Protein binding miniature proteins and uses thereof Number:7,393,918 from the United States Patent and Trademark Office (PTO) owispatent In certain aspects, the present invention provides miniature proteins resulted from a protein scaffold such as an avian pancreatic polypeptide that can be modified by substitution of at least one amino acid residue. In other aspects, the present invention provides diagnostic and therapeutic uses of these miniature proteins.<H miniature, proteins, Protein, binding, 7393918.html, Patent, pto, 7393918

Title: Protein binding miniature proteins and uses thereof

Abstract: In certain aspects, the present invention provides miniature proteins resulted from a protein scaffold such as an avian pancreatic polypeptide that can be modified by substitution of at least one amino acid residue. In other aspects, the present invention provides diagnostic and therapeutic uses of these miniature proteins.

Patent Number: 7,393,918 Issued on 07/01/2008 to Golemi-Kotra, et al.

Inventors: Golemi-Kotra; Dasantila (Thornhill, CA), Schepartz Shrader; Alanna S. (Wilton, CT)
Assignee: Yale University (New Haven, CT)

Appl. No.: 11/009,101

Filed: December 10, 2004

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
60529401	Dec., 2003

Current U.S. Class: 530/300

Current International Class: C07K 14/00 (20060101)

Field of Search: 435/69.1 514/2-21

References Cited [Referenced By]

Foreign Patent Documents


WO 0181375	Nov., 2001	WO
WO 03/053996	Jul., 2003	WO

Other References

Chin, J. W. et al., "Concerted evolution of structure and function in a Miniature Protein", Journal of the American Chemical Society, American Chemical Society, 123:2929-2930 (2001). cited by other .
Golemi-Kotra D. et al., "High Affinity, Paralog-specific Recognition of the Mena EVH1 Domain by a Miniature Protein," Journal of the American Chemical Society, 126(1):4-5 (2004). cited by other .
Rutledge, Stacey E. et al., "Molecular recognition of protein surfaces: high affinity ligands for the CBP KIX domain", Journal of the American Chemical Society, 125:14336-14347 (2003). cited by other .
Armstrong, K.M., et al., "The (i, i+4) Phe-His Interaction Studies in an Alanine-based .alpha.-Helix," J. Mol. Biol., 230:284-291, (1993). cited by other .
Aszodi, A., et al., "The vasodilator-stimulated phosphoprotein (VASP) is involved in cGMP-and cAMP-mediated inhibition of agonist-induced platelet aggregation, but is dispensable for smooth muscle function," Embo J., 18:37-48 (1999). cited by other .
Bachmann, C., et al., "The EVH2 Domain of the Vasodilator-stimulated Phosphoprotein Mediates Tetramerization, F-actin Binding, and Actin Bundle Formation," J. Biol. Chem., 274:23549-23557 (1999). cited by other .
Ball, L.J., et al., "EVH1 domains: structure, function and interactions," Febs Lett., 513:45-52 (2002). cited by other .
Blundell, T.L., et al., "X-ray analysis (1.4-A resolution) of avian pancreatic polypeptide: Small globular protein hormone," Proc. Natl. Acad. Sci., 78(7):4175-4179, USA, (1981). cited by other .
Callebaut, I., et al., "EVH1/WH1 domains of VASP and WASP proteins belong to a large family including Ran-binding domains of the RanBP1 family," Febs Lett. 441:181-185 (1998). cited by other .
Cameron, L.A., et al., "Secrets of actin-based motility revealed by a bacterial pathogen," Nat. Rev. Mol. Cell. Biol., 1:110-119 (2000). cited by other .
Carl, U.D., et al., "Aromatic and basic residues within the EVH1 domain of VASP specify its interaction with proline-rich ligands," Curr. Biol., 9:715-718 (1999). cited by other .
Chin et al., "Methodology for Optimizing Functional Miniature Proteins Based on Avian Pancreatic Polypeptide Using Phage Display", Bioorg. Med. Chem. Lett., 11:1501-1505 (2001). cited by other .
Chin, J.W., et al., "Design and Evolution of a Miniature Bcl-2 Binding Protein," Angew Chem. Int. Ed. Engl., 40(20):3806-3809 (2001). cited by other .
Cunningham, B.C. and Wells, J.A., "Minimized proteins," Curr. Opin. Struct. Biol., 7:457-462 (1997). cited by other .
Ermekova, K.S., et al., "The WW Domain of Neural Protein FE65 Interacts with Proline-rich Motifs in Mena, the Mammalian Homolog of Drosophila Enabled," J. Biol. Chem, 272(52):32869-32877 (1997). cited by other .
Gertler, F.B., et al., "enabled, a dosage-sensitive suppressor of mutations in the Drosophila Abl tyrosine kinase, encodes an Abl substrate with SH3 domain-binding properties," (abstract) Genes Dev., 9:521-533 (1995). cited by other .
Gertler, F.B., et al., "Genetic Suppression of Mutations in the Drosop abl Proto-Oncogene Homolog," Science, 248(4957):857-860 (1989). cited by other .
Gertler, F.B., et al., "Mena, a Relative of VASP and Drosophila Enabled, Is Implicated in the Control of Microfilament Dynamics," Cell, 87:227-239 (1996). cited by other .
Haffner, C., et al., "Molecular cloning, structural analysis and functional expression of the proline-rich focal adhesion and microfilament-associated protein VASP," Embo J., 14(1):19-27 (1995). cited by other .
Halbrugge, M. and Walter, U., "Analysis, purification and properties of a 50 000-dalton membrane-associated phosphoprotein from human platelets," J. Chromatogr., 521:335-343 (1990). cited by other .
Halbrugge, M., and Walter, U., "Purification of a vasodilator-regulated phosphoprotein from human platelets," Eur. J. Biochem, 185:41-50 (1989). cited by other .
Huttelmaier, S., et al., "Characterization of the actin binding properties of the vasodilator-stimulated phosphoprotein VASP," FEBS Lett., 451:68-74 (1999). cited by other .
Kocks, C., et al., "L. monocytogenes-Induced Actin Assembly Requires the actA Gene Product, a Surface Protein," Cell, 68:521-531 (1992). cited by other .
Koehler et al., "Discovery of an Inhibitor of a Transcription Factor Using Small Molecule Microarrays and Diversity-Oriented Syntheses", J. Am. Chem. Soc., 125:8420-8421 (2003). cited by other .
Lanier, L.M., et al., "Mena Is Required for Neurulation and Commissure Formation," Neuron, 22;313-325 (1999). cited by other .
Laurent, v., et al., "Role of Proteins of the Ena/VASP Family in Actin-based Motility of Listeria monocytogenes," J. Cell. Biol., 144(6):1245-1258 (1999). cited by other .
Loisel, T.P., et al., "Reconstitution of actin-based motility of Listeria and Shigella using pure proteins," Nature 401:613-616 (1999). cited by other .
Montclare, J.K. and Schepartz, A., "Miniature Homeodomains: High Specificity without an N-Terminal Arm," J. Am. Chem. Soc., 125:3416-3417 (2003). cited by other .
Niebuhr, K., et al., "A novel proline-rich motif present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand for the EVH1 domain, a protein module present in the Ena/VASP family," Embo J., 16(17):5433-5444 (1997). cited by other .
Pistor, S., et al., "The bacterial actin nucleator protein ActA of Listeria monocytogenes contains multiple binding sites for host microfilament proteins," Curr. Biol., 5:517-525 (1995). cited by other .
Prehoda, K.E., et al., "Structure of the Enabled/VASP Homology 1 Domain-Peptide Complex: A Key Component in the Spatial Control of Actin Assembly," Cell, 97:471-480 (1999). cited by other .
Reinhard, M., et al., "Identification, purification, and characterization of a zyxin-related protein that binds the focal adhesion and microfilament protein VASP (vasodilator-stimulated phosphoprotein)," Proc. Natl. Acad. Sci. USA, 92:7956-7960 (1995). cited by other .
Reinhard, M., et al., "The 46/50 kDa phosphoprotein VASP purified from human platelets is a novel protein associated with actin filaments and focal contacts," Embo J., 11(6):2063-2070 (1992). cited by other .
Smith, G.A., et al., "The Tandem Repeat Domain in the Listeria monocytogenes ActA Protein Controls the Rate of Actin-based Motility, the Percentage of Moving Bacteria, and the Localization of Vasodilator-stimulated Phosphoprotein and Profilin," J. Cell. Biol., 135(3):647-660 (1996). cited by other .
Southwick, F.S. and Purich, D.L., "Arrest of Listeria movement in host cells b a bacterial ActA analogue: Implications for actin-based motility," Proc. Natl. Acad. Sci. USA, 91:5168-5172 (1994). cited by other .
Suarez, M., et al., "A role for ActA in epithelial cell invasion by Listeria monocytogenes," Cell Microbiol. 3(12):853-864 (2001). cited by other .
Theriot, J.A., et al., "Listeria Monocytogenes-Based Assays for Actin Assembly Factors," Methods Enzymol. 298:114-122 (1998). cited by other .
Tonan, K., et al., "Conformations of Isolated Fragments of Pancreatic Polypeptide," Biochemistry, 29:4424-4429 (1990). cited by other .
Vita, C., et al., "Novel Miniproteins Engineered by the Transfer of Active Sites to Small Natural Scaffolds," Biopolymers, 47:93-100 (1998). cited by other .
Vita, C., et al., "Scorpion toxins as natural scaffolds for protein engineering," Proc. Natl. Acad. Sci. USA, 92:6404-6408 (1995). cited by other .
Williamson, M.P., "The structure and function of proline-rich regions in proteins," Biochem. J., 297:249-260 (1994). cited by other .
Wills, Z., et al., "The Tyrosine Kinase Abl and Its Substrate Enabled Collaborate with the Receptor Phosphatase Dlar to Control Motor Axon Guidance," Neuron, 22:301-312 (1999). cited by other .
Zarrinpar, A., et al., "The Structure and Function of Proline Recognition Domains," Sci. STKE RE8 (2003). cited by other .
Zondlo, et al., "Highly Specific DNA Recognition by a Designed Miniature Protein", J. Am. Chem. Soc., 121:6938-6939 (1999). cited by other.

