Three-dimensional structure of the neuronal-Sec1–syntaxin 1a complex

Syntaxin 1a and neuronal Sec1 (nSec1) form an evolutionarily conserved heterodimer that is essential for vesicle trafficking and membrane fusion. The crystal structure of the nSec1–syntaxin 1a complex, determined at 2.6 Å resolution, reveals that major conformational rearrangements occur in syntaxin relative to both the core SNARE complex and isolated syntaxin. We identify regions of the two proteins that seem to determine the binding specificity of particular Sec1 proteins for syntaxin isoforms, which is likely to be important for the fidelity of membrane trafficking. The structure also indicates mechanisms that might couple the action of upstream effector proteins to conformational changes in syntaxin 1a and nSec1 that lead to core complex formation and membrane fusion.

[1]  R. Hosono,et al.  Regulation of the UNC-18–Caenorhabditis elegansSyntaxin Complex by UNC-13 , 1999, The Journal of Neuroscience.

[2]  G. Webb,et al.  Genetic interactions between a pep7 mutation and the PEP12 and VPS45 genes: evidence for a novel SNARE component in transport between the Saccharomyces cerevisiae Golgi complex and endosome. , 1997, Genetics.

[3]  T. Südhof,et al.  Synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin , 1993, Nature.

[4]  Benedikt Westermann,et al.  SNAREpins: Minimal Machinery for Membrane Fusion , 1998, Cell.

[5]  G. Palade,et al.  Intracellular aspects of the process of protein synthesis. , 1975, Science.

[6]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[7]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[8]  Reinhard Jahn,et al.  Structure and Conformational Changes in NSF and Its Membrane Receptor Complexes Visualized by Quick-Freeze/Deep-Etch Electron Microscopy , 1997, Cell.

[9]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[10]  R. Scheller,et al.  SNARE Interactions Are Not Selective , 1999, The Journal of Biological Chemistry.

[11]  T. Südhof,et al.  Phosphorylation of Munc-18/n-Sec1/rbSec1 by Protein Kinase C , 1996, The Journal of Biological Chemistry.

[12]  G. Tall,et al.  The phosphatidylinositol 3-phosphate binding protein Vac1p interacts with a Rab GTPase and a Sec1p homologue to facilitate vesicle-mediated vacuolar protein sorting. , 1999, Molecular biology of the cell.

[13]  R. Scheller,et al.  Distinct domains of syntaxin are required for synaptic vesicle fusion complex formation and dissociation , 1995, Neuron.

[14]  D. James,et al.  Characterization of Munc-18c and Syntaxin-4 in 3T3-L1 Adipocytes , 1997, The Journal of Biological Chemistry.

[15]  K. Broadie,et al.  Syntaxin 1A Interacts with Multiple Exocytic Proteins to Regulate Neurotransmitter Release In Vivo , 1999, Neuron.

[16]  A. Brünger,et al.  A Structural Change Occurs upon Binding of Syntaxin to SNAP-25* , 1997, The Journal of Biological Chemistry.

[17]  E. Stuenkel,et al.  Regulation of Exocytosis by Cyclin-dependent Kinase 5 via Phosphorylation of Munc18* , 1999, The Journal of Biological Chemistry.

[18]  Josep Ubach,et al.  Three-Dimensional Structure of an Evolutionarily Conserved N-Terminal Domain of Syntaxin 1A , 1998, Cell.

[19]  Jonathan Pevsner,et al.  Specificity and regulation of a synaptic vesicle docking complex , 1994, Neuron.

[20]  R. Scheller,et al.  n-Sec1: a neural-specific syntaxin-binding protein. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[21]  J. Littleton,et al.  ROP, the Drosophila Sec1 homolog, interacts with syntaxin and regulates neurotransmitter release in a dosage‐dependent manner , 1998, The EMBO journal.

[22]  P. De Camilli,et al.  A rat brain Sec1 homologue related to Rop and UNC18 interacts with syntaxin. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[23]  Thomas C. Südhof,et al.  The synaptic vesicle cycle: a cascade of protein–protein interactions , 1995, Nature.

[24]  T. Südhof,et al.  Synaptic assembly of the brain in the absence of neurotransmitter secretion. , 2000, Science.

[25]  J. Littleton,et al.  Rop, a drosophila homolog of yeast Sec1 and vertebrate n-Sect/Munc-18 proteins, is a negative regulator of neurotransmitter release in vivo , 1994, Neuron.

[26]  Mark K. Bennett,et al.  A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion , 1993, Cell.

[27]  R. Scheller,et al.  Protein-protein interactions contributing to the specificity of intracellular vesicular trafficking. , 1994, Science.

[28]  W. Antonin,et al.  Mixed and Non-cognate SNARE Complexes , 1999, The Journal of Biological Chemistry.

[29]  S. Pfeffer.  Transport-vesicle targeting: tethers before SNAREs , 1999, Nature Cell Biology.

[30]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[31]  V. Lupashin,et al.  t-SNARE activation through transient interaction with a rab-like guanosine triphosphatase. , 1997, Science.

[32]  Sejal M. Patel,et al.  SNARE Complex Formation Is Triggered by Ca2+ and Drives Membrane Fusion , 1999, Cell.

[33]  R. Schekman.  Genetic and biochemical analysis of vesicular traffic in yeast , 1992, Current Biology.

[34]  J. Meldolesi,et al.  Blockade of membrane transport and disassembly of the Golgi complex by expression of syntaxin 1A in neurosecretion-incompetent cells: prevention by rbSEC1. , 1999, Journal of cell science.

[35]  P. Novick,et al.  Sec1p Binds to Snare Complexes and Concentrates at Sites of Secretion , 1999, The Journal of cell biology.

[36]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[37]  R. Scheller,et al.  Syntaxin 6 functions in trans-Golgi network vesicle trafficking. , 1997, Molecular biology of the cell.

[38]  G. Rubin,et al.  Mutations in the drosophila Rop gene suggest a function in general secretion and synaptic transmission , 1994, Neuron.

[39]  Frederick M. Hughson,et al.  Regulation of SNARE complex assembly by an N-terminal domain of the t-SNARE Sso1p , 1998, Nature Structural Biology.

[40]  T. Südhof,et al.  A conformational switch in syntaxin during exocytosis: role of munc18 , 1999, The EMBO journal.

[41]  K. Fiebig,et al.  Folding intermediates of SNARE complex assembly , 1999, Nature Structural Biology.

[42]  R. Scheller,et al.  Nsec1 Binds a Closed Conformation of Syntaxin1a , 2000, The Journal of cell biology.

[43]  A. Mayer,et al.  Sec18p (NSF)-Driven Release of Sec17p (α-SNAP) Can Precede Docking and Fusion of Yeast Vacuoles , 1996, Cell.

[44]  H. Erickson,et al.  Crystallization of a fragment of human fibronectin: Introduction of methionine by site‐directed mutagenesis to allow phasing via selenomethionine , 1994, Proteins.

[45]  D. Gallwitz,et al.  Identification and structure of four yeast genes (SLY) that are able to suppress the functional loss of YPT1, a member of the RAS superfamily , 1991, Molecular and cellular biology.

[46]  Reinhard Jahn,et al.  Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution , 1998, Nature.

[47]  W. Xiao,et al.  The synaptic SNARE complex is a parallel four-stranded helical bundle , 1998, Nature Structural Biology.