Browsing by Author "Martin, JL"

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    Cortactin adopts a globular conformation and bundles actin into sheets
    (American Society for Biochemistry and Molecular Biology, 2008-06-06) Cowieson, NP; King, GJ; Cookson, DJ; Ross, I; Huber, T; Hume, DA; Kobe, B; Martin, JL
    Cortactin is a filamentous actin-binding protein that plays a pivotal role in translating environmental signals into coordinated rearrangement of the cytoskeleton. The dynamic reorganization of actin in the cytoskeleton drives processes including changes in cell morphology, cell migration, and phagocytosis. In general, structural proteins of the cytoskeleton bind in the N-terminal region of cortactin and regulatory proteins in the C-terminal region. Previous structural studies have reported an extended conformation for cortactin. It is therefore unclear how cortactin facilitates cross-talk between structural proteins and their regulators. In the study presented here, circular dichroism, chemical cross-linking, and small angle x-ray scattering are used to demonstrate that cortactin adopts a globular conformation, thereby bringing distant parts of the molecule into close proximity. In addition, the actin bundling activity of cortactin is characterized, showing that fully polymerized actin filaments are bundled into sheet-like structures. We present a low resolution structure that suggests how the various domains of cortactin interact to coordinate its array of binding partners at sites of actin branching. © 2008, American Society for Biochemistry and Molecular Biology
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    Low resolution structural studies of Munc18c complexed with a Syntaxin-4/T4- Lysozyme Fusion
    (Australian Institute of Nuclear Science and Engineering, 2016-11-29) Whitten, AE; Rehman, AU; Hu, SH; Tnimov, Z; Christie, MP; King, GJ; Jarrott, RJ; Norwood, S; Alexandrov, K; Collins, BM; Martin, JL
    Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) act at every intracellular trafficking pathway. Cognate v-SNAREs (e.g. VAMP) and t-SNAREs (Syntaxin (Sx) and SNAP) form a high affinity SNARE ternary complex (Sx-SNAP-VAMP) that brings the membranes together, triggering fusion. Syntaxins consist of a SNARE motif, and a three-helix bundle. In an open confirmation, the SNARE motif is free to form the SNARE ternary complex (stimulating fusion), but in the closed confirmation fusion is inhibited. Sec1p/Munc18 (SM) proteins bind to Sx, regulating SNARE mediated fusion [1], but their exact role is not well understood [2-4]. In the cell, Sx is bound to the membrane, and it is possible that this tethering may influence the manner in which it interacts with other proteins. As a means of investigating structural changes arising due to tethering, here, we investigate how the addition of a C-terminal T4-Lysozyme (soluble) fusion to Sx4 modulates its interaction with Munc18c. Preliminary low-resolution models of the Munc18c-Sx4T4 complex optimized against small-angle scattering data will be presented.
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    Low-resolution solution structures of Munc18:Syntaxin protein complexes indicate an open binding mode driven by the Syntaxin N-peptide
    (National Academy of Sciences, 2012-06-19) Christie, MP; Whitten, AE; King, GJ; Hu, SH; Jarrott, RJ; Chen, KE; Duff, AP; Callow, P; Collins, BM; James, DE; Martin, JL
    When nerve cells communicate, vesicles from one neuron fuse with the presynaptic membrane releasing chemicals that signal to the next. Similarly, when insulin binds its receptor on adipocytes or muscle, glucose transporter-4 vesicles fuse with the cell membrane, allowing glucose to be imported. These essential processes require the interaction of SNARE proteins on vesicle and cell membranes, as well as the enigmatic protein Munc18 that binds the SNARE protein Syntaxin. Here, we show that in solution the neuronal protein Syntaxin1a interacts with Munc18-1 whether or not the Syntaxin1a N-peptide is present. Conversely, the adipocyte protein Syntaxin4 does not bind its partner Munc18c unless the N-peptide is present. Solution-scattering data for the Munc18-1:Syntaxin1a complex in the absence of the N-peptide indicates that this complex adopts the inhibitory closed binding mode, exemplified by a crystal structure of the complex. However, when the N-peptide is present, the solution-scattering data indicate both Syntaxin1a and Syntaxin4 adopt extended conformations in complexes with their respective Munc18 partners. The low-resolution solution structure of the open Munc18:Syntaxin binding mode was modeled using data from cross-linking/mass spectrometry, small-angle X-ray scattering, and small-angle neutron scattering with contrast variation, indicating significant differences in Munc18:Syntaxin interactions compared with the closed binding mode. Overall, our results indicate that the neuronal Munc18-1:Syntaxin1a proteins can adopt two alternate and functionally distinct binding modes, closed and open, depending on the presence of the N-peptide, whereas Munc18c:Syntaxin4 adopts only the open binding mode. © 2012, National Academy of Sciences.
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    SNARE-ing the structures of Sec1/Munc18 proteins
    (Elsevier BV, 2014-12-01) Archbold, JK; Whitten, AE; Hu, SH; Collins, BM; Martin, JL
    Membrane fusion is essential for cellular transport in eukaryotes. Abnormalities contribute to a wide range of diseases including diabetes and neurological disorders. A key regulator of SNARE-mediated membrane fusion is the Sec1/Munc18 (SM) protein family. Universal structural features of SM proteins have been identified that affect the way these interact with their partner Syntaxin SNARE proteins. Whilst the molecular basis for SM-regulated SNARE complex formation has been extensively studied, it remains poorly understood. Recent crystal structures of SM proteins alone or in complex have provided new insight. Here we examine the available structural information on SM proteins for clues to how these enigmatic proteins might regulate SNARE complex assembly and membrane fusion. © 2014, Elsevier Ltd.