A conformational switch in syntaxin during exocytosis: role of munc18.
Syntaxin 1, an essential protein in synaptic membrane fusion, contains a helical autonomously folded N-terminal domain, a C-terminal SNARE motif and a transmembrane region. The SNARE motif binds to synaptobrevin and SNAP-25 to assemble the core complex, whereas almost the entire cytoplasmic sequence participates in a complex with munc18-1, a neuronal Sec1 homolog. We now demonstrate by NMR spectroscopy that, in isolation, syntaxin adopts a 'closed' conformation. This default conformation of syntaxin is incompatible with core complex assembly which requires an 'open' syntaxin conformation. Using site-directed mutagenesis, we find that disruption of the closed conformation abolishes the ability of syntaxin to bind to munc18-1 and to inhibit secretion in PC12 cells. These results indicate that syntaxin binds to munc18-1 in a closed conformation and suggest that this conformation represents an essential intermediate in exocytosis. Our data suggest a model whereby, during exocytosis, syntaxin undergoes a large conformational switch that mediates the transition between the syntaxin-munc18-1 complex and the core complex.
Dulubova
I
I
Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235, USA.
Sugita
S
S
Hill
S
S
Hosaka
M
M
Fernandez
I
I
Südhof
T C
TC
Rizo
J
J
D000818:Animals; D000954:Antigens, Surface / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D019556:COS Cells; D005089:Exocytosis; D006801:Humans; D051938:Munc18 Proteins; D016296:Mutagenesis; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D016716:PC12 Cells; D011487:Protein Conformation; D011522:Protons; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins
D000954:Antigens, Surface; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D009587:Nitrogen Isotopes; D011522:Protons; D011993:Recombinant Fusion Proteins; C494212:STX1A protein, human; C497937:STXBP1 protein, human; C494213:Stx1a protein, rat; C497939:Stxbp1 protein, rat; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins