Molecular basis for the trafficking of transmembrane proteins through Ubiquitin, Syntenin-1 and Tollip complexes

In this research project the three dimensional structures and molecular interactions of a dynamic trafficking complex that internalizes transmembrane proteins will be characterized at a resolution that is useful for drug discovery. The tetraspanin trafficking (TETRAF) complex involves four human proteins. Tetraspanin proteins span the membrane four times, and link directly to a cytosplasmic adaptor protein called syntenin. We will determine how syntenin induces specific hetero-dimerization of receptors via their cytoplasmic tails in a phosphorylation-dependent manner. The recycling of this tetraspanin-syntenin complex is mediated by the ubiquitin protein, which exhibits a novel binding site near its C-terminus. The tollip protein interacts directly with phosphoinositide lipids, and we have additionally discovered its unique binding site for syntenin, revealing a previously unknown supermolecular complex. We propose that this membrane-associated protein assembly is responsible for controlling how tetraspanins are controlled within healthy cells and contributes to the migration and invasiveness of tumour cells. The targeting of the tetraspanin complexes presents promising therapeutic opportunities due to the key roles played in infection, tumour formation and metastasis. Our understanding of the underlying mechanisms however remains in its infancy, with no structures available for any TETRAF complexes despite its relevance to improving human health and well being. These multidomain targets remain challenging for analysis in terms of their solution structures and conformational dynamics under physiological conditions, warranting further fundamental biochemical and molecular research to define the key interactions and regulatory mechanisms.

We will apply a method known as nuclear magnetic resonance spectroscopy using our national facility's superconducting magnets, which can be used to detect a unique signal for each of the thousands of atomic nuclei in the molecular complex. 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. We have obtained resolved spectra of all four proteins that form the TETRAF complex, and plan to extend these to elucidate the molecular structures and functions, thus providing a mechanism for tetraspanin internalization. We have identified novel binding sites in syntenin and tollip that bind lipid molecules, and will use spin labels and computer methods to validate and define how membrane recruitment mediates traffic of the complex between plasma and endocytic compartments. Together with our collaborators we will provide the first comprehensive structural, functional and biochemical insights into how this assembly acts at a molecular level, allowing us to much more accurately manipulate its behaviour in vitro and in vivo. The endpoints of the project include structures of the complexes bound to lipid and protein ligands that regulate tetraspanin activity in cells, a description of the dynamics and structural determinants of these binding events, and, in the long term, a rational basis for designing inhibitors and mutations for in vitro and in vivo analysis of this system for drug discovery.

Our goal is to elucidate the trafficking pathway of transmembrane proteins in tetraspanin enriched microdomains (TERMs) mediated by the cytoplasmic proteins syntenin1, tollip and ubiquitin. We aim to address the following:

How do the structure and dynamics of full length syntenin-1 and its constituent domains and functional motifs influence binding to ubiquitin and regulate association with tetraspanins and receptors in TERMs in membranes? In particular we will investigate the specific interactions of syntenin-1 with ubiquitin, elucidate the ubiquitin-complexed structure of Tollip C2 domain and characterise the role of the tollip-syntenin-ubiquitin ternary complex. To elucidate the structures in physiological solution we will use NMR and small angle X-ray scattering as well as X-ray crystallography, allowing interaction sites and binding determinants to be defined at multiple levels of resolution.

How is the membrane-associated C2 domain of Tollip, an early endosomal protein involved in protein sorting, recruited to the syntenin-Ub complex to promote trafficking of tetraspanin associated receptors into late endosomes for degradation/recycling? The lipid complexes of syntenin and C2 domains will be characterized, and the bilayer inserted orientations calculated using spin labels, micelles and bilayers, allowing us to model the protein-membrane complex using PRE and RDC based NMR restraints.

Does the phosphorylation of syntenin1 by Ulk1 kinase affect its interaction with ubiquitin and Tollip and in turn regulate transmembrane protein trafficking? We have recently discovered that syntenin's PDZ domains prefer binding to phosphorylated receptor sequences, and will explore the ramifications of this unprecedented finding in the context of the TETRAF mechanism. This will inform the mutation of binding and regulatory residues for in vitro assays and determination of their specific effects on receptor trafficking and protein localization in cell-based systems.

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 35 manuscripts since 2006, including papers in JACS, EMBO J, PLOS and PNAS and scholarly reviews in Nature Reviews and Methods In Molecular Biology. Open access journals will continue to be the medium for communicating our research results. Overduin also operates websites at www.proteinexpress.org, www.lipidprism.org and www.nmr.bham.ac.uk to disseminate scientific knowledge and practice. The team will continue to presents at conferences, and will run focused meetings will be used to transfer knowledge and skills, with Overduin being the organizer of four BBSRC-funded JPA workshops on protein expression, annual EU-funded meetings on membrane-protein interactions, and Wellcome Trust-funded NMR workshops on software and data analysis 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 collaborations, with GSK and AstraZeneca supporting PhD studentships on academic research in Overduin's group. The tetraspanin superfamily includes targets for malaria (CD81), Hepatitis C virus (CD81) and HIV (CD9, CD63, CD151) infection, melanoma (CD63), tumour metastasis (CD151), B-cell leukemia and lymphoma growth (CD37), with companies and the Army requesting our constructs and expertise. Thus our research on the fundamental mechanisms of tetraspanin trafficking has broad applicability and value.

Wider Public: The PIs will continue to play an active role in promoting the public understanding of science, and have contributed to stories in the Birmingham Post, Business Desk, Guardian, Information Daily, Telegraph, BBC-WM and Research TV in the past several years. Overduin is a member of the steering group of the British Science Association, which held a Science Festival in Birmingham in Sept 2010, and every four years thereafter, and runs events for this and University Open Days. Lab and facility tours will continue to given by Overduin and members of his group to high school classes and the public, and several high school students have performed summer research projects in his lab recently through Nuffield bursories.

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 serves on its Executive Committee, helping 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 cancer, and demonstrating the benefits of new technologies and drug discovery through academic - industrial collaborations.

Business engagement: Overduin is a member of the University of Birmingham's Regional Advisory Group which is tasked with exploring strategic collaborations with regional partners. He established Science Capital as a nonprofit organization to connect internationally recognized scientists with the public and businesses to present and discuss innovations and priorities for growth and investment. For example, he presented his BBSRC-funded research alongside AstraZeneca and Sygnature Discovery, to an audience of 100 such experts on 19 Sept 2012 in order to provide awareness of opportunities being opened up for drug discovery.

The cost of these public engagement activities are estimated at 3 hours per month, as well as the associated local travel costs (~£20/month), all of which is given freely for this publicly funded research.