, b) selected 2D classes, c) FSC plot for the 3D refinement, d) final map, and e) local resolution estimation, showing that the map is mostly in the 2.8 Å – 3.3 Å range. Figure 5. Atomic model of the BeeR filament. a) Complete atomic model, coloured as in Figure 1, fitted in the electron potential map in grey. b) Map-to- model FSC plot for the complete atomic model. c) Close-up view of a BeeR monomer, in Rainbow colouring, in the EM map density. d) Representative density for various elements of the structure, including the nucleotide (top), a helix (left), 3-sheet strand (right), and C-terminal loop (bottom). Figure 6. Comparison between BeeR and other bacterial actins. a) A monomer of BeeR is shown, in rainbow colouring, on its own (left) and overlaid on a MamK monomer (middle) or actin monomer (right) in grey, with the RMSD indicated. b) Three adjacent BeeR subunits are shown in yellow, on its own and aligned to MamK and actin as in a). The helical parameters for each is indicated. c) Full filament structure for BeeR, MamK and actin, coloured as in Figure 1. Figure 7. The BeeR filaments bundle in the absence of the N-terminal unstructured domain. Negative-stain TEM micrograph of BeeR filaments obtained with the WT protein (top) and with a construct lacking the N-terminal domain (bottom). b) 2D classes of filaments from the corresponding proteins. Figure 8. The BeeRL2 nanocage. (a) Negative-stain TEM micrograph of BeeRL2 nanocages formed with ATP. (b) Preliminary Cryo-EM structure of the BeeRL2 nanocage. The dimensions of the internal cavity are indicated. Figure 9. Assembly and disassembly of the BeeRL2 nanocage. (a) DLS analysis of BeeRL2 nanocage assembly, and disassembly, in the presence of ATP. (b) Negative-stain TEM micrograph of the BeeRL2nanocage in the presence of ATP (top) or AMP-PNP (bottom), showing that disassembly correlates with ATP hydrolysis. c) and d) Dynamic Light Scattering analysis of BeeR and BeeRL2 oligomerization, respectively, in 50 mM NaCl. BeeR forms oligomers very rapidly upon addition of ATP, and no dissociation is observed over 24h. In contrast, BeeRL2assembles after ~ 3h in the presence of ATP, and disassembles after ~ 20h. Figure 10. Incorporation of cargo molecules into the BeeR and BeeRL2 nanoparticles. (a) UV absorption at the Doxorubicin peak (480 nm), for isolated BeeR or BeeRL2nanoparticles incubated with Doxorubicin prior to oligomer formation, demonstrating Doxorubicin incorporation. (b) Pelleting assay, showing that Lysozyme co-purified with the BeeR nanotubes. (c) BeeR pellets in the presence (left) or absence (right) of Doxorubicin, with an easily-recognizable bright red color. d) Titration of Doxorubicin against BeeR, demonstrating a plateau at around 17:1 ratio, indicative of the incorporation power of the BeeR nanotube. Figure 11. a) Pelleting assay for BeeR, Monomeric protein is present in the supernatant (S), whereas polymers are found in the Pellet fraction (P). At low salt concentration, BeeR is primarily monomeric in the absence of nucleotide, and polymerizes in the presence of ATP, as well as ADP. However, in physiological salt concentration, BeeR polymerization is only obtained in ATP, and is mostly monomeric with ADP. b) Cryo-electron micrograph of BeeRL2in the presence of ATP. c) Representative 2D classes of BeeRL2, demonstrating distinct views. d) Preliminary 3D reconstruction of the BeeRL2 nanocage, revealing a ~ 110 nm3cavity. Figure 12. Schematic representation of cell-based characterization of Doxorubicin delivery by the BeeR and BeeRL2 nanoparticles. Detailed Description The inventors have identified a novel protein, BeeR, which forms a nanotube in the presence of ATP. The inventors modified the sequence of BeeR to provide BeeRL2, which forms a nanocage in the presence of ATP. The following examples demonstrate that BeeR and BeeRL2, as well as proteins with a suitable sequence identity thereto, can be used as biological nanostructures, for example to encapsulate therapeutic or diagnostic agents in the presence of ATP (or suitable analogue) and deliver the cargo to a target site. The inventors have shown that the nanostructures have innate ATPase activity, and therefore disassemble after a certain period of time to release their cargo. Examples Example 1 The inventors report the structure of a previously unreported family of bacterial actin homologues, which forms a rigid, three-stranded, polar filament. This unusual architecture suggests a distinct mechanism of filament assembly, and provides new avenues to the understanding of the evolution of actin-like proteins. Results and discussion The inventors searched for previously-uncharacterized actin homologues in the bacterial metagenomics database, in order to identify novel actin-like proteins that could adopt unique filament architectures. The inventors identified a set of proteins, most closely related to MamK (13-16) (25-30 % sequence identity) (Figure 1a) but present in non-magnetotactic bacteria, and therefore presumably with a distinct function. Intriguingly, this protein is highly conserved in the Verrucomicrobiota phylum, a poorly characterized family of anaerobic bacteria ubiquitously present in soil samples, as well as