Primary Examiner: Monshipouri; Maryam
Assistant Examiner: Tsay; Marsha
Attorney, Agent or Firm: Wolf, Greenfield & Sacks, P.C.

Government Interests

FUNDING

Work described herein was funded, in part, by National Institutes of Health Grant GM 59843. The United States government has certain rights to the invention.

Parent Case Text

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application No. 60/529,401, filed Dec. 11, 2003, the disclosure of which is hereby incorporated by reference in its entirety.

Claims

We claim:

1. An isolated polypeptide selected from the group consisting of: (a) an isolated polypeptide comprising amino acid sequence SEQ ID NO: 1; and (b) an isolated polypeptide at least 95 percent identical to SEQ ID NO: 1, wherein the polypeptide binds to a protein selected from the group consisting of: Drosophila Enabled (Ena), mammalian Mena, vasodilator stimulated phosphoprotein (VASP), Enabled/VASP-like protein (Evl), and Wiskott-Aldrich syndrome protein (WASP).

2. An isolated avian pancreatic polypeptide (aPP) of SEQ ID NO: 1 modified by substitution of at least one and no more than three amino acid residues, wherein at least one of the substituted residues is a substitution of one of positions 1-8 of the aPP of SEQ ID NO: 1, wherein the modified polypeptide binds to a protein selected from the group consisting of: Drosophila Enabled (Ena), mammalian Mena, vasodilator stimulated phosphoprotein (VASP), Enabled/VASP-like protein (Evl), and Wiskott-Aldrich syndrome protein (WASP).

3. An isolated avian pancreatic polypeptide (aPP) of SEQ ID NO: 1 modified by substitution of at least one and no more than three amino acid residues, wherein at least one substituted residue is a substitution of position 2 with a hydrophobic residue selected from the group consisting of leucine, isoleucine, valine, or alanine; and wherein the modified polypeptide binds to a protein selected from the group consisting of: Drosophila Enabled (Ena), mammalian Mena, vasodilator stimulated phosphoprotein (VASP), Enabled/VASP-like protein (Evl), and Wiskott-Aldrich syndrome protein (WASP).

4. The modified polypeptide of claim 3, wherein at least two and no more than three amino acid residues on the type II polyproline (PPII) helix of the aPP are substituted.

5. The modified polypeptide of claim 3, wherein one of the substitutions is a substitution of at least one amino acid residue in the linker region between the PPII helix and the alpha helix domain of the aPP.

6. The modified polypeptide of claim 5, wherein at least two amino acid residues on the linker region are substituted.

7. The modified polypeptide of claim 3, wherein the modified polypeptide binds to one protein selected from the group consisting of: Drosophila Enabled (Ena), mammalian Mena, vasodilator stimulated phosphoprotein (VASP), Enabled/VASP-like protein (Evl), and Wiskott-Aldrich syndrome protein (WASP), but does not bind to the other proteins of this group.

8. The modified polypeptide of claim 3, wherein the at least one amino acid residue substitution is in positions 2, 5, or 8.

