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Laboratory of Molecular Biophysics
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Work in this group addresses structure-function relationships of medically important proteins from three different areas: adhesive cellular interactions, the eukaryotic cell cycle, and the enzyme arylamine N-acetyltransferase (NAT). We study these proteins by both experimental and theoretical approaches. Experimentally, proteins are subject to biochemical and biophysical characterisation, as well as structural analysis by X-ray crystallography and nuclear magnetic resonance. Development of theoretical methods has centred on developing tools for molecular analysis. These include programs to study protein surfaces in terms of their chemical character as indicated by sequence conservation, electrostatic potential, or other potentials calculated by the program GRID. We have also been exploring the use of this approach to identify probable sites of protein-protein interaction.
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| Figure 1: The 1.25 Å
structure of mouse CD44, drawn in ribbon trace, with anisotropic
thermal ellipsoids at atomic positions |
Adhesive interactions involving CD44, the cell surface receptor for hyaluronan, underlie fundamental processes such as limb morphogenesis, wound healing, leukocyte migration and tumor metastasis. Critical to these events, the regulation of CD44's hyaluronan-binding activity is known to be effected by changes in N-glycosylation, switching the receptor "on" under appropriate circumstances. How glycosylation influences CD44 function has until now been unclear. Like many hyaluronan-binding proteins found in extracellular matrix, CD44 contains a conserved lectin-like domain termed the Link module. However, CD44 is unique in that regions of the extracellular domain additional to the Link module are required for receptor function, and evidence suggests these "extensions" are involved in regulation. We have shown using X-ray crystallography and NMR spectroscopy that sequences flanking the Link module form a supplementary structural lobe that extends the main hyaluronan-binding surface. Moreover, the location of key N-glycosylation sites revealed for the first time how such glycans might regulate CD44 function. This year we have pursued crystallographic studies with mouse CD44 with a view to producing crystals that lend themselves to binding studies.
FAK is localised to focal adhesions via its C-terminal Focal Adhesion Targeting (FAT) domain. FAT performs this function by binding to paxillin and talin, both of which are in turn associated with the cytoplasmic tails of integrins. FAT is also required for binding of FAK substrates: a phospho-tyrosine motif within FAT binds to the Src homology (SH) 2 domain of the adapter protein Grb2. A proline-rich sequence immediately upstream of the FAT domain binds to the SH3 domains of p130 CAS, Graf, and the p85 subunit of phosphatinosytol-3 kinase.
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| Figure 2: A) Binding sites for paxillin LD peptides on FAK, and resulting models for association in B) bimolecular and C) multimolecular complexes |
This year we have established that the FAT domain binds to long-forms of all 5 LD motifs of paxillin, indicating a highly relaxed specificity, the implications of which we are exploring by further binding studies.
We have collaborated with other groups in the LMB on various aspects of cell-cycle regulation over recent years, the results of which are discussed elsewhere in this report (see reports from Prof. Louise Johnson, Dr. Jim McDonnell, and Prof. Jane Endicott).
This group has focussed this year on characterising the dynamic properties of CDK molecules. To this end, we have developed a web-based server that evaluates the essential dynamics of submitted proteins
This analysis, together with others based on molecular dynamics, have revealed a surprisingly dynamic structure for CDK2/cyclin A, and demonstrated an intrinsic preference for this complex to undergo a characteristic set of motions.
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| Figure 3: First Eigenvector of structural variability of cyclin-complexed CDK2. |
This year we have extended our structure function studies of the arylamine N-acetyltransferase family of enzymes by developing a structure activity relationship for M. smegmatis NAT with reference to the known structure for the complex of NAT with isoniazid. This has further offered insights into "handles" within the active site that might be exploited in inhibitor design.
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| Figure 4: A) Observed and B-F) putative substrate complexes of M. smegmatis NAT. |
This year we have developed and implemented algorithms for the analytical calculation of protein surfaces, and for the evaluation of electrostatic potential through solving the Poisson-Boltzmann equation by finite difference methods. These algorithms have been implemented in the programs AESOP, ccp4mg, and coot. We have also designed a novel algorithm for assigning amino-acid conservation from analysis of aligned sequences that promises to reveal important functional surfaces.
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| Figure 5: Analytical tools: Surfaces of A) cd44A with associated electrostatic potential map, and B) p38 kinase coloured by sequence conservation, and revealing the site of substrate recruitment. |
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