title: Syntaxin1a variants lacking an N-peptide or bearing the LE mutation bind to Munc18a in a closed conformation.
abstract: In neurons, soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins drive the fusion of synaptic vesicles to the plasma membrane through the formation of a four-helix SNARE complex. Members of the Sec1/Munc18 protein family regulate membrane fusion through interactions with the syntaxin family of SNARE proteins. The neuronal protein Munc18a interacts with a closed conformation of the SNARE protein syntaxin1a (Syx1a) and with an assembled SNARE complex containing Syx1a in an open conformation. The N-peptide of Syx1a (amino acids 1-24) has been implicated in the transition of Munc18a-bound Syx1a to Munc18a-bound SNARE complex, but the underlying mechanism is not understood. Here we report the X-ray crystal structures of Munc18a bound to Syx1a with and without its native N-peptide (Syx1aΔN), along with small-angle X-ray scattering (SAXS) data for Munc18a bound to Syx1a, Syx1aΔN, and Syx1a L165A/E166A (LE), a mutation thought to render Syx1a in a constitutively open conformation. We show that all three complexes adopt the same global structure, in which Munc18a binds a closed conformation of Syx1a. We also identify a possible structural connection between the Syx1a N-peptide and SNARE domain that might be important for the transition of closed-to-open Syx1a in SNARE complex assembly. Although the role of the N-peptide in Munc18a-mediated SNARE complex assembly remains unclear, our results demonstrate that the N-peptide and LE mutation have no effect on the global conformation of the Munc18a-Syx1a complex.
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: Karen N
authors.initials: KN
authors.lastname: Colbert
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Hattendorf
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Weiss
authors.forename: Pawel
authors.initials: P
authors.lastname: Burkhardt
authors.forename: Dirk
authors.initials: D
authors.lastname: Fasshauer
authors.forename: William I
authors.initials: WI
authors.lastname: Weis
mesh_terms: D019943:Amino Acid Substitution; D006801:Humans; D051938:Munc18 Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010455:Peptides; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D050600:SNARE Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D050827:Syntaxin 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D051938:Munc18 Proteins; D010455:Peptides; D050600:SNARE Proteins; C494212:STX1A protein, human; C497937:STXBP1 protein, human; D050827:Syntaxin 1
keywords: SM proteins; membrane trafficking; protein crystallography