9. The modified polypeptide of claim 3, wherein position 1 is proline.

10. The modified polypeptide of claim 3, wherein position 3 is proline.

11. The modified polypeptide of claim 3, wherein position 4 is proline.

12. The modified polypeptide of claim 3, wherein position 6 is proline.

13. The modified polypeptide of claim 3, wherein position 7 is proline.

Description

BACKGROUND

Biological interactions, such as protein:protein interactions, protein:nucleic acid interactions, and protein:ligand interactions are involved in a wide variety of processes occurring in living cells. For example, agonism and antagonism of receptors by specific ligands may effect a variety of biological processes such as gene expression, cellular differentiation and growth, enzyme activity, metabolite flow and metabolite partitioning between cellular compartments. Undesirable or inappropriate gene expression and/or cellular differentiation, cellular growth and metabolism may be attributable, at least in many cases, to biological interactions involving the binding and/or activity of proteinaceous molecules, such as transcription factors, peptide hormones, receptor molecules, and enzymes.

Peptides present potential therapeutic and prophylactic agents for many human and animal diseases, biochemical disorders and adverse drug effects, because they can interact highly specifically with other molecules. Thus, mimetic peptides have been designed and developed based on three dimensional protein structures. For example, many proteins recognize nucleic acids, other proteins or macromolecular assemblies using a partially exposed alpha helix. Within the context of a native protein fold, such alpha helices are usually stabilized by extensive tertiary interactions with residues that may be distant in primary sequence from both the alpha helix and from each other. With notable exceptions (Armstrong et al., 1993, J. Mol. Biol., 230: 284-291), removal of these tertiary interactions destabilizes the alpha helix and results in molecules that neither fold nor function in macromolecular recognition (Zondlo et al., 1999, J. Am. Chem. Soc., 121: 6938-6939). The ability to recapitulate or perhaps even improve on the recognition properties of an alpha helix within the context of a small molecule may find utility in the design of synthetic mimetics or inhibitors of protein function (Cunningham et al., 1997, Curr. Opin. Struct. Biol., 7:457-462) or new tools for proteomics research.

Proteins generally recognize each other using large and shallow complementary surfaces. Therefore, small proteins (miniature proteins) with well-defined three-dimensional structures and finely tuned functional properties are perhaps ideally suited for protein surface recognition and disruption of protein:protein interaction. Clearly, there is a need for developing the miniature proteins (in particular, those with high affinity and high specificity for a target molecule) as therapeutics and prophylactics.

SUMMARY OF THE INVENTION

In certain embodiments, the invention relates to an avian pancreatic polypeptide (aPP) modified by substitution of at least one amino acid residue, which is located within (as a component of) a type II polyproline (PPII) helix of the polypeptide when the polypeptide is in a tertiary form. In some embodiments, the modified polypeptide contains at least two or three substituted residues. Optionally, the residue substitutions can include modification of position 2 (e.g., F in SEQ ID NO:1) with other hydrophobic residues (L, I, V, A). In other embodiments, the modified polypeptide is further modified by substitution of at least one amino acid residue (e.g., two residues) of the linker region between the PPII helix and the alpha helix domain of the polypeptide. The modified polypeptide of the invention is also referred to as a miniature protein.

In certain cases, the substituted residue of the PPII helix is selected from a site on a first protein through which the first protein interacts with a second protein. The first protein can be a known protein such as, but are not limited to, a protein that interacts with EVH1 domains. Examples of the first proteins include zyxin, vinculin, and the ActA protein of Listeria monocytogenes. The second protein which interacts with the first protein includes but is not limited to any protein that contains an EVH1. For example, the second protein can be selected from the group consisting of: Drosophila Enabled (Ena), mammalian Mena, vasodilator stimulated phosphoprotein (VASP), Enabled/VASP-like protein (Evl), and Wiskott-Aldrich syndrome protein (WASP). In a preferred embodiment, the site on the first protein is a protein binding site (e.g., a polyproline helix). In some embodiments, the modified avian pancreatic polypeptide is capable of inhibiting the interaction between the first protein and the second protein, while in other embodiments, it is capable of enhancing this interaction.

In a specific embodiment, the miniature protein of the invention preferentially binds to one protein selected from the group consisting of: Drosophila Enabled (Ena), mammalian Mena, vasodilator stimulated phosphoprotein (VASP), Enabled/VASP-like protein (Evl), and Wiskott-Aldrich syndrome protein (WASP), but does not bind to the other proteins of the group.

In certain embodiments, the invention encompasses a phage-display library comprising a plurality of recombinant phage that express any of the aforementioned modified avian pancreatic polypeptides. In a related embodiment, the invention encompasses a phage-display library comprising a plurality of recombinant phage that express a protein scaffold modified by substitution of at least one amino acid residue, this residue being exposed on a type II polyproline helix of the polypeptide when the polypeptide is in a tertiary form. In some cases, the protein scaffold of the phage-display library comprises the avian pancreatic polypeptide. The invention also encompasses an isolated phage selected from the phage library of the invention.

In a specific embodiment, a miniature protein of the invention comprises the amino acid sequence PFPPTPPGEEAPVEDLIRFYNDLQQYLNVV (SEQ ID NO: 1). In other embodiments, the miniature protein may comprise any of the following amino acid sequence: PAPPTPPGEEAPVEDLIRFYNDLQQYLNVV (SEQ ID NO: 2); PFPPLPPGEEAPVEDLIRFYNDLQQYLNVV ((SEQ ID NO: 3); PLPPTPPGEEAPVEDLIRFYNDLQQYLNVV (SEQ ID NO: 4); PFPPTPPGEELPVEDLIRFYNDLQQYLNVV (SEQ ID NO: 5). Further, the present invention contemplates all the variants with A substituted at each non-A position of the avian pancreatic polypeptide, and all the variants with sarcosine substituted at positions 1-8 of the avian pancreatic polypeptide.

Further, the present invention provides an isolated polypeptide selected from the group consisting of: (a) an isolated polypeptide comprising any of the amino acid sequences as set forth in SEQ ID NOs: 1-5; (b) an isolated polypeptide comprising a fragment of at least twelve contiguous amino acids of any of SEQ ID NOs: 1-5; (c) an isolated polypeptide comprising one or more amino acid substitutions in any of the amino acid sequences as set forth in SEQ ID NOs: 1-5; and (d) an isolated polypeptide at least 95 percent identical to any of SEQ ID NOs: 1-5.

In a related embodiment, the invention also encompasses a nucleic acid encoding any one of the aforementioned miniature polypeptides of the invention.

