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

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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.