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

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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.