In certain embodiments, the invention encompasses a method of preparing a miniprotein that modulates the interaction between a first protein and a second protein, comprising the steps of: (a) identifying at least one amino acid residue that contributes to the binding between a first protein and a second protein; and (b) modifying an avian pancreatic polypeptide by substitution of said at least one amino acid residue, such that said at least one amino acid residue is exposed on a type II polyproline (PPII) helix of the polypeptide when the polypeptide is in a tertiary form. As used herein, the term "modulate" refers to an alteration (enhancement or inhibition) in the association between two molecular species, for example, the effectiveness of a biological agent to interact with its target by altering the characteristics of the interaction in a competitive or non-competitive manner.

In certain embodiments, the invention further encompasses a method of identifying a miniprotein that modulates the interaction between a first protein and a second protein, comprising a step of isolating at least one recombinant phage clone from the phage display library of the invention that displays a protein scaffold that modulates the association between a first protein and a second protein.

In certain embodiments, the invention provides a method of modulating (enhancing or inhibiting) cell migration, comprising contacting a cell with a modified polypeptide of the invention in an effective amount for modulating cell migration, wherein the modified polypeptide regulates signaling through a protein of the Ena/VASP family (an Ena/VASP protein). In this method, the Ena/VASP protein is preferably selected from the group consisting of: Drosophila Enabled (Ena), mammalian Mena, vasodilator stimulated phosphoprotein (VASP), Enabled/VASP-like protein (Evl), and Wiskott-Aldrich syndrome protein (WASP). Optionally, the modified polypeptide binds to an EVH1 domain of the Ena/VASP protein. A preferred cell of this method is a mammalian cell. In certain cases, the mammalian cell is a tumor cell.

In certain embodiments, the invention provides a method for inhibiting tumor cell metastasis in a subject, comprising administering to a subject having or at risk of developing a metastatic cancer a modified polypeptide of the invention in an effective amount for inhibiting cell migration such that tumor cell metastasis is inhibited, wherein the modified polypeptide inhibits signaling through an Ena/VASP protein. In this method, the Ena/VASP protein is preferably selected from the group consisting of: Drosophila Enabled (Ena), mammalian Mena, vasodilator stimulated phosphoprotein (VASP), Enabled/VASP-like protein (Evl), and Wiskott-Aldrich syndrome protein (WASP). Optionally, the modified polypeptide binds to an EVH1 domain of the Ena/VASP protein. A preferred subject of this method is a mammal, for example, a human.

In certain embodiments, the invention provides a method of modulating (enhancing or inhibiting) growth of a neuronal cell, comprising contacting a neuronal cell with a modified polypeptide of the invention in an effective amount for modulating growth of the neuronal cell, wherein the modified polypeptide regulates signaling through an Ena/VASP protein. In this method, the Ena/VASP protein is preferably selected from the group consisting of: Drosophila Enabled (Ena), mammalian Mena, vasodilator stimulated phosphoprotein (VASP), Enabled/VASP-like protein (Evl), and Wiskott-Aldrich syndrome protein (WASP). Optionally, the modified polypeptide binds to an EVH1 domain of the Ena/VASP protein.

In certain embodiments, the invention provides a method of inhibiting neurodegeneration in a subject, comprising administering to a subject at risk of a neurodegeneration disorder a modified polypeptide of the invention in an amount effective to prevent neurodegeneration, wherein the modified polypeptide regulates signaling through an Ena/VASP protein. In this method, the Ena/VASP protein is preferably selected from the group consisting of: Drosophila Enabled (Ena), mammalian Mena, vasodilator stimulated phosphoprotein (VASP), Enabled/VASP-like protein (Evl), and Wiskott-Aldrich syndrome protein (WASP). Optionally, the modified polypeptide binds to an EVH1 domain of the Ena/VASP protein. A variety of neurodegenerative disorders can be treated by this method, such as Down Syndrome; Parkinson's disease; amyotrophic lateral sclerosis (ALS), stroke, direct trauma, Huntington's disease, epilepsy, ALS-Parkinsonism-dementia complex; progressive supranuclear palsy; progressive bulbar palsy, spinomuscular atrophy, cerebral amyloidosis, Pick's atrophy, Retts syndrome; Wilson's disease, Striatonigral degeneration, corticobasal ganglionic degeneration; dentatorubral atrophy, olivopontocerebellar atrophy, paraneoplastic cerebellar degeneration; Tourettes syndrome, hypoglycemia; hypoxia; Creutzfeldt-Jakob disease; and Korsakoffs syndrome.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows protein grafting of the FP4 epitope on a miniature protein scaffold.

FIG. 1B shows sequences of peptides and miniature proteins described in this work. Residues important for aPP folding are in blue or yellow, residues important for Mena.sub.1-112 recognition are in red. These amino acid residues are shown in duplicate in black font in the lower panel of FIG. 1B.

FIG. 1C is a spectra showing the mean residue ellipticity (.THETA..sub.MRE) of 5 .mu.M pGolemi at 25.degree. C.

FIG. 1D shows the temperature dependence of the .THETA..sub.MRE at 222 nm.

FIG. 1E shows amino acid sequences of four additional miniature proteins of the invention.

FIGS. 2A-2D show interactions between EVH1 domains, miniature proteins and peptides measured by fluorescence perturbation (A, C, and D) or fluorescence polarization (B). FIG. 2A shows binding of pGolemi (magenta), ActA.sub.11 (red) or PPII7 (yellow). FIGS. 2B and 2C show binding of pGolemi.sup.Flu (25 nM, B) or pGolemi (C) to Mena.sub.1-112 (circle), VASP.sub.1-115 (triangle) or Evl.sub.1-115 (square). FIG. 1D shows binding of ActA.sub.11 to Mena.sub.1-112 (circle), VASP.sub.1-115 (triangle) or Evl.sub.1-115 (square). Fraction bound refers to fraction of EVH1 domain bound (A, C, D) or pGolemi.sup.Flu (B).

FIG. 3 shows median speed of L. monocytogenes observed in the absence (white) or presence of pgolemi (purple) and ActA .sub.11 (red). Errors bars show the intraquartile range.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based at least in part on protein grafting, an approach to protein minimization that was successfully developed by Applicants. The protein grafting approach has been used for the identification of highly functional miniature proteins by stabilization of .alpha.-helical binding epitopes on a protein scaffold (Zondlo et al., 1999, J. Am. Chem. Soc., 121:6938-6939; Chin et al., 2001, Bioorg. Med. Chem. Lett., 11:1501-1505; Chin et al., 2001, J. Am. Chem. Soc., 123:2929-2930; Chin et al., 2001, Angew Chem. Int. Ed. Engl., 2001, 40:3806-3809; Montclare et al., 2003, J. Am. Chem. Soc., 125:3416-3417; Rutledge et al., 2003, J. Am. Chem. Soc., 125:14336-47). In these methods, protein grafting involves removing residues required for molecular recognition from their native alpha helical context and grafting them on the scaffold provided by small yet stable proteins.

