Laboratory of Molecular Biophysics
Laboratory Journal 2003
Martin Noble

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Martin Noble

Adhesive interactions, the cell cycle, and NAT enzymes

Research topics and recent results

Work in my 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. On the theoretical side, we have been developing tools to visualise and analyse protein structures. These include (i) the CCP4 molecular graphics viewer (CCP4MG), (ii) a novel protocol for determining and mapping hydrophobicity at the surface of a protein, and (iii) a simple server for the prediciotn of preferred concerted modes of motion in a protein structure. In addition, we have been developing and applying experimental methods of structure based inhibitor design to the areas of biology in which we are interested. We continue to be grateful for support from the MRC, BBSRC.

Adhesion and signalling from adhesive complexes

The cell membrane is a location of bidirectional signalling. External events may activate signalling networks within the cell, while internal signals may be transduced through modulation of the properties of cell-surface proteins that mediate cellular adhesion to the extracellular matrix or to other cells. We are studying three prototypical signalling systems that involve both outside-in and inside-out signalling. These are (i) CD44, a transmembrane protein found on the surface of many cell-types in mammals, where it acts as the major receptor for the glycosaminoglycan hyaluronan (HA), (ii) focal adhesions, the cellular substructure that forms where integrins bind to proteinaceous components of the extracellular matrix, and (iii) Signalling from ZAP70.

CD44

(With Dr. D. Jackson, IMM, Dr. A.J. Day, MRC Immunochemistry)

Figure 1: Electrostatic potential distribution around a postulated HA binding site on CD44

Adhesive interactions involving CD44, the cell surface receptor for hyaluronan, underlie fundamental processessuch 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 reveals for the first time how such glycans might regulate CD44 function.

FAT

Maria Hoellerer, Eugene Valkov (With Dr. S. Arold, CNRS Montpellier, Prof. I.Campbell, Dr. J. Werner, Dr. M. Ginsberg Scripps, San Diego)

Focal adhesions (FAs) are large submembrane signalling complexes formed at sites of cellular attachment to the extracellular matrix. FAs process and transduce integrin-mediated signalling events and are implicated in the regulation and metastasis of cancer cells. The interaction of so-called LD-motifs with their targets plays an important role in the assembly of FAs. We have determined the molecular basis for the recognition of two paxillin LD-motifs when bound to the FA targeting domain (FAT) of FA kinase using a combination of X-ray crystallography, solution NMR, isothermal titration calorimetry and homology modelling. The four-helical FAT domain displays two LD binding sites on opposite sites of the helical bundle. One of the sites overlaps with the phosphorylation and Grb2 binding site of FAT, and has to be liberated to allow signalling after FA recruitment. Binding of LD peptides stabilises the FAT domains. Consideration of known LD-interacting structures indicates that helix-helix interactions are likely to predominate in LD recognition. Moreover, threading studies have suggested that the LD-interacting domain of p95PKL may share a common four-helical core with the FAT domain and the tail of vinculin, defining a structural family responsible for some, but not all, examples of LD-motif binding.

ZAP70 and tandem SH2 units

Dr. Ewa Pilka

Structures determined for the intact common parts of Scr-family members Src, Hck, and Abl have revealed a joint role for their non-catalytic domains in substrate selection and the regulation of intrinsic kinase activity. This offers a paradigm that we are hoping to explore by analysis of the protein kinase ZAP70, which features tandem SH2 domains as well as a kinase catalytic somain. To compliment this work, we are looking at structural studies of other examples of tandem SH2 domains.

Cell cycle

ATP competitive inhibitors of CDK2

David Pratt (With Dr. Jane Endicott, Biochemistry, Prof. Louise Johnson, Biochemistry, AstraZeneca PLC, and members of the ADDI, Newcastle University).

Cyclin-dependent kinase 2 (CDK2) is an important target for structure-based design of anti-tumour agents. We have used an analysis of inhibitor binding to monomeric CDK2 and binary CDK2/cyclin A to inform the design of potent CDK2 inhibitors in collaboration with AstraZeneca and the Anticancer Drug Discovery Initiative at Newcastle University. To date, structural information has lead to the design of an inhibitor with a Ki value of 3nM, approximately 5000-fold better than that of the lead compound.

Competitors of the MDM2/p53 interaction

Jan Gruber, Ben Hall, Jen-Francois Trempe (With Dr. Jane Endicott, Dr. Jim McDonnell and members of the ADDI, Newcastle University).

MDM2 is a ubiquitin ligase that is partly responsible for the short half life of the tumor-supressor gene product p53. We are using a range of biophysical and structural techniques to explore inhibition of the p53/MDM2 interaction as a potential approach to anti-cancer therapy. These include attempts to crystallize the protein with inhibitors identified by screening, and computational simulation to learn about the behaviour of the p53-binding domain of MDM2.

