Protein-protein interactions and methods for identifying interacting proteins and the amino acid sequence at the site of interaction

Abstract

The invention relates to protein-protein interactions and methods for identifying interacting proteins and the amino acid sequence at the site of interaction. Using overlapping hexapeptides that encode for the entire amino acid sequences of the linker domains of human P-glycoprotein gene 1 and 3 (HP-gp1 and HP-gp3), a direct and specific binding between HP-gp1 and 3 linker domains and intracellular proteins was demonstrated. Three different stretches ( 617 EKGIYFKLVTM 627 , (SEQ ID NO: 1) 658 SRSSLIRKRSTRRSVRGSQA 677 (SEQ ID NO: 2) and 694 PVSFWRIMKLNLT 706 (SEQ ID NO: 3) for HP-gp1 and 618 LMKKEGVYFKLVNM 631 (SEQ ID NO: 4), 648 KAATRMAPNGWKSRLFRHSTQKNLKNS 674 (SEQ ID NO: 5), and 695 PVSFLKVLKLNKT 707 (SEQ ID NO: 6) for HP-gp3) in linker domains bound to proteins with apparent molecular masses of ˜80 kDa, 57 kDa and 30 kDa. The binding of the 57 kDa protein was further characterized. Purification and partial N-terminal amino acid sequencing of the 57 kDa protein showed that it encodes the N-terminal amino acids of alpha and beta-tubulins. The method of the present invention was further validated with Annexin. The present invention thus demonstrates a novel concept whereby the interactions between two proteins are mediated by strings of few amino acids with high and repulsive binding energies, enabling the identification of high affinity binding sites between any interacting proteins.

1 . A method of identifying a compound that modulates the binding of a polypeptide to a peptide in a chosen protein, the peptide region being adjacent to a repulsive peptide region of the chosen protein, the method comprising: 
 (a) providing a set of short overlapping peptides spanning a complete sequence of at least a domain of the chosen protein, the set of short overlapping peptides being covalently attached to a support;    (b) contacting the support to which the overlapping peptides are covalently attached with a candidate compound and the polypeptide under conditions enabling binding between the peptide attached to the support and the polypeptide;    (c) washing the support to remove unbound polypeptides of the mixture; and    (d) detecting binding of the polypeptide to the peptide attached to the support,    wherein a change in the binding of the polypeptide to the peptide attached to the support in the presence of the candidate compound compared to the binding of the polypeptide to the peptide attached to the support in the absence of the candidate compound identifies the candidate compound as a compound that modulates binding of the polypeptide to the peptide in the chosen protein.    
     
     
         2 . The method of  claim 1 , wherein the domain of the chosen protein is a high affinity domain of the chosen protein.  
     
     
         3 . The method of  claim 1 , wherein the polypeptide is known to bind to the chosen protein.  
     
     
         4 . The method of  claim 1 , wherein the support is selected from the group consisting of a chip, a bead, and a plate.  
     
     
         5 . The method of  claim 1 , wherein the overlapping peptides attached to the support are synthesized synthetically using the amino acid sequence of the chosen protein.  
     
     
         6 . The method of  claim 1 , wherein each of the overlapping peptides attached to the support is from about 5 amino acids to about 15 amino acids in length.  
     
     
         7 . The method of  claim 1 , wherein each of overlapping peptides attached to the support is from about 5 amino acids to about 12 amino acids in length.  
     
     
         8 . The method of  claim 1 , wherein each of the overlapping peptides attached to the support is from about 5 amino acids to about 10 amino acids in length.  
     
     
         9 . The method of  claim 1 , wherein each of the overlapping peptides attached to the support is from about 5 amino acids to about 7 amino acids in length.  
     
     
         10 . The method of  claim 1 , wherein the chosen protein is human P-glycoprotein 1.  
     
     
         11 . The method of  claim 10 , wherein the polypeptide is tubulin.  
     
     
         12 . The method of  claim 1 , wherein the chosen protein is human P-glycoprotein 3.  
     
     
         13 . A method of identifying an agent which modulates an interaction between high-affinity interacting domains between a set of overlapping peptides spanning a complete sequence of a chosen protein, domain thereof or part thereof, covalently bound to a support, and a mixture of proteins wherein said mixture of proteins is a mixture of cellular proteins, comprising: 
 (a) incubating the set of overlapping peptides with the mixture in the presence of at least one agent, under conditions enabling the binding between the high-affinity interacting domain in a peptide of the set and one or more proteins of the mixture to occur;    (b) washing off any protein-protein interaction which is not a high-affinity interaction in step (a); and    (c) identifying which peptide from step (a) interacts with high-affinity to a protein of the mixture in the presence of the agent as compared to in the absence of the agent,    thereby identifying the agent as a modulator of high-affinity interaction when the interaction in the presence of the agent is measurably different from the interaction in the absence of the agent.    
     
     
         14 . The method of  claim 13 , wherein said mixture of proteins and/or the mixture of peptides contains a label.  
     
     
         15 . The method of  claim 13 , wherein the set of overlapping peptides is synthesized synthetically using the sequence of the chosen protein.  
     
     
         16 . The method of  claim 13 , wherein the support is chosen from a chip, a bead, or a plate.