Biology subjects

Eitel, A. R.

Publications and source records attributed to Eitel, A. R..

3 recordsLinked to original sources

bioRxiv2026

Membrane-gated SNARE zippering focuses energy for fusion

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) drive stagewise membrane fusion by zippering into membrane-bridging four-helix bundles. Yet the conformations underlying successive fusion stages and the coupling of folding energy to bilayer remodeling remain unclear. Using optical tweezers, we measured the intermediates, energetics, kinetics, and force dependence of individual synaptic SNARE complexes assembled in cis on single membranes and in trans between apposed membranes. Membrane-anchored cis-SNAREs assembled through N-terminal and cooperative C-terminal/linker-domain transitions, whereas their transmembrane domains showed little intrinsic dimerization. Syntaxin retained membrane-dependent helical continuity through its linker domain before zippering was complete. PIP2 strengthened but slowed late zippering. In trans, membrane repulsion arrested single trans-SNARE complexes in a half-zippered state. G{beta}{gamma} further clamped this intermediate and inhibited late zippering; G-GDP, but not G-GTP{gamma}S, relieved the clamp, revealing a nucleotide-dependent mechanism for GPCR-mediated inhibition of neurotransmitter release. Modeling suggests that cooperative late zippering, syntaxin linker helicity, and concerted action of multiple SNAREs focus folding energy released over a long distance onto short-range membrane apposition. Thus, mechanically gated SNARE zippering is regulated by membrane forces, lipids, and regulatory proteins.

cell biology↗

bioRxiv2026

Architecture of the Gβγ-prefusion SNARE complex reveals the molecular mechanism of inhibition of vesicle fusion

Presynaptic inhibitory GPCR (Gi/o GPCR) signaling is an essential regulatory mechanism in vertebrate physiology. Near the presynaptic active zone, Gi/o GPCR activation releases G-protein {beta}{gamma} heterodimers (G{beta}{gamma}) which act to inhibit synaptic vesicle fusion through either modulation of Ca2+ entry via voltage-gated Ca2+ channels, or by direct interactions with the core exocytotic machinery comprised of the ternary SNARE complex downstream of Ca2+ influx. The precise molecular mechanism underlying G{beta}{gamma}-SNARE mediated inhibition has remained unclear due to lack of structural data for the G{beta}{gamma}-SNARE complex. We address this long-standing question here by stabilizing the interaction between G{beta}1{gamma}2 and a pre-fusion ternary SNARE mimetic and determining the structure using single-particle cryo-EM. We used our cryo-EM envelope to build an atomic level prediction of the interaction interface. We validated key interaction sites predicted by our model at the C-terminus of SNAP-25 using site directed mutagenesis and biochemical affinity measurements. Additionally, we found that G{beta}1{gamma}2 and a fragment of the regulatory protein complexin can engage the pre-fusion SNARE complex simultaneously. On the basis of these results, we propose a model in which G{beta}1{gamma}2 acts on the partially zipped SNARE complex at a late stage in the vesicle docking and priming cycle. In the model, the amino-terminal coiled-coil of G{beta}1{gamma}2 forms an interface with the C-terminus of the target membrane SNARE (t-SNARE) complex to prevent complete incorporation of the vesicle SNARE (v-SNARE) into the core SNARE helical bundle, thus blocking vesicle approach to the plasma membrane. The {beta}-propeller domain of G{beta}1 may also sterically hinder vesicle approach. Together these results provide crucial structural insights into the mechanism of binding of G{beta}{gamma} to the SNARE complex, and lends essential insights into the critical role of GPCR signaling to the SNARE complex in modulating synaptic vesicle fusion.

biophysics↗

bioRxiv2021

Specificity of Loxosceles α clade phospholipase D enzymes for choline-containing lipids: role of a conserved aromatic cage

Spider venom GDPD-like phospholipases D (SicTox) have been identified to be one of the major toxins in recluse spider venom. They are divided into two major clades: the clade and the {beta} clade. Most clade toxins present high activity against lipids with choline head groups such as sphingomyelin, while activities in {beta} clade toxins vary and include preference for substrates containing ethanolamine headgroups (Sicarius terrosus, St_{beta}IB1). A structural comparison of available PLDs structures reveals a conserved aromatic cage in the clade. To test the potential influence of the aromatic cage on membrane-lipid specificity we performed molecular-dynamics (MD) simulations of the binding of several PLDs onto lipid bilayers containing choline headgroups; two SicTox from the clade, Loxosceles intermedia IA1 (Li_IA) and Loxosceles laeta III1 (Ll_III1), and one from the {beta} clade, St_{beta}IB1. The simulation results reveal that the aromatic cage captures a choline-headgroup and suggest that the cage plays a major role in lipid specificity. We also simulated an engineered St_{beta}IB1, where we introduced the aromatic cage, and this led to binding with choline-containing lipids. Moreover, a multiple sequence alignment revealed the conservation of the aromatic cage among the clade PLDs. Here, we confirmed the membrane binding site of and {beta} clade PLDs on choline and ethanolamine-containing bilayers, respectively. Furthermore, our results suggest a major role in choline lipid recognition of the aromatic cage of the clade PLDs. The MD simulation results are supported by in vitro liposome binding assay experiments.

bioinformatics↗