Numerous researchers have engineered protein scaffolds to present binding residues on a relatively small peptide carrier. These scaffolds are small polypeptides onto which residues critical for binding to a selected target can be grafted. The grafted residues are arranged in particular positions such that the spatial arrangement of these residues mimics that which is found in the native protein. These scaffolding systems are commonly referred to as miniproteins (miniature proteins). A common feature is that the binding residues are known before the miniprotein is constructed.

Examples of these miniproteins include the thirty-seven amino acid protein charybdotoxin (Vita et al., 1995, Proc. Natl. Acad. Sci. USA, 92: 6404-6408; Vita et al., 1998, Biopolymers, 47: 93-100) and the thirty-six amino acid protein, avian pancreatic peptide (Zondlo et al., 1999, Am. Chem. Soc., 121: 6938-6939). Avian pancreatic polypeptide (aPP) is a polypeptide in which residues fourteen through thirty-two form an alpha helix stabilized by hydrophobic contacts with an N-terminal type II polyproline (PPII) helix formed by residues one through eight. Because of its small size and stability, aPP is an excellent scaffold for protein grafting of alpha helical recognition epitopes (Zondlo et al., 1999, Am. Chem. Soc., 121: 6938-6939).

Miniature Proteins

The present invention provides engineered miniature proteins that associate with (bind to) specific sequences (DNA or other proteins) and also provides methods for designing and making these miniature proteins. As used herein, the term "miniature protein" or "miniprotein" refers to a relatively small protein containing at least a protein scaffold and one or more additional domains or regions that help to stabilize its tertiary structure. Preferably, these miniature proteins bind to a target molecule (e.g., DNA or other proteins) with high affinity and selectivity.

As used herein, the term "binding" or "bind to" refers to the specific association or other specific interaction between two molecular species, such as protein-protein interactions. It is contemplated that such association is mediated through specific binding sites on each of the two interacting molecular species. As used herein, the term "binding site" refers to the reactive region or domain of a molecule that directly participates in its specific binding with another molecule. For example, when referring to the binding site on a protein, binding occurs as a result of the presence of specific amino acid sequence that interacts with the other molecule.

Schematically, the invention involves a technique that the inventors have designated as protein grafting (see, e.g., FIG. 1). In one aspect, this technique identifies critical binding site residues from a protein that participate in binding-type association between that protein and its specific binding partners. Then these residues are grafted onto a small but stable protein scaffold. As used herein, the term "protein scaffold" refers to a region or domain of a relatively small protein, such as a miniature protein, that has a conserved tertiary structural motif which can be modified to display one or more specific amino acid residues in a fixed conformation. The preferred protein scaffolds of the invention comprise members of the pancreatic fold (PP fold) protein family, particularly the avian pancreatic polypeptide.

The PP fold protein scaffolds of the invention generally contain thirty-six amino acids and are the smallest known globular proteins. Despite their small size, PP fold proteins are stable and remain folded under physiological conditions. The preferred PP fold protein scaffolds of the invention consist of two anti-parallel helices, an N-terminal type II polyproline helix (PPII) between amino acid residues two and eight, and an alpha-helix between residues 14 and 31 and/or 32. The stability of the PP fold protein scaffolds of the invention derives predominantly from interactions between hydrophobic residues on the interior face of the alpha-helix at positions 17, 20, 24, 27, 28, 30, and 31 and the residues on the two edges of the polyproline helix at positions 2, 4, 5, 7, and 8. In general, the residues responsible for stabilizing its tertiary structure are not substituted in order to maintain the tertiary structure of the miniature protein or are compensated for using phage display.

In certain embodiments, at least one of the critical binding site residues of a selected protein is grafted onto the protein scaffold in positions which are not essential in maintaining tertiary structure, preferably on the type II polyproline helix. In one preferred embodiment, two or three of such binding site residues are grafted onto the protein scaffold (e.g., aPP). Preferred positions for grafting these binding site residues on the protein scaffold include, but are not limited to, positions on the type II polyproline helix of aPP. Substitutions of binding site residues may be made, although they are less preferred, for residues involved in stabilizing the tertiary structure of the miniature protein.

A skilled artisan will readily recognize that it is not necessary that actual substitution of the grafted residues occur on the protein scaffold. Rather it is necessary that a peptide be identified, through, for example, phage display, that comprises a polypeptide constituting a miniature protein having the association characteristics of the present invention. Such peptides may be produced using any conventional means, including, but not limited to synthetic and recombinant techniques.

Members of the PP fold family of protein scaffolds which are contemplated by the present invention include, but are not limited to, avian pancreatic polypeptide (aPP), Neuropeptide Y, lower intestinal hormone polypeptide, and pancreatic peptide. In the most preferred embodiment, the protein scaffold comprises the PP fold protein, avian pancreatic polypeptide (see, e.g., Blundell et al., 1981, Proc. Natl. Acad. Sci. USA, 78: 4175-4179; Tonan et al., 1990, Biochemistry, 29: 4424-4429). aPP is a PP fold polypeptide characterized by a short (eight residue) amino-terminal type II polyproline helix linked through a type I beta turn (also referred to herein as the linker region) to an eighteen residue alpha-helix. Because of its small size and stability, aPP is an excellent protein scaffold for, e.g., protein grafting of polyproline helix recognition epitopes.

In certain embodiments, the present invention encompasses miniature proteins that bind to a target protein. Optionally, the binding of the miniature proteins modulates protein-protein interaction between the target protein and its binding partner (protein). In one embodiment, making the protein-binding miniature proteins of the invention involves identifying the amino acid residues which are essential to binding of the target protein to its binding partner. In some embodiments, these essential residues are identified using three-dimensional models of a target protein or a protein complex which binds to or interacts with another protein based on crystallographic studies, while in other embodiments, they are identified by studies of deletion or substitution mutants of the target protein. The residues that participate in binding of the protein to its binding partner are then grafted onto those positions which are not necessary to maintain the tertiary structure of the protein scaffold to form the protein-binding miniature protein.

The structure of any protein which binds to another protein can be used to derive the protein-binding miniature proteins of the invention. Preferred embodiments include proline rich sequences on some proteins that are folded into type II polyproline (PPII) helices. The PPII helical structures can be recognized and bound to by certain protein motifs, such as EVH1 domains, SH3 domains, and WW domains.