in the gut microbiome (17). Sequence analysis of this protein family confirmed that it possessed the hallmarks of actin-like homologues, including conserved residues for ATP binding and hydrolysis (Figure 3), and structure prediction confirmed that it likely adopts an actin-like fold. Nonetheless, one distinctive feature of this family was the presence of an additional 30-50 amino-acid extension at the N-terminus (NTD) (Figure 3), which is predicted to be unstructured and is not conserved in sequence, but not found in other actin-like proteins. In order to characterize this newly-identified family, the inventors purified one representative orthologue, from the bacterium Opitutus terrae (18). A pelleting assay confirmed that it is soluble in isolation, but polymerizes in the presence of nucleotide (Figure 1b), confirming its actin-like property. Intriguingly, under the condition used (low salt conditions), the inventors observed a similar propensity for polymerization in the presence of both ATP and ADP. This is distinct to that of most observed actin homologues, where polymerization is vastly more efficient with ATP compared to ADP. However, in more physiological conditions (250 mM NaCl), polymerization is only observed in the presence of ATP, similar to that of most observed actin homologues (Figure 11a). Negative-stain Electron Microscopy (EM) analysis of the obtained polymers revealed that they consist of long, well-ordered filamentous structures, which appeared as twisted rail tracks (Figure 1c), very distinct from that of any other actin-like proteins. Notably, these filaments are highly rigid, very long (up to several mm), and visual inspection suggested that it adopts a rail tracks-like architecture, consisting of two strand rotating on their individual axis. This prompted the inventors to name this family the Bacterial Elongated Entwined Rail- like protein (BeeR). The inventors next used cryo-EM to gain further insights into the molecular structure of this BeeR filament. As shown on Figure 4a, these were readily incorporated in ice within cryo-EM grids, presumably due to their length and rigidity. 2D classification of the resulting particles confirmed that they were highly ordered, with readily identifiable secondary structure elements (Figure 4b). Using particles within the well-resolved 2D classes, the inventors were then able to determine its structure by helical reconstruction, to 3.1 Å resolution (Figures 4 and 5). Unexpectedly, this unambiguously revealed that BeeR adopts a three-stranded, parallel, right-handed, staggered filament architecture (Figure 1d-e). The diameter of this filament, at ~ 80 Å, is significantly larger than most other actin- like proteins, and it includes a large cavity at its core (~ 25 Å-wide), responsible for its rail track-like appearance by negative-stain EM. Accordingly, this helical arrangement can be described in multiple ways: (1) as a 3-start helix, with a rise of one subunit (~ 52 Å) in a right-handed filament with a twist of ~ 19 °; (2) as a 2-start helix in a left-handed filament, with a rise of ~ 35 Å and a twist of ~ -177 °; or (3) as a 1-start helix in a right-handed filament, with a rise of ~ 17 Å and a twist of ~ 126 ° (Figure 2a). These are equivalent in terms of subunit arrangement, but the inventors note that representation (3) was used as helical parameters for the cryo-EM map reconstruction, as it has the lowest rise and therefore could potentially maximize the number of particles used in our reconstruction. The structure of the BeeR monomer is consistent with that of an actin-like protein, with the canonical domains Ia, Ib, IIa and IIb adopting a U-shaped arrangement (Figures 5c and 6a), and the nucleotide buried between domains Ib and IIb (19). It is most similar to its closest homologue of known structure, MamK (13, 14), with a RMSD of 1.3 Å for aligned atoms (5.1 Å for all atoms). Within the BeeR filament, the longitudinal interface along strands is largely similar to other actin-like filaments (Figure 6b and 6c), with domains Ib and IIb making extensive contacts with domains IIa of the subunit above. Notably, its strand arrangement resembles mostly that of MamK, but also actin, both of which have similar interfaces, but with a slightly more twisted filament in the later. The N- terminal domain, which the inventors identified as a hallmark of the BeeR family (see Figure 3) is not resolved in their map, supporting its intrinsically disordered nature. In contrast, the cross-strand contacts in the BeeR filament is strikingly distinct to that of all other actin-like filaments. Each protomer makes contact with two consecutive subunits of the adjacent strand, mainly alongside the 2-start helix interface, as shown in Figure 2b. Contacts with the top subunit are minimal, with an interface area of ~35 Å2, and consist of a hydrogen bond network between Arg 157 of one subunit, and Thr 307 and Gln 310 in the adjacent subunit. In contrast, the contact with the bottom subunit are extensive, forming a contact surface area of ~ 450 Å2,and consist of multiple hydrogen bonds, between Glu 149 / Glu 153 and Asn 246, and between Arg 242 / Arg 252 and Asn 380. In addition, a hydrophobic residue at