Elucidation of the mechanism of SHP-2 phosphatase localisation and activity

In this research project the three dimensional solution structures, molecular interactions and interdomain flexibility of the human SHP-2 protein will be characterized at levels of resolution needed for understanding cell signaling and informing drug discovery. We focus on the detailed mechanisms of a signalling enzyme which acts as a protein tyrosine phosphatase and in which mutations have been found to cause Noonan's syndrome. This is one of the most important phosphatase targets, with mutations afflicting 1 in 1-2 thousand live births worldwide, and has also been directly implicated in leukaemias and solid tumours. The molecular mechanism revolves around the two SH2 domain and the attached phosphatase enzyme, which removes phosphates from substrate proteins on tyrosine residues and regulated a system of intra- and inter-molecular interactions. The phosphatase domain is a positive transducer of signaling pathways which control cell growth and differentiation. Mutations known to be involved in disease generally hyperactivate SHP2 and lead to cell transformation, but the exact mechanism are unclear as it involves changes in local and global structure and flexibility, hence there is a real need for further research to inform accurate diagnosis and the design of specific and tailored agents. Phosphatase like SHP-2 remain challenging targets for drug discovery and molecular analysis, particularly in terms of resolving their solution structures and conformational dynamics under physiological conditions, warranting further fundamental research and technological developments to render them more amenable to experimental investigation and uncover their mechanisms at a predictive level. Our analysis relies on a method known as nuclear magnetic resonance spectroscopy, which can be used to detect a unique signal for the thousands of individual atomic nuclei in the SHP-2 and ligand molecules. The method provides an unprecedented level of information about the shape, conformation, motions and chemical interactivity of a protein in three dimensional space and over a range of timescales from picoseconds to seconds, and will yield a valuable insights for understanding the proteins behavious in cells and amenability for new drug discovery approaches. We have obtained promising spectra of SHP2's catalytic and regulatory tandem SH2 domains, and have just begun to assign its NMR signals, to identify novel putative ligands and map unprecedented binding sites. We are now best positioned to help provide a comprehensive understanding of its solution mechanisms, better than that which is available for any other human PTP target. We have predicted a unique site that could direct SHP-2 to plasma membrane sites, and will use spin labels, bilayers and computational modeling to define and validate the nature of the proposed membrane binding mechanism. We will study the interactions with receptor ligands that locallize and alter SHP-2's signaling activity, and are on the road to discover completely new classes of inhibitors. Together with our collaborators we will develop a much more comprehensive molecular mechanism for SHP-2 which includes structural, functional dynamical and chemical dimensions, explaining how this phosphatase behaves in physiological contexts, allowing us to more accurately manipulate its behaviour in cells and in vivo. This project will deliver solution structures of the protein bound to the lipid micelles and ligands responsible for localising and regulating activity of wild-type and mutated states in cells, a deeper understanding of the role of flexibility in these binding events, and a rational basis for designing novel inhibitors and transgenic models for in vitro and in vivo analysis which together could help to unlock the therapeutic potential of SHP-2 and has broader applications across phosphatase superfamily.

Our specific aims are to: 1. Assign the 1H, 15N and 13C resonances of SHP2's phosphatase and tandem SH2 domains, and using the results to pinpoint the lipid and ligand interaction and transduction sites including for specific recognition of soluble signals and ITIM and competing motifs. The solution structures will be elucidated using high field NMR and synchrotron SAXS data, allowing the flexibility between domains and within the full length protein to be defined. 2. Characterize the binding properties and conformational changes induced by signaling molecules and ITAM, ITIM and hemITAM motifs, allowing the identification and exploitation of new sites and ligands that modulate SHP2's activities. The HADDOCK program will be used to rapidly calculate complexed structures, defining the binding modes and specific contacts responsible for selective recognition and ligand competition within physiological mixtures. 3. Prove our proposed membrane interaction sites in the cSH2 and C-terminal polybasic modules, defining lipid binding specificities and micelle insertion depth and angle, validating with novel membrane systems including bicelles and bilayer discs, and modelling the protein-micelle complex using PRE and RDC based NMR restraints in order to design mutations that specifically abolish lipid binding and membrane insertion. 4. Elucidate how the regulatory and catalytic domains of the SHP-2 interact in the presence of mixed micelles and bilayers inducing the plasma membrane receptor-colocalized state, and modelling the structure of their complexed full length form on the membrane, detailing how the enzyme is regulated by membrane-dependent repositioning of the nSH2 and cSH2 domains.

Broader Scientific Community: Scientific groups will be engaged by primary journal articles, reviews, web sites, exchange visits and access to research services. Overduin's group has produced over 30 manuscripts since 2004, including papers in high impact journals like JACS, EMBO J, EMBO Reports and PNAS USA as well as accessible scholarly reviews including Nature Reviews and Methods In Molecular Biology. Open access journals will continue to be the primary medium for communicating our research results. Overduin also operates websites at proteinexpress.org, lipidprism.org and nmr.bham.ac.uk to disseminate scientific knowledge and practice on protein production, lipid signaling and NMR, respectively. Focussed meetings will be used to transfer knowledge and skills, with Overduin being the organizer of four BBSRC-funded JPA workshops on protein expression (2007-2010) as well as annual meetings for the LipidPrism project on membrane-protein interactions, and through HWB-NMR on NMR applications and methods including software, sample preparation and challenging systems. Commercial Sector: Pharmaceutical and biotech companies will learn about the tools and methods being developed here through their visits to Birmingham as HWB-NMR users, as well as through active collaborations supporting a PhD student and joint research activities. The PTP superfamily includes drug discovery targets for cancer, diabetes, obesity, and SHP2 mutations are critically involved in Noonan and Leopard Syndromes. Our research here on the mechanisms of PTP activities and targeting will thus have direct applicability to drug discovery and the development of therapeutic agents and diagnostics for diverse genetic diseases. Wider Public: Overduin will continue to play an active role in promoting the public understanding of science, and contributed to articles in the Birmingham Post (June 2010, Mar 2009, Nov 2006), Public Service and Parliament magazines (2009), Guardian (Nov 2007), Telegraph (Nov 2005) and Research TV (Nov 2004). Overduin is a member of the steering group of the British Science Association, and is organising two British Science Festival events in Birmingham in Sept 2010. Lab and facility tours will continue to given by Overduin and members of his group to politicians and the public, e.g. high school classes, several science students have performed summer research projects in his lab in recent years. Overduin also volunteers as Chair of the Science and Medicine Forum of the Lunar Society, a scientific body which was originally founded in the West Midlands in 1775. He also serves as its Honorary Secretary, helping to to organize monthly lectures and public events with attendances of up to 600 people. Recent speakers he has hosted include the Nobel Laureate Paul Nurse, President, Rockefeller University and Sir Liam Donaldson, Chief Medical Officer. This provides an avenue to present results on pharmaceutical research and genetics, proteomics and systems biology of disease, helping to overcome public anxiety about human mutations and breast cancer, and demonstrating the benefits of new technologies and drug discovery through academic - industrial collaborations. As requested by the guidance, the costs of these public engagement activities are estimated at 2 hours per month of Overduin's time and effort, as well as the associated local travel costs (~£20/month), all of which is given freely for this publicly funded research. Business engagement: Overduin is a member of the University of Birmingham's Regional Advisory Group which explores strategic collaborations with major partners. With the support of Birmingham City Council he founded Science Capital in 2010 as a grassroots membership organization that connects internationally recognized scientists with business and financial experts through discussions of innovative technologies, policies and legal frameworks, thus stimulating new partnerships.