In a specific embodiment, the invention provides miniature proteins that bind to a protein of the Ena/VASP family (herein referred to as "an Ena/VASP protein"). For example, the protein grafting procedure described herein was applied to the PPII helix of the ActA protein of Listeria monocytogenes to design a miniature protein capable of binding to an EnaNASP protein. In this procedure, the primary sequence of a PPII helix of a protein is aligned with residues in the PPII helix of aPP. Alignments with a large number of conflicts are eliminated as they would force selection between sequences that were well folded or have high affinity, but make it difficult to isolate a molecule with both these properties. Structural models of the aPP based peptides that are associated or complexed with the EVH1 domain of an Ena/VASP protein in each of the alignments are evaluated for unfavorable interactions or steric clashes between the VanderWaals surface of the Ena/VASP protein and the backbone of the aPP scaffold. Structural models with multiple unfavorable interactions or steric clashes are eliminated from further consideration.

Examples of the protein-binding miniature proteins which bind to an Ena/VASP protein include, but are not limited to, the amino acid sequence depicted in SEQ ID NOs: 1-5 (FIG. 1).

Variants of Miniature Proteins

The miniature proteins of the present invention further include conservative variants of the miniature proteins herein described. As used herein, a conservative variant refers to alterations in the amino acid sequence that do not substantially and adversely affect the binding or association capacity of the protein. A substitution, insertion or deletion is said to adversely affect the miniature protein when the altered sequence prevents or disrupts a function or activity associated with the protein. For example, the overall charge, structure or hydrophobic-hydrophilic properties of the miniature protein can be altered without adversely affecting an activity. Accordingly, the amino acid sequence can be altered, for example to render the peptide more hydrophobic or hydrophilic, without adversely affecting the activities of the miniature protein.

These variants, though possessing a slightly different amino acid sequence than those recited above, will still have the same or similar properties associated with the miniature proteins depicted in SEQ ID NOs: 1-5. Ordinarily, the conservative substitution variants, will have an amino acid sequence having at least ninety percent amino acid sequence identity with any of the miniature sequences set forth in SEQ ID NOs: 1-5, more preferably at least ninety-five percent, even more preferably at least ninety-eight percent, and most preferably at least ninety-nine percent. Identity or homology with respect to such sequences is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the known peptides, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. N-terminal, C-terminal or internal extensions, deletions, or insertions into the peptide sequence shall not be construed as affecting homology.

Thus, the miniature proteins of the present invention include molecules comprising any of the amino acid sequences of SEQ ID NOs: 1-5; fragments thereof having a consecutive sequence of at least about 20, 25, 30, 35 or more contiguous amino acid residues of the miniature proteins of the invention; amino acid sequence variants of such sequences wherein at least one amino acid residue has been inserted N- or C-terminal to, or within, the disclosed sequence; amino acid sequence variants of the disclosed sequences, or their fragments as defined above, that have been substituted by another residue. Contemplated variants further include those derivatives wherein the protein has been covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid (for example, a detectable moiety such as an enzyme or radioisotope).

Nucleic Acid Molecules Encoding Miniature Proteins

The present invention further provides nucleic acid molecules that encode the subject miniature proteins (e.g., comprising any of the amino acid sequences of SEQ ID NOs: 1-5) and the related miniature proteins herein described, preferably in isolated form. As used herein, "nucleic acid" includes cDNA and mRNA, as well as nucleic acids based on alternative backbones or including alternative bases whether derived from natural sources or synthesized. As used herein, a nucleic acid molecule is said to be "isolated" when the nucleic acid molecule is substantially separated from contaminant nucleic acid encoding other polypeptides from the source of nucleic acid.

The present invention further provides fragments of the encoding nucleic acid molecule. As used herein, a "fragment of an encoding nucleic acid molecule" refers to a portion of the entire protein encoding sequence of the miniature protein. The size of the fragment will be determined by the intended use. For example, if the fragment is chosen so as to encode an active portion of the protein, the fragment will need to be large enough to encode the functional region(s) of the protein. The appropriate size and extent of such fragments can be determined empirically by persons skilled in the art.

Modifications to the primary structure itself by deletion, addition, or alteration of the amino acids incorporated into the protein sequence during translation can be made without destroying the activity of the miniature protein. Such substitutions or other alterations result in miniature proteins having an amino acid sequence encoded by a nucleic acid falling within the contemplated scope of the present invention.

The present invention further provides recombinant DNA molecules that contain a coding sequence. As used herein, a recombinant DNA molecule is a DNA molecule that has been subjected to molecular manipulation. Methods for generating recombinant DNA molecules are well known in the art, for example, see Sambrook et al., (1989) Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory Press. In the preferred recombinant DNA molecules, a coding DNA sequence is operably linked to expression control sequences and vector sequences.

The choice of vector and expression control sequences to which one of the protein family encoding sequences of the present invention is operably linked depends directly, as is well known in the art, on the functional properties desired (e.g., protein expression, and the host cell to be transformed). A vector of the present invention may be at least capable of directing the replication or insertion into the host chromosome, and preferably also expression, of the structural gene included in the recombinant DNA molecule.

Expression control elements that are used for regulating the expression of an operably linked miniature protein encoding sequence are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, and other regulatory elements. Preferably, the inducible promoter is readily controlled, such as being responsive to a nutrient in the host cell's medium.

In one embodiment, the vector containing a coding nucleic acid molecule will include a prokaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra-chromosomal in a prokaryotic host cell, such as a bacterial host cell, transformed therewith. Such replicons are well known in the art. In addition, vectors that include a prokaryotic replicon may also include a gene whose expression confers a detectable marker such as a drug resistance. Typical of bacterial drug resistance genes are those that confer resistance to ampicillin or tetracycline.

Vectors that include a prokaryotic replicon can further include a prokaryotic or bacteriophage promoter capable of directing the expression (transcription and translation) of the coding gene sequences in a bacterial host cell, such as E. coli. A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention. Any suitable prokaryotic host can be used to express a recombinant DNA molecule encoding a protein of the invention.

Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can also be used to form a recombinant DNA molecules that contains a coding sequence. Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired DNA segment.

Eukaryotic cell expression vectors used to construct the recombinant DNA molecules of the present invention may further include a selectable marker that is effective in an eukaryotic cell, preferably a drug resistance selection marker. A preferred drug resistance marker is the gene whose expression results in neomycin resistance, i.e., the neomycin phosphotransferase (neo) gene. (Southern et al., (1982) J. Mol. Anal. Genet. 1, 327-341). Alternatively, the selectable marker can be present on a separate plasmid, the two vectors introduced by co-transfection of the host cell, and transfectants selected by culturing in the appropriate drug for the selectable marker.

