title: Synaptotagmin 1 and SNAREs form a complex that is structurally heterogeneous.
abstract: Synaptotagmin 1 (syt1) functions as a Ca(2+)-sensor for neuronal exocytosis. Here, site-directed spin labeling was used to examine the complex formed between a soluble fragment of syt1, which contains its two C2 domains, and the neuronal core soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Changes in electron paramagnetic resonance lineshape and accessibility for spin-labeled syt1 mutants indicate that in solution, the assembled core SNARE complex contacts syt1 in several regions. For the C2B domain, contact occurs in the polybasic face and sites opposite the Ca(2+)-binding loops. For the C2A domain, contact is seen with the SNARE complex in a region near loop 2. Double electron-electron resonance was used to estimate distances between the two C2 domains of syt1. These distances have broad distributions in solution, which do not significantly change when syt1 is fully associated with the core SNARE complex. The broad distance distributions indicate that syt1 is structurally heterogeneous when bound to the SNAREs and does not assume a well-defined structure. Simulated annealing using electron paramagnetic resonance-derived distance restraints produces a family of syt1 structures where the Ca(2+)-binding regions of each domain face in roughly opposite directions. The results suggest that when associated with the SNAREs, syt1 is configured to bind opposing bilayers, but that the syt1/SNARE complex samples multiple conformational states.
authors.affiliation: Department of Chemistry, Biophysics Program and Center for Membrane Biology at the University of Virginia, Charlottesville, VA 22904-4319, USA.
authors.forename: Alex L
authors.initials: AL
authors.lastname: Lai
authors.forename: Hao
authors.initials: H
authors.lastname: Huang
authors.forename: Dawn Z
authors.initials: DZ
authors.lastname: Herrick
authors.forename: Natalie
authors.initials: N
authors.lastname: Epp
authors.forename: David S
authors.initials: DS
authors.lastname: Cafiso
mesh_terms: D000818:Animals; D008958:Models, Molecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D050600:SNARE Proteins / Q000737:chemistry* / Q000378:metabolism; D050863:Synaptotagmin I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D050600:SNARE Proteins; D050863:Synaptotagmin I