the C-terminus, Leu 385, is buried in a hydrophobic pocket of the adjacent subunit consisting of Ile 277 and Leu 284. It is noteworthy that most of the aforementioned residues are conserved among BeeR orthologues, but not in MamK or MreB (Figure 3). This suggests that the three-stranded filament architecture is a feature of this protein family, and not unique to the particular O. terrae orthologue characterized here. Most notably, the majority of the cross-strand interaction occurs between helix a3 and the C- terminal ~ 10 residues. In contrast, the inventors note that the loop forming the cross-strand interface in MamK (residues 80-85) is not present in any of the BeeR orthologues, confirming that they cannot adopt a similar 2-stranded architecture. As mentioned above, a hallmark of the BeeR family is the presence of a predicted disordered NTD. The inventors sought to assess if this NTD was involved in filament formation; as shown in Figure 7a, a construct lacking the NTD still retained its capacity to polymerize, and the filaments adopt the same overall rail- track-like morphology (Figure 7b). However, the inventors observed that those filaments have a very high propensity to form larger bundles, consisting of multiple filaments sticking together. These were never seen with the WT protein. Without wishing to be bound by theory, the inventors propose that the NTD of BeeR acts as a “repellant”, preventing the formation of bundles that might alter their cellular function. The diameter of the cavity of BeeR (~ 2.5 nm), with gaps all along the structure, makes it unlikely that it is involved in solute or macromolecular transport within the tube. Instead, the inventors propose that the three-stranded tubular structure formed by BeeR provides a much more rigid filament than that of other actin homologues, as supported by the 2D classes shown above, as well as the fact that various Z-lengths used for the reconstruction does not significantly affect the obtained resolution. Nonetheless, the structure of the BeeR filament provides novel insights into the molecular diversity of actin-like proteins, and adds to the diverse structural architectures that can be formed by this family. It uncovers novel paths towards our understanding of the evolution of the actin fold. Materials and methods Sequence analysis: The BeeR sequence was identified from scanning the metagenomics database (3) for MamK orthologues in non-magnetotactic bacteria, using BLAST (4). Sequences were aligned with ClustalW (5), and a non-rooted genealogic tree was constructed with PhyML (6). Protein expression and purification: The BeeR sequence, codon-optimised for expression in E. coli, was cloned into pET21a. The protein was over-expressed in BL21a cells, expression was induced when the cells reach mid-log phase with 1 mM IPTG, for 16h at 20°C. Cells were then harvested at 6,000 rpm for 10 min, and lysed by sonication. Debris were then removed by centrifugation at 20,000 rpm for 45 min. BeeR was purified from the supernatant using two successive rounds of Ammonium Sulphate precipitation (15% saturation), in 25 mM KCL, 10 mM HEPES pH 7.0, 2 mM EDTA, 5 mM DTT, followed by size-exclusion chromatography using a Superdex 200 column (Cytiva), in the same buffer. Pelleting assay: Purified BeeR protein was concentrated to ~ 10 mg / ml in the aforementioned buffer supplemented with 5 mM MgCl2. ATP or ADP were added to 5 mM, and 30 ml of the corresponding samples were spun using an Airfuge 347855 ultracentrifuge (Beckman Coulter) at 100,000rpm for 10 min. The supernatant was extracted by pipetting, and the pellet was resuspended in 30 ml of the aforementioned buffer. In further experiments (Fig 11a), purified BeeR protein was concentrated to ~ 10 mg / ml in the aforementioned buffer supplemented with 5 mM MgCl2. ATP or ADP were added to 5 mM, and 30 ml of the corresponding samples were spun using a SciSpin Micro centrifuge at 14,000rpm for 30 min. The supernatant was extracted by pipetting, and the pellet was resuspended in 30 ml of the aforementioned buffer. Negative-stain TEM: Purified BeeR protein was diluted to ~ 0.02 mg / ml in the aforementioned buffer, ATP and MgCl2were added to 1 mM, and the resulting sample was applied to carbon-coated TEM grids. Micrographs were collected on a FEI Morgagni TEM operated at 100 kV, and equipped with a Gatan Orius camera. Cryo-EM data collection: Following size-exclusion chromatography, BeeR was concentrated to ~2 mg / ml, and ATP and MgCl2 were added to a final concentration of 1 mM. The sample was applied to holey carbon grids, which were imaged in a Glacios TEM (Thermo Fisher) operated at 200 kV and equipped with Flacon IV camera. A dataset of ~ 4,000 micrographs was collected using EPU, with a pixel size of 1.5 Å, and a total dose of 40 e / Å (4). EM data processing: Cryo-EM data was processed with CryoSPARC (7). Frame alignment and CTF estimation were performed using default parameters. An initial set of ~ 1,000 particles with a box side of 512 pixels were picked manually from 10 micrographs at different defocus, and used to generate initial 2D classes. These were then used as templates for single-particle template picking, leading to a set of ~240,000 particles. 2D classification was employed to select the best particles (~ 140,000), which were then used for ab-initio structure determination. This led to a map with clear structural features that allowed to determine the helical symmetry. Following this, helical refinement was applied, using a rise of 17.5 Å and a twist of 126.5 °. This led to final map that was refined to 3.6 Å resolution. Model building and structure analysis: An initial atomic model of BeeR was generated with AlphaFold (8), and 36 copies were placed in the corresponding density of the EM map. ATP and Mg molecules were placed manually with Coot (9). The obtained model was subject to real- space refinement in Phenix (10). Map and model were displayed using ChimeraX (11) and PyMol. Interface contacts were analyzed with PISA (12). Example 2 As indicated above, the closest homologue to BeeR of known structure, is the magnetosome-associated bacterial actin homologue MamK (15-16). The inventors (and others) have previously reported the structure of the MamK filament (13- 14), demonstrating that it adopts a two-stranded architecture, with the cross- strand interface being exclusively formed by a single loop, termed L2. This loop is unique to MamK, and not found in BeeR or other bacterial actin homologues (Figure 3). The inventors therefore sought to investigate the effect of inserting this loop within the BeeR sequence. The corresponding protein (termed BeeRL2) was expressed and purified as described above for BeeR; gel filtration confirmed that it was monomeric in the absence of nucleotide. However, when ATP and magnesium were included in the buffer, BeeRL2 did not form filaments; instead, the inventors observed that this protein adopted a cage-like architecture, as observed by negative-stain TEM analysis (Figure 8a) and cryo-EM analysis (Figure 11b). Preliminary 3D reconstruction of this oligomeric assembly, based on negative- stain TEM data, indicated that it possesses a ~900 nm3cavity. 2D classification confirmed that they possess an ordered architecture, with a central cavity of ~ 6nm diameter (Figure 11c). Preliminary 3D reconstruction of this oligomeric assembly confirmed this architecture, although preferred orientation of the particles have precluded the inventors from obtaining a high- resolution structure. To further characterize the assembly and disassembly of the BeeRL2 nanocage, the inventors used dynamic light scattering (DLS) to monitor oligomerization over time, using different nucleotides. As shown in Figure 9a, in the absence of nucleotide the sample remained largely monomeric over ~24h. In contrast, oligomerization was observed rapidly after the addition of nucleotide, with full assembly obtained after ~6h, followed by slow depolymerization starting around ~15h. When ADP was used, slow assembly was observed, and the complex was not fully assembled within the timescale of this experiment. The DLS experiments were repeated for both BeeR (Figure 9c) and BeeRL2 (Figure 9d). As shown in Figure 9c, in the absence of nucleotide BeeR remains largely monomeric over ~24h. Oligomerization was observed rapidly after the addition of nucleotide, with full assembly obtained within ~1h. No significant depolymerization was observed within the timeframe of this experiment. Similar oligomerization propensity was observed with ADP. In contrast, BeeRL2polymerizes more slowly, with full oligomerization obtained after ~4h, and followed by slow depolymerization starting around ~15h (Figure 9d). When ADP was used, slow assembly was observed, and the complex was not fully assembled within the timescale of this experiment. To verify that depolymerization of the BeeRL2 nanocage correlated with ATP hydrolysis, the inventors employed negative-stain TEM to characterize the BeeRL2samples in the presence of either ATP or the non-hydrolysable ATP analogue AMP- PNP. As shown in Figure 9b, in the presence of ATP the BeeRL2 nanocages were readily identifiable after 1h of incubation with nucleotide. In contrast, no oligomers were observed in the same sample after 24h incubation at room temperature. When AMP-PNP was employed as the nucleotide, nanocage complexes were observed after 1h as well as after 24h incubation, demonstrating that nanocage disassembly was due to ATP hydrolysis. Finally, the inventors sought to establish is cargo molecules could be incorporated in the BeeR or BeeRL2nanoparticles. In the first instance, the anticancer drug Doxorubicin was used as the cargo. Doxorubicin is readily tracked by its UV absorption spectrum, with a peak at 480 nm (20). Using the pelleting assay described in Example 1, the inventors pelleted BeeR and BeeRL2, incubated with 5 mM Doxorubicin, with or without ATP. As demonstrated in Figure 10a, UV absorbance for Doxorubicin was found in the pellet for both BeeR and BeeRIL2, in the presence of ATP, but not in the absence of nucleotide. This demonstrated that Doxorubicin specifically co-pellets with both BeeR and BeeRL2 nanoparticles. The inventors next sought to quantify this, exploiting the UV absorption of Doxorubicin, to confirm that both BeeR and BeeRL2nanoparticles incorporated Doxorubicin in this assay. As shown in Figure 10a, a low background