Transformed Host Cells

The present invention further provides host cells transformed with a nucleic acid molecule that encodes a miniature protein of the present invention. The host cell can be either prokaryotic or eukaryotic. Eukaryotic cells useful for expression of a miniature protein of the invention are not limited, so long as the cell line is compatible with cell culture methods and compatible with the propagation of the expression vector and expression of the gene product.

Transformation of appropriate cell hosts with a recombinant DNA molecule encoding a miniature protein of the present invention is accomplished by well known methods that typically depend on the type of vector used and host system employed. With regard to transformation of prokaryotic host cells, electroporation and salt treatment methods can be employed (see, for example, Sambrook et al., (1989) Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory Press; Cohen et al., (1972) Proc. Natl. Acad. Sci. USA 69, 2110-2114). With regard to transformation of vertebrate cells with vectors containing recombinant DNA, electroporation, cationic lipid or salt treatment methods can be employed (see, for example, Graham et al., (1973) Virology 52, 456-467; Wigler et al., (1979) Proc. Natl. Acad. Sci. USA 76, 1373-1376).

Successfully transformed cells (cells that contain a recombinant DNA molecule of the present invention), can be identified by well known techniques including the selection for a selectable marker. For example, cells resulting from the introduction of a recombinant DNA of the present invention can be cloned to produce single colonies. Cells from those colonies can be harvested, lysed and their DNA content examined for the presence of the recombinant DNA using a method such as that described by Southern, (1975) J. Mol. Biol. 98, 503-517 or the proteins produced from the cell assayed via an immunological method.

Production of Recombinant Miniature Proteins

The present invention further provides methods for producing a miniature protein of the invention using nucleic acid molecules herein described. In general terms, the production of a recombinant form of a protein typically involves the following steps: a nucleic acid molecule is obtained that encodes a miniature protein of the invention, for example, the nucleic acid molecule encoding the miniature protein depicted in any of SEQ ID NOs: 1-5. The nucleic acid molecule is then preferably placed in operable linkage with suitable control sequences, as described above, to form an expression unit containing the protein open reading frame. The expression unit is used to transform a suitable host and the transformed host is cultured under conditions that allow the production of the recombinant miniature protein. Optionally the recombinant miniature protein is isolated from the medium or from the cells; recovery and purification of the protein may not be necessary in some instances where some impurities may be tolerated.

Each of the foregoing steps can be done in a variety of ways. The construction of expression vectors that are operable in a variety of hosts is accomplished using appropriate replicons and control sequences, as set forth above. The control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene. Suitable restriction sites, if not normally available, can be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors. A skilled artisan can readily adapt any host/expression system known in the art for use with the nucleic acid molecules of the invention to produce a recombinant miniature protein.

The present invention further contemplates making the miniature proteins by chemical synthesis.

Production of Miniature Proteins Using Phage Display

In some embodiments, the present invention contemplates producing and selecting a miniature protein using a phage display method (McCafferty et al., (1990) Nature 348, 552-554). In this method, display of recombinant miniature proteins on the surface of viruses which infect bacteria (bacteriophage or phage) make it possible to produce soluble, recombinant miniature proteins having a wide range of affinities and kinetic characteristics. To display the miniature proteins on the surface of phage, a synthetic gene encoding the miniature protein is inserted into the gene encoding a phage surface protein (e.g., pIII) and the recombinant fusion protein is expressed on the phage surface (McCafferty et al., 1990, Nature, 348: 552-554; Hoogenboom et al., 1991, Nucleic Acids Res., 19: 4133-4137). Variability is introduced into the phage display library to select for miniature proteins which not only maintain their tertiary, helical structure but which also display increased affinity for a preselected target because the critical (or contributing but not critical) binding residues are optimally positioned on the helical structure.

Since the recombinant proteins on the surface of the phage are functional, phage bearing miniature proteins that bind with high-affinity to a particular target molecule (e.g., a protein) can be separated from non-binding or lower affinity phage by antigen affinity chromatography. Mixtures of phage are allowed to bind to the affinity matrix, non-binding or lower affinity phage are removed by washing, and bound phage are eluted by treatment with acid or alkali. Depending on the affinity of the miniature protein for its target, enrichment factors of twenty-fold to a million-fold are obtained by a single round of affinity selection. By infecting bacteria with the eluted phage, however, more phage can be grown and subjected to another round of selection. In this way, an enrichment of a thousand-fold in one round becomes a million-fold in two rounds of selection. Thus, even when enrichments in each round are low (Marks et al., 1991, J. Mol. Biol, 222: 581-597), multiple rounds of affinity selection leads to the isolation of rare phage and the genetic material contained within which encodes the sequence of the domain or motif of the recombinant miniature protein that binds or otherwise specifically associates with it binding target.

In various embodiments of the invention, the methods disclosed herein are used to produce a phage expression library encoding miniature proteins capable of binding to protein that has already been selected using the protein grafting procedure described above. In these embodiments, phage display can be used to identify miniature proteins that display an even higher affinity for a particular target protein than that of the miniature proteins produced without the aid of phage display. In yet another embodiment, the invention encompasses a universal phage display library that can be designed to display a combinatorial set of epitopes or binding sequences to permit the recognition of target molecules (e.g., nucleic acids, proteins or small molecules) by a miniature protein without prior knowledge of the natural epitope or specific binding residues or motifs natively used for recognition and association.

Various structural modifications are also contemplated for the present invention that include the addition of restriction enzyme recognition sites into the polynucleotide sequence encoding the miniature protein that enable genetic manipulation of these gene sequences. Accordingly, the re-engineered miniature proteins can be ligated, for example, into an M13-derived bacteriophage cloning vector that permits expression of a fusion protein on the phage surface. These methods allow for selecting phage clones encoding fusion proteins that bind to a target molecule and can be completed in a rapid manner allowing for high-throughput screening of miniature proteins to identify the miniature protein with the highest affinity and selectivity for a particular target.

According to the methods of the invention, a library of phage displaying modified miniature proteins is incubated with the immobilized target molecule (e.g., a Mena protein) to select phage clones encoding miniature proteins that specifically bind to or otherwise specifically associate with the immobilized protein. This procedure involves immobilizing a polypeptide sample on a solid substrate. The bound phage are then dissociated from the immobilized polypeptide and amplified by growth in bacterial host cells. Individual viral plaques, each expressing a different recombinant miniature protein, are expanded to produce amounts of protein sufficient to perform a binding assay. The DNA encoding this recombinant binding protein can be subsequently modified for ligation into a eukaryotic protein expression vector. The modified miniature protein, adapted for expression in eukaryotic cells, is ligated into a eukaryotic protein expression vector.

