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abstracts
Retromer is a peripheral membrane protein complex that plays a critical role in a broad range of physiological, developmental and pathological processes including Wnt signalling, toxin transport and amyloid production in Alzheimer's disease. The classical mammalian retromer complex consists of a core heterotrimeric cargo recognition sub-complex (VPS26, VPS29 and VPS35) associated with a dimer of proteins from the SNX–BAR sorting nexin family that drives membrane deformation and tubulation. By recruiting the cargo-selective sub-complex to the forming tubules, the SNX–BAR coat complex mediates the retrograde transport of proteins from endosomes to the trans-Golgi network. Recent studies, however, have highlighted the molecular and functional diversity of retromer and the identification of new interacting proteins has revealed that the role of retromer extends to aspects of endosome-to-plasma membrane sorting and regulation of signalling events. Emerging evidence indicates that cargo specificity is mediated by specific sorting nexins. These include SNX3, involved in the trafficking of the Wntless/MIG-14 protein, and SNX27, a PX-FERM protein that mediates the retrieval of the β2-adrenergic receptor.Using the MX and SAXS/WAXS beamlines at the Australian Synchrotron, we have acquired crystallographic and small angle scattering data to determine how the core cargo recognition sub-complex assembles and to characterise the retromer-associated sorting nexins. We are using this structural information in combination with biochemical and biological studies in a synergistic approach to understand retromer-mediated endosomal protein sorting and how this fascinating protein complex contributes to a diverse set of cellular processes. The retromer complex is conserved across all eukaryotes. We are also currently exploring the structure of these proteins in the thermophilic fungus Chaetomium thermophilum and initial crystallisation experiments have produced some promising results.
abstracts
BAMLET and HAMLET represent a new class of compound having unrealised potential for treating a broad spectrum of cancers and some multi-drug resistant bacterial infections (Brinkmann et al. 2013; Marks et al. 2013). These compounds are composed of protein (14 to 84 kDa) and oleic acid (282 Da), the latter being the main active component. Hypothesised molten-globularity makes structural determination by NMR and X-ray crystallography very challenging. We carried out small angle X-ray scattering (SAXS) on BAMLET at pH 12 (Rath et al. 2014), the pH at which the complex can be prepared. SAXS showed that the protein component was an ensemble of extended, irregular, partially-unfolded conformations that varies with the amount of oleic acid incorporated into the complex. Increases in oleic acid concentration (from 1 to 20 molecules of oleate per protein molecule) correlate with increasing radius of gyration (from 21 to 29 Ang) without an increase in maximum particle dimension, indicating decreasing protein density. Three-dimensional models were generated that satisfy the probability distribution function that was derived by indirect Fourier transform of the SAXS data (Figure A). Models for the highest oleic acid content BAMLET (Figure B) indicate a partially unfolded conformation with the majority of the protein mass distributed around the periphery of the complex. Our results suggest that oleic acid inhibits the folding or collapse of the protein component of BAMLET to the globular form. SAXS was not able to identify the structure of the oleic acid component due to the very weak X-ray scattering contrast. However, the results support a model in which BAMLET retains oleic acid by non-specific association in the core of the partially unfolded protein. This represents a new type of lipid-binding protein structure. The structure of BAMLET will guide efforts to incorporate BAMLET into a delivery vehicle with the aim of realising the significant clinical potential of BAMLET.