of Doxorubicin was obtained in the absence of nucleotide, for both proteins. The inventors note that for BeeR, the background level is ~ 10x higher than that of BeeRL2, which may be due to some low level of polymerization in the absence of nucleotide, as suggested in the pelleting assay (see Example 1). In the presence of nucleotide, ~10x higher level of absorbance was observed for BeeR, and 100x higher for BeeRL2. This demonstrates that both proteins can readily incorporate Doxorubicin, upon oligomerization. Exploiting this assay, the inventors sought to determine the maximum amount of Doxorubicin that could be incorporated into the BeeR nanoparticles. To this end, the inventors performed the aforementioned experiment using different Doxorubicin concentrations, at a constant concentration of BeeR (0.02 mM). As shown in Figure 10d, the OD480increased linearly with increasing Doxorubicin concentration, until it reached a value of ~1, where it then remained constant. Standard curve demonstrated that OD480=1 for a Doxorubicin concentration of 0.34 mM, demonstrating that this is the maximum concentration that can be incorporated in this assay. From this, the inventors conclude that 0.02 mM of BeeR can incorporate a maximum of 0.34 mM of Doxorubicin, corresponding to a 17:1 Doxorubicin:BeeR ratio. In addition, to verify if the nanoparticles were able to incorporate larger cargo, the inventors also performed the pelleting assay described above, in the presence of the protein lysozyme, a 14 kDa enzyme that catalyzes the degradation of bacterial cell wall. As shown in Figure 10b (top gel), lysozyme was exclusively soluble in the pelleting assay, regardless of the presence of nucleotide. In contrast, when mixed with BeeR, lysozyme was found in the pellet fraction in the presence of nucleotide, demonstrating that it co-pellets with the BeeR nanotubes. To verify that the co-pelleting of lysozyme with the BeeR nanotube was specifically due to its incorporation within the cavity of the nanotubes, the inventors repeated the aforementioned assay, but they pre-incubated BeeR with ATP for 1h prior to adding lysozyme, thus inducing nanotube formation before adding the cargo protein. As shown in Figure 10b (bottom gel), in this configuration, lysozyme did not co-pellet with BeeR in the presence of ATP. This demonstrated that in the co-pelleting result described above is specifically due to lysozyme being incorporated within the assembled nanotube. Materials and Methods Cloning: The amino-acid sequence for BeeR (SEQ ID NO:1; WP_012375946.1) was reverse-transcribed into DNA with the codon usage optimized for expression in E. coli. The resulting DNA sequence was synthesized and cloned into the pET29a vector (Bio Basic). For BeeRL2, a 6-amino acid peptide was inserted in the BeeR sequence by site-directed mutagenesis (Agilent). The amino acid sequence for BeeRL2is provided in SEQ ID NO:2. Protein expression and purification: Plasmids for BeeR or BeeRL2were transformed into BL21(DE3)pLysS competent cells (Novagen). Transformants were grown in LB media containing 25 µg / ml Chloramphenicol and 50 µg / ml Kanamycin, shaking at 220 rpm at 37 °C, until they reach a OD600~ 0.6. Protein expression was induced by adding 1 mM IPTG, and caried on at 37°C for 4 hours. Cells were harvested at 5000 g for 15 minutes, and stored at -20°C. BeeR and BeeRL2-containing E. coli cell pellets were resuspended in lysis buffer (10 mM HEPES pH 7.0, 25 mM KCl, 2 mM EDTA, 5 mM DTT, EDTA-free protease inhibitors (Roche)), using 50 ml per L of culture. The resuspended cells were sonicated for 5 minutes, at 4°C, and cell debris were pelleted at 20,000 g for 40 minutes. BeeR and BeeRL2 were purified from the supernatant with two cycles of Ammonium precipitation in lysis buffer, at 25% saturation, with the final pellet resuspended in lysis buffer, using 2 ml / L of culture. This yielded proteins that were > 99% pure (as estimated by SDS-PAGE gel), with yields of ~ 32 mg / L of culture (BeeR), or ~ 18 mg / L of culture (BeeRL2). Pelleting Assay: Protein samples were diluted to ~ 0.5 mM in assembly buffer (10mM HEPES pH 7.0, 25 mM KCl, 5 mM MgCl2), to 30 µl. ATP or ADP were added as required, to 5 mM concentration. These were spun at either 15,000 rpm using a benchtop centrifuge (for BeeR) for 30 min, or at 120,000 g using an air drive ultracentrifuge for 20 min (for BeeRL2). Pellets were resuspended in 30 µl assembly buffer. For Lysozyme incorporation, lysozyme powder was resuspended in assembly buffer to 10 mM, and added to the pelleting mix to a final concentration of 0.5 mM. Following the pelleting assay, 5 µl of supernatant and pellets were run in a 18% SDS-PAGE gel. For Doxorubicin incorporation, Doxorubicin at 100 mM in water was diluted to 5 mM in the pelleting mix. Following the pelleting assay, the pellet was resuspended in 200 µL assembly buffer, and UV absorption at 480 nm was measured with a BioDrop spectrophotometer (Biochrom). Light Scattering: Samples containing BeeR or BeeRL2at ~ 0.1 mM in assembly buffer, and with ADP or ATP at 5 mM were diluted to 100 µl in assembly buffer, and added to a Greiner 96 well Sensoplate microplate. Dynamic Light Scattering was measured using a WYATT