OCMS Current Research Programme

OCMS Research Programme 2001-2005

OCMS research focuses on the exploration of macromolecular complexes and their chemistry. The complexes will be investigated by studying the individual components, mapping protein-protein interactions, identifying structure-function relationships and performing time-resolved studies, as well as by isolating and characterising the complexes themselves. We will try to exploit the information obtained, in terms of therapeutic strategies using the strong links now developed between Chemistry and Clinical Medicine. New methodologies will be developed, where necessary. OCMS has a good track record in technique development, having pioneered new applications of X-ray diffraction, mass spectrometry and NMR.

Structural genomics of secondary metabolism: towards structures for multi-enzyme complexes
Structure and interactions of large macromolecular assemblies involving membranes
Structural aspects of intracellular signalling

There are clear technological and scientific links between these three programmes; all require extensive facilities for X-ray diffraction, electron microscopy, mass spectrometry, NMR and protein production.

Programme A: Structural Genomics of Secondary Metabolism: Towards Structures for Multi-Enzyme Complexes

Participating OCMS members: JE Baldwin, ID Campbell, K Harlos, CJ Schofield (section coordinator), RC Wilmouth

Key collaborators: SE Jensen (University of Alberta, Canada), J Hajdu, I Anderson (Uppsala), CV Robinson (Cambridge), S Hasnain (Daresbury)

Objectives

To determine high resolution crystal structures or provide structural models (at least 10 new crystal structures) for all the proteins involved in the biosynthesis of b-lactams including clavams, penicillins, and cephalosporins in S. clavuligerus.
To investigate the role of multi-protein complexes and intermediate channelling in secondary metabolism.
To provide the necessary structural framework to modify the biosynthetic enzymes to develop fermentation routes to clinically useful new antibiotics and broad-spectrum b-lactamase inhibitors.
To employ a combined 'chemical structural genetics' approach to define the roles of all the proteins encoded for by the b-lactam biosynthesis operons in S. clavuligerus (including regulatory and transport proteins).
To foster an environment in which the knowledge and skills of trained chemists can be utilised to exploit structural genomics for scientific advances and medicinal benefit.

Figure: Crystals of several gene products of the clavulanic acid and cephalosporin C biosynthetic pathways

Programme B: Structure and Interactions of Large Macromolecular Assemblies Involving Membranes

Participating OCMS members: ID Campbell, J Grimes, S Fuller, EY Jones, DI Stuart and C Venién-Bryan

Key collaborators: S Davis (Nuffield Dept. Medicine), P A van der Merwe (Dunn School of Pathology), G Screaton (Institute for Molecular Medicine), D Bamford (Helsinki University)

Objectives

How are cytokine/receptor signalling assemblies arranged at the cell surface?
What molecular interactions govern T cell signalling through formation of an immunological synapse?
How can crystallographic analyses be extended to viruses containing a lipid envelope?

Programme of work

For the first two objectives, cryo-EM will place, in a larger scale context, systems whose individual components and interactions are being analysed by x-ray crystallography and solution NMR. Conversely, for the last objective, we will develop methods to move from cryo-EM studies to higher resolution structural analyses by x-ray crystallography. The methodologies to be used and developed are described in B.3.

Figure 1: X-ray crystallographic studies on the lipid envelope containing prokaryotic virus bacteriophage PRD1

Programme C: Structural Aspects of Intracellular Signalling

Participating OCMS members: JA Endicott, EF Garman, LN Johnson, J McDonnell, MEM Noble

Key collaborators: X Xu (Institute of Molecular Medicine), P Rugman (Roche Pharmaceuticals), M Sansom (Dept. Biochemistry), R Ravelli & S McSweeney (ESRF, Grenoble), DH Newell (Cancer Research Unit, University of Newcastle), C Nave (SRS Daresbury)

Objectives

Control by protein phosphorylation, especially of proteins that regulate the cell cycle, has been a major focus of our research within OCMS in the last decade. We now have a better understanding of the regulation of enzymatic activity by association with regulatory partners and by phosphorylation. We have examined how protein kinases and protein phosphatases recognise their substrates by study of protein-peptide interactions (Brown et al. (1999) Nat Cell Biol 1 438-443) and, more recently, intact protein-protein complexes (Song et al. (2001) Molecular Cell 7 615-626). The protein production strategies that we have invented to achieve these results are in considerable demand from both academics and industry.

Our focus on biological problems has generated structural targets that present technical challenges in sample handling, requiring technological developments in the area of cryo-crystallography. To address these challenges we have designed tools and apparatus, now marketed by Oxford Cryosystems, that are widely used in the crystallographic research community to allow data collection from small and radiation-sensitive samples.

Our objective is to use our accumulated experience to achieve three new goals:

To explore the assembly of large macromolecular complexes in which protein kinases are often found. We will broaden our research to include protein kinase complexes which act at the cell surface. In particular we will study the interplay of protein association and phosphorylation that results in intracellular signalling from the T-cell receptor through the non-receptor tyrosine kinase ZAP-70.
To extend our knowledge of the activity and regulation of protein kinases into the kinetic domain. We will combine information derived from CDK2 structure determination with molecular dynamic simulations of this protein family.
To understand the physical principles which limit crystal life-time in an X-ray beam. We will apply this understanding to the development of improved practices and tools for sample preparation, in anticipation of highly automated structure determination programs, especially in connection with the ESRF and Diamond synchrotrons.

Figure: Multiple conformations in crystals of SH3-SH2 module pairs from Src-family kinases Src, Hck, Fyn, and Lck. Structures are superimposed on the basis of their SH2 domains (lower right). Src Hck and Fyn fall into one family (upper right), while Lck differs (left).

See also