Award details

Cooperativity of SH3 domains in Cin85: a NMR and thermodynamics study

Reference	BB/D018994/1
Principal Investigator / Supervisor	Dr Mark Pfuhl
Co-Investigators / Co-Supervisors	Professor John Ladbury
Institution	University of Leicester
Department	Biochemistry
Funding type	Research
Value (£)	189,724
Status	Completed
Type	Research Grant
Start date	01/11/2006
End date	31/10/2008
Duration	24 months

Abstract

The recognition of polyproline sequences by SH3 domains is a key feature in the assembly of protein complexes involved in the regulation of signal transduction, membrane trafficking and cytoskeletal rearrangements. The recruitment of the correct proteins into such a complex is critically dependent on the specificity of the protein-protein interactions on which the assembly is based. The limited number of possible variations on the theme of interactions of SH3 domains with polyproline sequences is clearly not sufficient to provide this exquisite specificity alone. Instead it is assumed that the necessary specificity will be created by multiple, different protein interaction motifs acting together in the formation of a single complex. This is supported by the simultaneous occurrence of interaction motifs such as SH2 and SH3 domains in the same adapter or signalling protein, allowing for multiple interactions based on different interaction motifs. A recent thermodynamic characterisation of the polyproline binding of the three SH3 domains of the adapter protein Cin85 has revealed - besides a new SH3 specific polyproline motif PxxxPR - an unexpected and so far unexplained cooperativity of polyproline motif binding by adjacent SH3 domains. The isolated SH3 domains bind with an affinity of ~10 uM while the two SH3 domain fragment bound the same peptide with nanomolar affinity. The manipulation of binding affinities for polyproline sequences by combining several SH3 domains in the same adapter protein could provide a hitherto unknown mechanism to enhance specificity of protein protein interactions. We plan to characterise various fragments of Cin85 with respect tostructure, dynamics and peptide binding by solution state NMR spectroscopy and perform a more detailed thermodynamic analysis of the peptide binding of these constructs. These studies are expected to explain the base of the novel cooperativity of SH3-polyproline interactions on the atomic level.

Summary

The ability for a cell to receive and act on external stimuli is fundamental to life. An external stimulus to a cell results in the activation of a cell surface receptor protein. This protein is responsible for transmitting the signal into the cell. Once this has occurred a series of interactions are initiated resulting in a signalling pathway. Ultimately, this pathway evokes a cellular response either in the form of transcription of genes in the nucleus or via activation of other, secondary pathways. When these pathways malfunction disease states such as cancer, immunodeficiency and diabetes can prevail. As a result it is important to understand how these pathways work and how they can go wrong. In this work we are interested in understanding the structure and binding mechanism of a key protein, Cin85 . This protein functions to down-regulate fundamental signalling pathways. Many of the proteins involved in these pathways are made up of domains, which are common to a large number of proteins. These domains are sequences of amino acids which fold into independent structures, with identical function within the context of the intact protein. The Cin85 protein contains a region in which there are three very similar domains known as SH3 domains. These domains are responsible for making interactions with other proteins and are fundamental to the function. These domains have recently been shown to interact with novel sites on these other proteins, the amino acid sequences of which are distinct from interactions normally observed for SH3 domains. Furthermore, the domains have been shown to act in a cooperative manner. Cooperative binding of this type can be likened to a sort of Velcro whereby once one interaction is made it is easy for the rest of the interactions to follow. Recent studies have however pointed out that in Cin85 this might be a very effective way of getting specificity in interactions, i.e. the creation of a superVelcro by combining more than one SH3 domains in a protein to bind to proline rich parts of another protein. This molecular superVelcro binds 1000 times stronger than even the best examples of molecular Velcro studied so far. To find out exactly how this superVelcro works we plan to determine its structure on the atomic level. This will involve determining the structures of the independent domains, and subsequently all the domains together. Establishing the principles of its function in one protein will then allow us to look for other, similar proteins in the newly established genome databases to see if this superVelcro might also exist elsewhere. This will then allow us to understand the way in which cells regulate their activities.

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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