Technology DynaPro Plate Reader III, with measurements performed every 30 minutes. Negative-stain TEM: BeeRL2 oligomers were prepared as described above, and diluted to ~0.1 mg / ml, in assembly buffer. Samples were applied onto glow-discharged, carbon-coated TEM grids for 2 min, and washed in H2O, followed by staining in 2% Uranyl Acetate. Grids were imaged on a Morgagni TEM (Thermo Fisher) equipped with an Orius camera (Gatan). Cryo-EM data collection: BeeRL2was concentrated to ~2 mg / ml, and ATP and MgCl2were added to a final concentration of 1 mM. The sample was applied to holey carbon grids, which were imaged in a Glacios TEM (Thermo Fisher) operated at 200 kV and equipped with Flacon IV camera. A dataset of ~ 3,000 micrographs was collected using EPU, with a pixel size of 1.5 Å, and a total dose of 40 e / Å (4). EM data processing: Cryo-EM data was processed with CryoSPARC (7). Frame alignment and CTF estimation were performed using default parameters. An initial set of ~ 500 particles with a box side of 256 pixels were picked manually from 80 micrographs at different defocus, and used to generate initial 2D classes. These were then used as templates for single-particle template picking, leading to a set of ~2,000,000 particles. 2D classification was employed to select the best particles (~ 42,000), which were then used for ab-initio structure determination. This led to a map with clear structural features that resembled the 2D classes. Nonetheless, the 2D classes also indicated the presence of preferred orientation, which precluded refining this map to high resolution. Example 3 Whether BeeR and BeeRL2nanoparticles can readily release Doxorubicin in the vicinity of cancer cells is tested as follows. Doxorubicin-loaded BeeR / BeeRL2 nanoparticles are isolated by ultracentrifugation and added to cultures of the breast cancer model cell line MCF-7 (see Chen et al. “Breast Cancer MCF-7 Cell Spheroid Culture for Drug Discovery and Development.” J Cancer Ther. 2022 Mar;13(3):117-130. doi: 10.4236 / jct.2022.133009. Epub 2022 Mar 9. PMID: 36311820; PMCID: PMC9611733). Cytotoxicity is monitored over time, to assess the capacity of the nanoparticles to induce cell death. Empty nanoparticles are used as a control. Doxil is used as an additional control (Doxil is a commercially available Doxorubicin formulation in lipid nanoparticles; see Vaage et al “Therapy of mouse mammary carcinomas with vincristine and doxorubicin encapsulated in sterically stabilized liposomes.” Int J Cancer. 1993 Jul 30;54(6):959-64. doi: 10.1002 / ijc.2910540616. PMID: 8335404). Doxorubicin-resistant MCF-7 cells are employed as a negative control to verify that cell death is caused by the release of Doxorubicin (see Lemieux and Pagé. “Sensitivity of multidrug-resistant MCF-7 cells to a transferrin-doxorubicin conjugate.” Anticancer Res. 1994 Mar- Apr;14(2A):397-403. PMID: 8017839). A pilot experiment suggested that 1 mM Doxil kills MCF-7 cell cultures within 24h, whereas cell death is only observed after 72h for equivalent Doxorubicin concentration in BeeR nanoparticles. No significant cell death was observed for empty BeeR nanoparticles within this timeframe. The toxicity and immunogenicity of the BeeR and BeeRL2nanoparticles is assessed in a mouse model. Three 6-mice cohorts are injected (two IV injections, at 0 and 3 weeks, of 10 mg protein / mouse) with either the assembled nanoparticles (cohort 1), the nanoparticle proteins in their monomeric form (cohort 2), or a negative- control, and animal survival is monitored. The level of immune response induced for the nanoparticles is determined. The same regimen of assembled nanoparticles (BeeR and BeeRL2) is injected in 6-mice cohorts of naïve vs humanized mice models, with the same cohorts employed using a negative control. Lymph node analysis and antibody response is employed to monitor immune response. Sequences SEQ ID NO:1 (BeeR amino acid sequence) MIPASTEKPVPSAAIPANAPNVAVSKPAAPGTRTKTVLVGFDFGTNKSCVLAGTAGATDIAIS KIVPTVVGYVKEGIVDGIVAGNRSVLFGDDALQNRLHARLVAPMEHGVIAHPDAARDFVQH LRSLADPSGQAEIRAVVGVPANATEQAREDVRRCAFGIFDRILLIPEPFLAALGYRDDARLGQ SNYIDPVVNSLFIDIGGGTSDICLVQGYFPGPDDQISIPFAGDAIDQLLQEELNRTYPNNGLS LHKVREIKEAHGYVGPSRKPLDVKVVIGGKAHTLELGDTLARACNALIDKIYPALTTLIQRAS SDSVVTLLQNIIITGGGSQIKGIDTLLQKKLTEDGFESPKVRLAGHDYKRYVALGALKAARAA RENQWQVLLG SEQ ID NO:2 amino acid sequence) MIPASTEKPVPSAAIPANAPNVAVSKPAAPGTRTKTVLVGFDFGTNKSCVLAGTAGATDIAIS KIVPTVVGYVKEGIVDGIVAGNRSVLFGDDALQNRLHARLVAPMEHGVIAEISDRDIHPDAA RDFVQHLRSLADPSGQAEIRAVVGVPANATEQAREDVRRCAFGIFDRILLIPEPFLAALGYRD DARLGQSNYIDPVVNSLFIDIGGGTSDICLVQGYFPGPDDQISIPFAGDAIDQLLQEELNRTY PNNGLSLHKVREIKEAHGYVGPSRKPLDVKVVIGGKAHTLELGDTLARACNALIDKIYPALTT LIQRASSDSVVTLLQNIIITGGGSQIKGIDTLLQKKLTEDGFESPKVRLAGHDYKRYVALGAL KAARAARENQWQVLLG SEQ ID NO:3 (BeeR DNA sequence) atgattccggcaagcaccgaaaaaccggttccgagcgcagcaattcctgcaaatgcaccgaatgttgcagttagca aaccggcagcaccgggtacacgtaccaaaaccgttctggttggttttgattttggcaccaataaaagctgtgttctgg caggtacagccggtgcaaccgatattgccattagcaaaattgttccgaccgttgtgggttatgtgaaagaaggtatt gttgacggtattgtggcaggtaatcgtagcgttctgtttggtgatgatgcactgcagaatcgtctgcatgcacgtctg gtggcaccgatggaacatggtgttattgcacatcctgatgcagcacgtgattttgttcagcatctgcgtagcctggca gatccgagcggtcaggcagaaattcgtgcagttgttggtgttccggctaatgcaaccgaacaggcacgtgaagatg ttcgtcgttgtgcatttggtatttttgatcgcattctgctgattccggaaccgtttctggcagcactgggttatcgtgatg acgcacgcctgggtcagagcaactatattgatccggttgttaacagcctgttcattgatattggtggtggtacaagcg atatttgtctggttcagggttattttccgggtccggatgatcagattagcattccgtttgccggtgatgcaattgatcag ctgctgcaagaagaactgaatcgtacctatccgaataatggtctgagcctgcataaagtgcgtgaaattaaagaag cacacggttacgttggtccgagccgtaaaccgctggatgttaaagttgtgattggtggtaaagcacataccctggaa ctgggtgataccctggcacgtgcatgtaatgcactgattgataaaatctatccggcactgaccaccctgattcagcgt gcaagcagcgatagcgttgttaccctgctgcagaatattatcattaccggtggtggttcacagatcaaaggtattga cacactgctgcagaaaaaactgaccgaagatggttttgaaagcccgaaagttcgcctggcaggtcatgattataaa cgttatgttgcactgggtgcactgaaagcagcccgtgccgcacgtgaaaatcagtggcaggttctgctgggttaa SEQ ID NO:4 (BeeRL2 DNA sequence) atgattccggcaagcaccgaaaaaccggttccgagcgcagcaattcctgcaaatgcaccgaatgttgcagttagca aaccggcagcaccgggtacacgtaccaaaaccgttctggttggttttgattttggcaccaataaaagctgtgttctgg caggtacagccggtgcaaccgatattgccattagcaaaattgttccgaccgttgtgggttatgtgaaagaaggtatt gttgacggtattgtggcaggtaatcgtagcgttctgtttggtgatgatgcactgcagaatcgtctgcatgcacgtctg gtggcaccgatggaacatggtgttattgcagccgagcccggccccaacgaccatcctgatgcagcacgtgattttgt tcagcatctgcgtagcctggcagatccgagcggtcaggcagaaattcgtgcagttgttggtgttccggctaatgcaa ccgaacaggcacgtgaagatgttcgtcgttgtgcatttggtatttttgatcgcattctgctgattccggaaccgtttctg gcagcactgggttatcgtgatgacgcacgcctgggtcagagcaactatattgatccggttgttaacagcctgttcatt gatattggtggtggtacaagcgatatttgtctggttcagggttattttccgggtccggatgatcagattagcattccgtt tgccggtgatgcaattgatcagctgctgcaagaagaactgaatcgtacctatccgaataatggtctgagcctgcata aagtgcgtgaaattaaagaagcacacggttacgttggtccgagccgtaaaccgctggatgttaaagttgtgattgg tggtaaagcacataccctggaactgggtgataccctggcacgtgcatgtaatgcactgattgataaaatctatccgg cactgaccaccctgattcagcgtgcaagcagcgatagcgttgttaccctgctgcagaatattatcattaccggtggtg gttcacagatcaaaggtattgacacactgctgcagaaaaaactgaccgaagatggttttgaaagcccgaaagttcg cctggcaggtcatgattataaacgttatgttgcactgggtgcactgaaagcagcccgtgccgcacgtgaaaatcagt ggcaggttctgctgggttaa SEQ ID NO:5 (peptide inserted into BeeR to provide EISDRDI Source of Genetic Material Opitutus terrae, an uncultivated anaerobic bacterium isolated from rice paddy soil obtained from rice fields of the Italian Rice Research Institute in Vercelli, Italy. See Chin et al., Int. J. Syst. Evol. Microbiol., 2001, 51(6):1965-1968. References 1. Maeda H, Khatami M. Analyses of repeated failures in cancer therapy for solid tumors: poor tumor-selective drug delivery, low therapeutic efficacy and unsustainable costs. Clin Transl Med. 2018 Mar 1;7(1):11. doi: 10.1186 / s40169- 018-0185-6. PMID: 29541939; PMCID: PMC5852245. 2. Cristie-David and Marsh. Metal-dependent assembly of a protein nano-cage. Protein Sci. 2019 Sep;28(9):1620-1629. doi: 10.1002 / pro.3676. Epub 2019 Aug 6. PMID: 31278804; PMCID: PMC6699099. 3. Wang, D. Metagenomics Databases for Bacteria. Methods Mol Biol 2649, 55-67 (2023). 4. Johnson, M. et al. NCBI BLAST: a better web interface. Nucleic Acids Res 36, W5-9 (2008). 5. Thompson, J.D., Higgins, D.G. & Gibson, T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673-80 (1994). 6. Guindon, S. et al. New algorithms and methods to estimate maximum- likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59, 307-21 (2010). 7. Punjani, A., Rubinstein, J.L., Fleet, D.J. & Brubaker, M.A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nature Methods 14, 290-296 (2017). 8. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583-589 (2021). 9. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallographica Section D 60, 2126-2132 (2004). 10. Afonine, P.V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr D Struct Biol 74, 531-544 (2018). 11. Pettersen, E.F. et al. UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci 30, 70-82 (2021). 12. Krissinel, E. Stock-based detection of protein oligomeric states in jsPISA. Nucleic Acids Research 43, W314-W319 (2015). 13. Bergeron, J.R. et al. Structure of the magnetosome-associated actin-like MamK filament at subnanometer resolution. Protein Sci 26, 93-102 (2017). 14. Löwe, J., He, S., Scheres, S.H. & Savva, C.G. X-ray and cryo-EM structures of monomeric and filamentous actin-like protein MamK reveal changes associated with polymerization. Proc Natl Acad Sci U S A 113, 13396-13401 (2016). 15. Ozyamak, E., Kollman, J., Agard, D.A. & Komeili, A. The bacterial actin MamK: in vitro assembly behavior and filament architecture. J Biol Chem 288, 4265-77 (2013). 16. Komeili, A., Li, Z., Newman, D.K. & Jensen, G.J. Magnetosomes are cell membrane invaginations organized by the actin-like protein MamK. Science 311, 242-5 (2006). 17. Fuerst, J.A. The PVC superphylum: exceptions to the bacterial definition? Antonie Van Leeuwenhoek 104, 451-66 (2013). 18. Chin, K.J., Liesack, W. & Janssen, P.H. Opitutus terrae gen. nov., sp. nov., to accommodate novel strains of the division 'Verrucomicrobia' isolated from rice paddy soil. Int J Syst Evol Microbiol 51, 1965-1968 (2001) 19. Dominguez, R. & Holmes, K.C. Actin structure and function. Annu Rev Biophys 40, 169-86 (2011). 20. Sohail M, Sun Z, Li Y, Gu X, Xu H. Research progress in strategies to improve the efficacy and safety of doxorubicin for cancer chemotherapy. Expert Rev Anticancer Ther. 2021 Dec;21(12):1385-1398.

