Membrane Topologies of Neuronal SNARE Folding Intermediates

Abstract

Assembly of the SNARE complex is essential for neurotransmitter release at synapses. Target plasma membrane SNAREs (t-SNAREs) syntaxin 1A and SNAP-25 form the t-SNARE complex that serves as an intermediate toward final SNARE assembly with vesicle-associated SNARE (v-SNARE). Membrane topologies of syntaxin 1A and the t-SNARE complex were investigated using site-directed spin labeling EPR. EPR analysis revealed that the basic region at the membrane-water interface is unstructured but inserted into the membrane. Such membrane insertion leaves no gap between the t-SNARE core and the membrane. Yet the lack of structure could provide the flexibility necessary for the t-SNARE core. Further, the insertion of the basic interfacial region into the membrane may have profound implications for the mechanism of SNARE-induced membrane fusion.

Insertion of the Membrane-proximal Region of the Neuronal SNARE Coiled Coil into the Membrane

“…The saturation EPR analysis suggested that those three positions are immersed in the acyl chain region. In parallel, previous EPR studies demonstrated the adhesion of the polybasic linker region (Arg 262 , Arg 263 , Lys 264 , and Lys 265 ) of Syntaxin 1A onto the membrane (18,19). It was found that this region is laterally inserted into the membrane, similarly pulling the SNARE core toward the membrane.…”

“…In fact, EPR spectra for these three positions closely resemble those observed for the nitroxide attached to the membrane-inserted linker region of Syntaxin 1A (18,19).…”

“…1 The abbreviations used are: SNARE, soluble N-ethylmaleimidesensitive factor attachment protein receptor; 6,7-Br 2 -PC, 1-palmitoyl-2-stearoyl-(6,7)-dibromo-sn-glycero-3-phosphocholine; 11,12-Br 2 -PC, 1-palmitoyl-2-stearoyl-(11,12)-dibromo-sn-glycero-3-phosphocholine; DOPS, 1,2-dioleoyl phosphatidylserine; EPR, electron paramagnetic resonance; NiEDDA, nickel (II)-ethylenediamine-N,NЈ-diacetic acid; OG, n-octylglucoside; PBST-Met, phosphate-buffered saline pH 7.4, 0.05% (v/v) Tween 20, 10 mM L-methionine; POPC, 1-palmitoyl-2-oleoyl phosphatidylcholine; SNAP-25, 25-kDa, soluble N-ethylmaleimidesensitive factor attachment protein; TMD, transmembrane domain(s); VAMP2, vesicle-associated membrane protein 2; MTSSL, (1-oxyl-2,2,5,5-tetramethylpyrrolinyl-3-methyl) methanethiosulfonate spin label. acid residues, is unstructured but laterally inserted into the membrane, tightly coupling the coiled coil to the membrane (18,19). Importantly, clusters of basic amino acid residues are found in the linker regions of all transmembrane SNAREs (20), raising the possibility that SNARE linker regions generally insert into the membrane.…”

A Partially Zipped SNARE Complex Stabilized by the Membrane

“…The structure in the N-terminal region might be due to the self-association of Sso1pHT. Previously, it was shown that Syntaxin, the neuronal counterpart of Sso1p, self-associates to form dimers (30,31). To verify whether the N-terminal structure of Sso1pHT was due to a similar oligomerization, we collected the low temperature EPR spectra (32) and found that some mutants exhibited apparent spin-spin interactions (data not shown), supporting the oligomeric state of Sso1pHT.…”

“…One possibility is that membranes might no longer be mutually repulsive at the fusion site where several t-and vSNAREs are presumably clustered. Both t-and v-SNAREs carry basic membrane-proximal regions that have a strong propensity for the membrane surface (24,31,37). The insertion of several membrane-proximal regions into the small patch of the membrane might activate the surfaces to become no longer mutually repulsive.…”