Figure 2: HSQC spectral changes associated with inhibitor binding to the p53 binding domain of MDM2

NAT enzymes

Dr. Simon Holton (With Prof. E. Sim, Pharmacology)

A growing problem in global health care is the incidence of antibiotic-resistant bacteria. Acetylation is one mechanism employed by bacteria to evade antibiotics. The family of enzymes that acetylate arylamines and hydrazines (the arylamine N-acetyltransferase or NAT family of enzymes) have a novel fold and a novel mechanism. NAT enzymes are highly active in certain Salmonella strains, where they have been exploited in the 'Ames' test of carcinogenicity. NAT enzymes have also been recently identified in Mycobacterium tuberculosis, where they may be responsible for some instances of tubercular resistance to such antibiotics as isoniazid. Recent work by Professor Sim has identified defects in cell-wall synthesis in nat knockout strains of M. tuberculosis. In order to understand their catalytic mechanism, and to design specific inhibitors of pathogenic isoforms, NAT enzymes constitute an interesting target for structural characterisation
In 1999 we published the structure of NAT from S. typhimurium at 2.7 Å resolution, both alone and in complex with a bromacetanilid suicide inhibitor which has confirmed the location of the active site. Subsequently, we have solved the structure of NAT from M. smegmatis, a close relative of the potential drug target M. tuberculosis. M. smegmatis NAT crystallises in three crystal forms, one with space group P41212, one P212121 and one P6222. These crystals diffract beyond 2.0 Å resolution, giving rise to a much more well defined structure. We hope to take advantage of this improved resolution to refine our understanding of the catalytic mechanism of NAT. We have begun this by studying the structure of NAT in complex with substrates such as 5-aminosalicylate, a drug that targets Crohn's disease, and that is metabolised by endogenous NAT in humans. This year, we have also recently solved the strucure of Pseudomonas aeruginosa NAT.

Methods

Ligand design/libraries

Giles Robertson (With Dr. J. McDonnell, Dr. J. Endicott and Prof. L. Johnson).

Cyclin dependent kinases direct the progression of the eukaryotic cell cycle. Phases of this cycle are characterised by the activity of specific pairings of kinase subunits (CDKs) with cognate cyclin activators. The cyclin molecule imposes a partially active conformation upon the CDK, and further modulates its activity by recruiting substrates and inhibitor proteins. The cyclin recruitment site interacts with a degenerate "Cy- motif", which has the concensus sequence R/KXL.We will explore the character of interacting surfaces of CDK and cyclin molecules, by use of redundant peptide libraries. Crystallographic structures will be determined for complexes of tightly binding peptides with CDK and/or cyclin molecules. This will contribute to an understanding of the role of peptide motifs in the formation of macromolecular assemblies. It will also produce a body of structural information which may inform the design of CDK inhibitors. In addition, this project will test the use of crystallography as a high-throughput technique in combination with chemical libraries.

Visualisation/analysis

Jan Gruber

Progress towards structure determination that is both high throughput and high value is dependent on the development of integrated and automatic tools for electron density map interpretation and the analysis of the resulting atomic models. Advances in algorithms of map interpretation are extending the resolution regime in which fully automatic tools can work reliably, but, as yet, human intervention is required to interpret poor regions of macromolecular electron density, particularly where crystallographic data is available to only modest resolution (e.g. I/s(I) < 2.0 for dmin < 2.5 Å). In such cases, a set of manual and semi-manual model building molecular graphics tools is needed. At the same time, converting the knowledge encapsulated in a molecular structure into understanding is dependent upon visualisation tools, which must be able to communicate that understanding to others by means of both static and dynamic representations. CCP4MG is a program designed to meet these needs in a way that is closely integrated with the on-going development of CCP4 as a program suite suitable for both low and high intervention computational structural biology. As well as providing a carefully designed user interface to advanced algorithms of model building and analysis, CCP4MG is intended to present a graphical toolkit to developers of novel algorithms in these fields.

Protein motions

Dr. Paul Barrett

We have developed a public web-based facility to generate dynamic structural data for proteins, starting from a static structure (a PDB file). Most of the computational techniques used to achieve this are well established but take time to learn. Our service means that workers no longer need to learn these techniques if a simple dynamic analysis is all that they want. The user provides a protein coordinate file, selects a few options and clicks 'start'. The dynamics of the protein are then simulated. The user subsequently downloads the simulation trajectory and associated analyses, such as animations and "principal component" summaries. The service will allow a much wider community to benefit from the insights that dynamic data provides.

Figure 3: Concerted motions of the p53 binding domain of MDM2