Claims

Claims 1. An isolated polypeptide having the ability to form a nanostructure in the presence of ATP, wherein the isolated polypeptide comprises: (a) an amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2; or (b) an amino acid sequence having at least 70% sequence identity to SEQ ID NO:1 or SEQ ID NO:

2.

2. The isolated polypeptide according to claim 1, comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:1 or SEQ ID NO:

2.

3. The isolated polypeptide according to claim 1, comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1 or SEQ ID NO:

2.

4. The isolated polypeptide according to claim 1, comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1 or SEQ ID NO:

2.

5. The isolated polypeptide according to claim 1, comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:

2.

6. The isolated polypeptide according to claim 1, consisting of an amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:

2.

7. A fusion protein having the ability to form a nanostructure in the presence of ATP, wherein the fusion protein comprises the isolated polypeptide according to any one of claims 1-6 and at least one unrelated polypeptide; optionally wherein the at least one unrelated polypeptide is selected from the group consisting of: a label, a purification tag, a therapeutic moiety, an antigen, a targeting peptide, an antibody fragment, a peptide that enhances the biocompatibility, solubility, secretion, or half-life of the fusion protein or nanostructure, a conjugation domain, and any combination thereof.

8. An isolated nucleic acid molecule encoding the isolated polypeptide according to any one of claims 1-6 or the fusion protein according to claim 7.

9. An isolated nucleic acid molecule encoding a polypeptide or a fusion protein having the ability to form a nanostructure in the presence of ATP, wherein the isolated nucleic acid molecule comprises a nucleotide sequence shown in SEQ ID NO:3 or SEQ ID NO:4, or a codon-optimised version thereof.

10. The isolated nucleic acid molecule according to claim 9, consisting of a nucleotide sequence shown in SEQ ID NO:3 or SEQ ID NO:

4.

11. A nucleic acid vector comprising the isolated nucleic acid molecule according to any one of claims 8-10.

12. A host cell comprising the nucleic acid vector according to claim 11.

13. A nanostructure formed by the multimerisation of the isolated polypeptide according to any one of claims 1-6 or the fusion protein according to claim 7 in the presence of ATP or an analogue thereof.

14. The nanostructure according to claim 13, further comprising at least one chemically conjugated functional ligand, optionally wherein the functional ligand is selected from the group consisting of: biotin, polymers, diagnostic agents, therapeutic agents, masking peptides, targeting peptides, antigens, aptamers, hormones, vitamins, nanobodies, and antibodies or fragments thereof.

15. A composite nanostructure comprising the nanostructure according to claim 13 or claim 14 and at least one cargo molecule encapsulated therein.

16. The composite nanostructure according to claim 15, wherein the at least one cargo molecule is selected from: (a) a diagnostic agent, optionally wherein the diagnostic agent is an imaging agent such as a dye, a contrast agent, or a metal nanoparticle; and / or (b) a therapeutic agent, optionally wherein the therapeutic agent is selected from the group consisting of: a drug, a protein, a protein fragment, a peptide, an antigen, an antibody, an antibody fragment, an enzyme, a nucleic acid, an oligonucleotide, or an extract of biological material.

17. The composition nanostructure according to claim 16, wherein the at least one cargo molecule is an anti-neoplastic agent, optionally wherein theantineoplastic agent is selected from the group consisting of: alkylating agents, antimetabolites, topoisomerase inhibitors, antibiotics, mitotic inhibitors, protein kinase inhibitors, enzymes (e.g., L-asparaginase), proteasome inhibitors, PARP inhibitors, monoclonal antibodies, or a combination thereof.

18. A pharmaceutical composition comprising the nanostructure according to claim 13 or claim 14 or the composite nanostructure according to any one of claims 15-17.

19. A method of treating a disease or disorder comprising administering a therapeutically effective amount of the nanostructure according to claim 13 or claim 14, the composite nanostructure according to any one of claims 15-17, or the pharmaceutical composition according to claim 18 to a subject in need thereof, wherein the nanostructure, the composite nanostructure or the pharmaceutical composition comprises a therapeutic agent.

20. The method according to claim 19, wherein the disease or disorder is selected from the group consisting of infectious diseases, acute and chronic inflammatory diseases, cancer, and transplantation and autoimmune diseases.

21. The method according to claim 19 or claim 20, further comprising co- administering an additional therapeutic agent to the subject.

22. The method according to any one of claims 19-21, wherein the subject is a mammal, optionally wherein the subject is human.

23. The isolated polypeptide according to any one of claims 1-6, the fusion protein according to claim 7, the nanostructure according to claim 13 or claim 14, the composite nanostructure according to any one of claims 15-17, or the pharmaceutical composition according to claim 18 for use in treating a disease or disorder.

24. Use of the isolated polypeptide according to any one of claims 1-6, the fusion protein according to claim 7, the nanostructure according to claim 13 or claim 14, the composite nanostructure according to any one of claims 15-17, or the pharmaceutical composition according to claim 18 for the manufacture of a medicament for treating a disease or disorder.

25. A method of forming a nanostructure, wherein the method comprises combining isolated polypeptides according to any one of claims 1-6 or fusion proteins according to claim 7 with ATP or an analogue thereof, such that the isolated polypeptides or fusion proteins multimerise into a nanostructure.

26. A method of forming a composite nanostructure, wherein the method comprises combining isolated polypeptides according to any one of claims 1-6 or fusion proteins according to claim 7 with a cargo molecule and ATP or an analogue thereof, such that the isolated polypeptides or fusion proteins multimerise into a nanostructure with the cargo molecule encapsulated therein.

27. The method of forming a nanostructure according to claim 25 or the method of forming a composite nanostructure according to claim 26, further comprising conjugating at least one functional ligand to the assembled nanostructure.

28. The method of forming a nanostructure according to claim 25 or the method of forming a composite nanostructure according to claim 26 or claim 27, further comprising preventing or delaying the disassembly of the nanostructure by: (i) freezing the assembled nanostructure; (ii) utilising a poorly hydrolysable ATP analogue.

29. The method of forming a nanostructure according to claim 25 or the method of forming a composite nanostructure according to any one of claims 26- 28, wherein the ATP is photocaged ATP, and wherein the method further comprises inducing the disassembly of the nanostructure by a laser pulse.

30. The method of forming a nanostructure according to claim 25 or the method of forming a composite nanostructure according to any one of claims 26-28, further comprising inducing the disassembly of the nanostructure by exposing the nanostructure to a lower pH.

Images