Structural Basis for Assembly and Membrane Modulating Properties of the Retromer Protein Coat Complex

Daniel Shaw (2011). Structural Basis for Assembly and Membrane Modulating Properties of the Retromer Protein Coat Complex PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

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Author Daniel Shaw
Thesis Title Structural Basis for Assembly and Membrane Modulating Properties of the Retromer Protein Coat Complex
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2011-06
Thesis type PhD Thesis
Supervisor Brett Collins
Total pages 224
Total colour pages 43
Total black and white pages 181
Language eng
Subjects 03 Chemical Sciences
Abstract/Summary Retromer is a peripheral membrane protein coat complex, named to reflect its role in conducting the ‘retrograde’ trafficking of transmembrane proteins from the endosome to the trans-Golgi network (TGN). The function of this coat complex is highly conserved in all eukaryotes from simple amoeba to plants and mammals with critical roles in diverse physiological, developmental and pathological processes. Retromer is a heteropentameric complex composed of two discrete subcomplexes: a core, cargo-binding, trimeric subcomplex and a membrane targeting and remodelling subcomplex. The core subcomplex is composed of the subunits vacuolar protein sorting (VPS)35, VPS29 and VPS26 while the membrane binding subcomplex consists of a homo- or heterodimer from the sorting nexin (SNX) family containing some combination of SNX1, SNX2, SNX5 and SNX6. However, unlike other coat complexes such as clathrin and the ESCRTs the molecular mechanisms by which retromer controls endosome-to-TGN sorting remain poorly understood. Crystal structures of the core subunits VPS29 and VPS26 are currently available, as well as the crystal structure for the C-terminal domain of VPS35 (476-780) bound to VPS29. However, a structure for the full intact trimeric core remains elusive. In addition, while crystal structures exist for the PX-BAR homodimer of SNX9 that functions in endocytosis, there are no structures for the intact PX-BAR unit of the retromer-associated SNX molecules. This thesis furthers the molecular details of both the cargo-loading, trimeric, core and membrane remodelling sorting nexin subcomplexes of retromer. In chapter 2 the mechanism of assembly of the core retromer subunit has been investigated using isothermal titration calorimetry (ITC). Thermodynamic characterisation of retromer assembly has revealed that VPS35 forms the central scaffold to which VPS26 and VPS29 bind in an independent, non-cooperative manner. In agreement with the thermodynamics of retromer assembly, solution structure studies of the retromer core using small angle X-ray scattering (SAXS) have revealed a model whereby VPS35 forms an extended scaffold with VPS26 and VPS29 bound at distal ends, with the potential for forming symmetric higher order-assemblies. The results presented in chapter 3 provide a direct assessment of the ability of SNX1, SNX2, SNX5 and SNX6 to form homo- and heterodimer species using co-purification and biophysical techniques. These results demonstrate that SNX1 and SNX2 are able to form homodimers while SNX5 exists as a monomer in solution. SNX1 is able to form stable heterodimers with SNX5 and SNX6, but not with SNX2. Investigation of the solution structures of SNX1 and SNX2 homodimers by SAXS reveal a relatively rigid membrane interaction superdomain that displays a striking resemblance to the SNX9 crystal structure where a yoke domain maintains the inter-domain orientation. Using ITC the results presented in chapter 4 provide a quantitative assessment of the lipid binding specificities of the retromer-related SNXs. These results reveal that full length SNX1, SNX2 and SNX5 show a general preference for lipids phosphorylated at the 3 and 5 position of the inositol ring, a property that is in line with cell biology experiments revealing a localisation of these molecules to membranes of the early and late endosomes. However, the results presented here also indicate that these SNX proteins possess an ability to bind a relatively broad spectrum of phosphoinositides (PIs). Interestingly, these results also suggest that both the PX and BAR domains of these SNX molecules impart PI-binding properties. Electron microscopy studies of the membrane structures formed by addition of SNX1 and SNX2 proteins to Folch lipids reveal tubular structures with an average diameter of 10 – 15 nm, which is close in dimensions to the 20 nm tubular structures formed by SNX9. Such results are consistent with the SAXS studies presented in chapter 3 that showed SNX1 and SNX2 display similar molecular geometries to the SNX9 crystal structure. The NMR results presented in chapter 5 indicate that mammalian SNX1 directly associates with mammalian VPS29, albeit with a low affinity (Kd > 150 M) and binding could not be reproduced in ITC experiments. Despite the weak and/or transient nature of this interaction we show that binding is specific, mediated by a hydrophobic surface on VPS29 that is on the opposite face to the metal/VPS35-binding interface. This mimics the mode of interaction between the yeast retromer components Vps29p and Vps5p that our group has previously characterised. The last chapter of the thesis reports all attempts to determine crystal structures of the retromer-related SNXs. While these attempts were unsuccessful they provide a strong foundation for further experiments aimed at elucidating high resolution structures of these proteins.
Keyword Retromer
Sorting nexins
PX domains
BAR domains
X-ray crystallography
Additional Notes Colour Pages- 15, 19, 30, 31, 32, 39, 40, 43, 44, 46, 48, 49, 51, 58, 61, 63, 64, 65, 80, 81, 106, 107, 108, 109, 110, 111, 113, 146, 157, 158, 159, 160, 161, 162, 174, 179, 211, 212, 213, 214, 216, 219, 220.

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Created: Wed, 07 Dec 2011, 17:50:49 EST by Mr Daniel Shaw on behalf of Library - Information Access Service