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

Abstract

In the neuron, soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins assemble into an ␣-helical coiled coil that bridges the synaptic vesicle to the plasma membrane and drives membrane fusion, a required process for neurotransmitter release at the nerve terminal. How does coiled coil formation drive membrane fusion? To investigate the structural and energetic coupling between the coiled coil and membrane, the recombinant SNARE complex in the phospholipid bilayer was studied using fluorescence quenching and site-directed spin labeling EPR. Fluorescence analysis revealed that two native Trp residues at the membrane-proximal region of the coiled coil are inserted into the membrane, tightly coupling the coiled coil to the membrane. The EPR results indicate that the coiled coil penetrates into the membrane with an oblique angle, providing a favorable geometry for the basic residues to interact with negatively charged lipids. The result supports the proposition that core complex formation directly leads to the apposition of two membranes, which could facilitate lipid mixing. Trp residues and basic residues are abundant at the membrane-proximal region of transmembrane SNARE proteins, suggesting the generality of the proposed mechanism for the SNARE complex-membrane coupling.

Conformation of the synaptobrevin transmembrane domain

“…The other three constructs (L93, L99, and L107) each show a satellite peak centered near 1,615 cm Ϫ1 , indicative of a 40-cm Ϫ1 shift from the ␣-helical region of the spectrum. Thus, the transmembrane ␣-helix continues into the polybasic, juxtamembrane region, but this helix does not extend into the cytoplasmic domain in agreement with previous results (22).…”

“…The synaptobrevin transmembrane domain is relatively short, such that the apolar residues (S115 and T116) cannot protrude from the interior leaflet of the membrane, assuming that the thickness of the bilayer is undisturbed by synaptobrevin or other factors. The observed tilt angle allows the tryptophan side chains to be buried in the bilayer as predicted by EPR studies (22) and allows basic residues to ''snorkel'' out of the bilayer to interact favorably with the phosphate region of the lipid bilayer (22,58). The need for snorkeling is particularly evident for K94, which is a full helical turn farther into the bilayer than W90.…”

“…The cytoplasmic domain construct was identical to that used for crystallization of the SNARE complex (residues 1-96) (13,33). However, some of these residues actually insert into the membrane (22). The transmembrane construct contained the predicted transmembrane domain as well as the first 23 aa of the juxtamembrane region (residues 74-116).…”

“…The conserved juxtamembrane region (residues 77-90) binds phospholipids and calmodulin (21). Tryptophan residues in the juxtamembrane region cause portions of the SNARE motif to insert into the bilayer (22). These interactions have been suggested to regulate SNARE complex formation and play a role in vesicle fusion (23)(24)(25).…”

Determinants of Synaptobrevin Regulation in Membranes

“…The lack of reactivity was attributed to the membrane proximal linker region of synaptobrevin (aa 85-92, Figure 1), which connects the SNARE motif with the Cterminal transmembrane domain. Spin-labeling experiments suggested this region to form an amphipatic helix that is tilted at an angle of 33°, with two highly conserved tryptophan residues (Trp89 and Trp90) dipping into the hydro- phobic core of the bilayer (Kweon et al, 2003a). When these Trp-residues were replaced with serine, SNARE binding was restored (Kweon et al, 2003b).…”