Phage display methods that can be used to make the miniature proteins of the present invention include those disclosed in Brinkman et al., (1995) J. Immunol. Methods 182, 41-50; Ames et al., (1995) J. Immunol. Methods 184:177-186; Kettleborough et al., (1994) Eur. J. Immunol. 24, 952-958; Persic et al., (1997) Gene 187, 9-18; Burton et al., (1994) Adv. Immunol. 57, 191-280; U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743, 5,837,500 & 5,969,108.

Methods to Identify Binding Partners

In certain embodiments, the present invention relates to methods for use in isolating and identifying binding partners of the miniature proteins of the invention. In some aspects, a miniature protein of the invention is mixed with a potential binding partner or an extract or fraction of a cell under conditions that allow the association of potential binding partners with the miniature protein of the invention. After mixing, peptides, polypeptides, proteins or other molecules that have become associated with a miniature protein of the invention are separated from the mixture. The binding partner bound to the protein of the invention can then be removed and further analyzed. To identify and isolate a binding partner, the entire miniature protein can be used. Alternatively, a fragment of the miniature protein which contains the binding domain can be used.

As used herein, a "cellular extract" refers to a preparation or fraction which is made from a lysed or disrupted cell. A variety of methods can be used to obtain an extract of a cell. Cells can be disrupted using either physical or chemical disruption methods. Examples of physical disruption methods include, but are not limited to, sonication and mechanical shearing. Examples of chemical lysis methods include, but are not limited to, detergent lysis and enzyme lysis. A skilled artisan can readily adapt methods for preparing cellular extracts in order to obtain extracts for use in the present methods. Once an extract of a cell is prepared, the extract is mixed with a miniature protein of the invention under conditions in which association of the miniature protein with the binding partner can occur. A variety of conditions can be used, the most preferred being conditions that closely resemble conditions found in the cytoplasm of a human cell. Features such as osmolarity, pH, temperature, and the concentration of cellular extract used, can be varied to optimize the association of the protein with the binding partner.

After mixing under appropriate conditions, the bound complex is separated from the mixture. A variety of techniques can be utilized to separate the mixture. For example, antibodies specific to a protein of the invention can be used to immunoprecipitate the binding partner complex. Alternatively, standard chemical separation techniques such as chromatography and density-sediment centrifugation can be used. After removal of non-associated cellular constituents found in the extract, the binding partner can be dissociated from the complex using conventional methods. For example, dissociation can be accomplished by altering the salt concentration or pH of the mixture.

To aid in separating associated binding partner pairs from the mixed extract, the miniature protein of the invention can be immobilized on a solid support. For example, the miniature protein can be attached to a nitrocellulose matrix or acrylic beads. Attachment of the miniature protein to a solid support aids in separating peptide-binding partner pairs from other constituents found in the extract. The identified binding partners can be either a single protein or a complex made up of two or more proteins. Alternatively, binding partners may be identified using the Alkaline Phosphatase fusion assay according to the procedures of Flanagan & Vanderhaeghen, (1998) Annu. Rev. Neurosci. 21, 309-345 or Takahashi et al., (1999) Cell 99, 59-69; the Far-Western assay according to the procedures of Takayama et al., (1997) Methods Mol. Biol. 69, 171-184 or Sauder et al., J. Gen. Virol. (1996) 77, 991-996 or identified through the use of epitope tagged proteins or GST fusion proteins.

Alternatively, the nucleic acid molecules encoding a miniature protein of the invention can be used in a yeast two-hybrid system. The yeast two-hybrid system has been used to identify other protein partner pairs and can readily be adapted to employ the nucleic acid molecules herein described (see, e.g., Stratagene Hybrizap.RTM. two-hybrid system).

EVH1 Domains and Ena/VASP Protein Family

In certain embodiments, miniature proteins of the invention binds to an Ena/VASP protein with high affinity and high specificity. The evolutionarily-conserved Ena/VASP protein family has been implicated in the regulation of cell migration (Gertler et al., 1996, Cell, 87: 227-39). Enabled (Ena) was identified as a genetic suppressor of loss-of-function mutations in Drosophila Ableson tyrosine kinase (D-Ab1) (Gertler et al., 1990, Science, 248: 857-60). Loss-of-function mutations in Ena ameliorated the embryonic central nervous system defects associated with loss of D-Ab1 in combination with mutations in any of several known D-Ab1 modifier genes (Gertler, et al., 1995, Genes Dev, 9: 521-33). VASP was identified biochemically as an abundant substrate for cyclic-nucleotide dependent kinases in mammalian platelets (Halbrugge et al., 1990, J Chromatogr, 521: 335-43). Two other mammalian members of this protein family, Mena (mammalian Enabled) and EVL (Ena/VASP like), were identified by sequence similarity (Gertler et al., 1996, Cell, 87: 227-39).

All Ena/VASP family members share a conserved domain structure. The N-terminal third of the protein, called the EVH1 (Ena VASP Homology) domain, mediates subcellular targeting of Ena/VASP proteins to focal adhesions by binding to proteins containing a motif whose consensus is D/E FPPPPX D/E (Niebuhr et al., 1997, Embo J, 16: 5433-44). Mutational analysis indicated that the phenylalanine residue, along with flanking acidic residues on either side, are critical for optimal binding (Carl et al., 1999, Curr Biol, 9: 715-8). The EVH1 ligand motif is found in a number of cellular proteins, including the focal adhesion proteins zyxin and vinculin. The central portion of Ena/VASP proteins contains proline-rich stretches, which have been reported to be binding sites for three types of proteins: the G-actin binding protein profilin, SH3 domain-containing proteins, and WW domain-containing proteins (Ermekova et al., 1997, J Biol Chem, 272: 32869-77; Gertler et al., 1996, Cell, 87: 227-39). The C-terminal third of Ena/VASP proteins contains the EVH2 domain that binds in vitro to F-actin and has a putative coiled-coil region reported to be important for multimerization (Bachmann et al., 1999, J Biol Chem, 274: 23549-57; Huttelmaier et al., 1999, FEBS Lett, 451: 68-74).

In addition to their capacity to bind profilin and actin, the localization of Ena/VASP proteins suggests that they may be involved in regulating actin dynamics and/or adhesion (Reinhard et al., 1992, Embo J., 11: 2063-70; Gertler et al., 1996, Cell, 87: 227-39; Lanier et al., 1999, Neuron, 22: 313-25). Genetic analyses of Ena/VASP family members in flies and mice demonstrated that these proteins function in processes that involve cell shape change, and movement including platelet aggregation and axon guidance (Aszodi et al., 1999, Embo J., 18: 37