Prediction and Validation Tools for Novel Membrane Interaction Surfaces from Protein Structures

It is generally thought that about one quarter of genomes encodes transmembrane proteins, many of which are important receptors and drug targets. Most of the remainder are typically assumed to be soluble proteins including signalling and metabolic enzymes. However an unknown number of these actually bind reversibly to membrane surfaces, and these interactions determine where these proteins are located inside cells, and regulate their enzymatic and signaling activities. In fact, it can be argued that a protein's location is just as important as its intrinsic activity, restricting its access to locally concentrated substrates, cofactors and ligands within the viscous and compartmentalized cell. Without an effective way to identify and test these membrane interaction surfaces, our knowledge of protein function will continue to be limited and our research progress in molecular biology and biochemistry will be restricted. Hence we are developing new computational and biophysical methods to accurately detect and validate protein-membrane interactions which localize proteins inside cells, providing new insights and tools for understanding cellular processes and disease mechanisms. A variety of protein modules including BAM, FYVE, PH and PX domains are known to bind membrane surfaces in response to changes cell stimulation, growth and differentiation. We propose that by analyzing the structural properties of such proteins, the principles of membrane binding can be elucidated and generalized, and entirely new classes of PMPs can be found. The interactions are diverse. Some proteins bind membranes specifically yet dynamically by reversible recognition of individual phospholipid headgroups, yet others bind tightly, being anchored to the bilayer where they help assemble molecular complexes and catalyze reactions. Yet our studies have revealed common themes including exposed hydrophobic loops, basic patches and polarized surfaces. These properties are integrated here by an algorithm that automatically identifies membrane binding sites from structures in seconds. Once suitably trained, this method will allow users to accurately predict the new types of PMPs, and experimental methods and lipid/micelle libraries will be available to allow researchers to efficiently validate such discoveries. The lack of fast and accurate tools to detect protein surfaces that interact with membranes has impeded progress in the fields of molecular and cellular biology, and has limited interactions between the fields of proteomics and lipidomics. The dearth of understanding about membrane protein interactions is compounded by technical difficulties of studying 'sticky' membrane interacting domains and delicate bilayer structures. Thus there is a real need for convenient and insightful computational and new experimental tools to analyze protein membrane recognition. Our solution aims to provide sufficient information to allow users to design and test how proteins are targeted to specific membrane domains, to predict their spatial orientations on membrane surfaces, and to reveal whether conformational changes could accompany binding events. Broad applicability is ensured by the fact that protein membrane interactions determine the organization and regulated activities of so many cellular organelles and molecular complexes. Some lipid binding domains influence cell proliferation, differentiation, survival, migration, adhesion and invasion. Others are involved in neurogenesis, angiogenesis, wound healing, immunity and developmental diseases. Our research will enable a deeper understanding of their interactions in sufficient detail to aid in the design of ligands and inhibitors, and may aid in the design of therapeutic agents where lipids normally bind. The tools will be developed and applied using selected human proteins to achieve high impact and disease relevancy, and will be standardized where possible to maximize applicability to any protei

We aim to train and validate a new tools and resources for discovery of novel membrane interacting surfaces. We have created a prototype that recapitulates known membrane interacting sites on 20 protein structures. The algorithm will be tested for its ability to predict novel membrane binding sites by screening the protein data bank for new peripheral membrane proteins (PMPs). This will allow expansion of the training set to include new folds and sites. The methods could be used to predict novel biological functions, and may even allow estimation of the fraction of the proteome that binds reversibly to membranes. Expansion of the training set by addition of newly validated PMPs will allow improvement of the predictive power of the algorithm and will allow recognition of diverse membrane binding modes and lipid binding features. The functional properties of the novel PMPs will be validated by defining lipid binding profiles, micelle interacting surfaces and mapping residues which interact specifically with phospholipids by thermal shift and NMR assays. Using the precedents of phosphoinositide recognition by modules including FYVE, PH and PX domains, the membrane and lipid interacting hydrophobic groups, basic and polar residues, and conformational changes associated with membrane binding will be identified, allowing these features to be incorporated into a refined energy function for discriminating any interaction sites for intracellular and extracellular membranes. Biochemical and cellular validation will be based on surface plasmon resonance (SPR) and confocal microscopy analysis of full length proteins associating with liposomes and cell membranes. The functional implications of membrane targeting will be based on further collaborative studies of the signalling, phosphorylation and protein complexation of transfected PMP proteins following mutation of key binding site residues.

Broader Scientific Community: Scientific groups will be engaged by primary journal articles, reviews, web sites, exchange visits and access to research products and services. Overduin's group has produced over 30 manuscripts since 2004, including papers in high impact journals like JACS, EMBO J and PNAS and scholarly reviews including Nature Reviews and Methods In Molecular Biology articles in 2009. Open access journals will continue to be the primary medium for communicating our research results. Overduin also operates websites at www.proteinexpress.org, www.lipidprism.org and www.nmr.bham.ac.uk to upload results and disseminate scientific knowledge and practice. Conferences and meetings will be used to transfer knowledge and skills, with Overduin being the organizer of four BBSRC-funded JPA workshops on protein expression and analysis (2007-2010) as well as several meetings of EU projects on lipid signaling proteins, and with HWB-NMR staff offering training for new NMR applications and associated computational methods including protein ligand screening. 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 collaborations, with GlaxoSmithKline supporting a PhD studentship focussed on academic research activities on signaling enzymes in Overduin's group. Peripheral membrane proteins include many drug targets including phosphatases and cyclo-oxygenases, and their membrane interaction mechanisms remain largely unknown and unexploited despite the need for novel sites for design and screening of new lead molecules and scaffolds. Wider Public: Overduin will continue to play an active role in promoting the public understanding of science, and has contributed to articles in the Guardian (Nov 2007), Birmingham Post (Mar 2009, Nov 2006), Telegraph (Nov 2005) and Research TV (Nov 2004). His research on membrane protein solubilization in lipid nanoparticles was covered by 20 magazines in 2009 following a press release by the BBSRC. Overduin is a member of the steering group of the British Science Association, which will be holding a Science Festival in Birmingham in Sept 2010, and will host a tour and demo in HWB-NMR. Lab and facility tours will continue to given by Overduin and members of his group to school groups and the public throughout the year, and local high school students generally performed summer research projects in his lab. 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 volunteers as its Honorary Secretary, helping to to organize monthly lectures and public events with attendances of hundreds of 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 engaging talks demonstrating the benefits of new technologies and research discoveries. 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. Business engagement: Overduin was appointed to the University of Birmingham's Regional Advisory Group in 2008, this body being tasked with exploring strategic collaborations with regional partners. He is helping to establish Science Capital as a new organization to connect entrepeneurial scientists with the public and businesses to present and discuss innovations and potential commercial applications.