Biology subjects

Jaczynska, K.

Publications and source records attributed to Jaczynska, K..

3 recordsLinked to original sources

bioRxiv2024

A lever hypothesis for Synaptotagmin-1 action in neurotransmitter release

Neurotransmiter release is triggered in microseconds by Ca2+-binding to the Synaptotagmin-1 C2 domains and by SNARE complexes that form four-helix bundles between synaptic vesicles and plasma membranes, but the coupling mechanism between Ca2+-sensing and membrane fusion is unknown. Release requires extension of SNARE helices into juxtamembrane linkers that precede transmembrane regions (linker zippering) and binding of the Synaptotagmin-1 C2B domain to SNARE complexes through a primary interface comprising two regions (I and II). The Synaptotagmin-1 Ca2+-binding loops were believed to accelerate membrane fusion by inducing membrane curvature, perturbing lipid bilayers or helping bridge the membranes, but SNARE complex binding orients the Ca2+-binding loops away from the fusion site, hindering these putative activities. Molecular dynamics simulations now suggest that Synaptotagmin-1 C2 domains near the site of fusion hinder SNARE action, providing an explanation for this paradox and arguing against previous models of Sytnaptotagmin-1 action. NMR experiments reveal that binding of C2B domain arginines to SNARE acidic residues at region II remains after disruption of region I. These results and fluorescence resonance energy transfer assays, together with previous data, suggest that Ca2+ causes reorientation of the C2B domain on the membrane and dissociation from the SNAREs at region I but not region II. Based on these results and molecular modeling, we propose that Synaptotagmin-1 acts as a lever that pulls the SNARE complex when Ca2+ causes reorientation of the C2B domain, facilitating linker zippering and fast membrane fusion. This hypothesis is supported by the electrophysiological data described in the accompanying paper. Significance statementNeurotransmiter release requires SNARE complexes that fuse synaptic vesicles with the plasma membrane and the Ca2+-sensor synaptotagmin-1, which was thought to facilitate membrane fusion directly through its Ca2+-binding loops. However, binding of Synaptotagmin-1 to SNARE complexes orients these loops away from the fusion site. Using molecular dynamics simulations, we show that placing Synaptotagmin-1 at the fusion site hinders the action of SNARE complexes. Spectroscopic studies show that Ca2+ binding to Synaptotagmin-1 can change its interactions with SNARE complexes and, together with molecular modeling, suggest that Synaptotagmin-1 acts as a lever, pulling SNARE complexes and thus facilitating their action on the membranes to induce fusion. Functional studies described in the accompanying paper support this hypothesis.

biophysics↗

bioRxiv2023

Molecular mechanism underlying SNARE-mediated membrane fusion enlightened by all-atom molecular dynamics simulations

The SNARE proteins syntaxin-1, SNAP-25 and synaptobrevin mediate neurotransmiter release by forming tight SNARE complexes that fuse synaptic vesicles with the plasma membranes in microseconds. Membrane fusion is generally explained by the action of proteins on overall membrane properties such as curvature, elastic modulus and tension, and a widespread model envisions that the SNARE motifs, juxtamembrane linkers and C-terminal transmembrane regions of synaptobrevin and syntaxin-1 form continuous helices that act mechanically as semi-rigid rods, squeezing the membranes together as they assemble ( zipper) from the N- to the C-termini. However, the mechanism underlying fast SNARE-induced membrane fusion remains unknown. We have used all-atom molecular dynamics simulations to investigate this mechanism. Our results need to be interpreted with caution because of the limited number and length of the simulations, but they suggest a model of membrane fusion that has a natural physicochemical basis, emphasizes local molecular events over general membrane properties, and explains extensive experimental data. In this model, the central event that initiates fast (microsecond scale) membrane fusion occurs when the SNARE helices zipper into the juxtamembrane linkers which, together with the adjacent transmembrane regions, promote encounters of acyl chains from both bilayers at the polar interface. The resulting hydrophobic nucleus rapidly expands into stalk-like structures that gradually progress to form a fusion pore, aided by the SNARE transmembrane regions and without clearly discernible intermediates. The propensity of polyunsaturated lipids to participate in encounters that initiate fusion suggests that these lipids may be important for the high speed of neurotransmiter release.

biophysics↗

bioRxiv2023

Oxidative regulation of TDP-43 self-association by a beta-to-alpha conformational switch

An evolutionarily conserved region of the TDP-43 low complexity domain twenty residues in length can adopt either an -helical or {beta}-strand conformation. When in the latter conformation, TDP-43 self-associates via the formation of a labile, cross-{beta} structure. Self-association can be monitored via the formation of phase separated protein droplets. Exposure of droplets to hydrogen peroxide leads to oxidation of conserved methionine residues distributed throughout the low complexity domain. Oxidation disassembles the cross-{beta} structure, thus eliminating both self-association and phase separation. Here we demonstrate that this process reciprocally enables formation of -helical structure in precisely the same region formerly functioning to facilitate {beta}-strand mediated self-association. We further observe that the -helical conformation allows interaction with a lipid-like detergent, and that exposure to lipids enhances the {beta}-to- conformational switch. We hypothesize that regulation of this oxidative switch will prove to be important to the control of localized translation within vertebrate cells. The experimental observations reported herein were heavily reliant on studies of 1,6-hexanediol, a chemical agent that selectively dissolves labile structures formed via the self-association of protein domains of low sequence complexity. This aliphatic alcohol is shown to exert its dissociative activity primarily via hydrogen bonding interactions with carbonyl oxygen atoms of the polypeptide backbone. Such observations underscore the central importance of backbone-mediated protein:protein interactions that facilitate the self-association and phase separation of low complexity domains. Significance StatementThe TDP-43 protein is a constituent of RNA granules involved in regulated translation. TDP-43 contains a C-terminal domain of 150 amino acids of low sequence complexity conspicuously decorated with ten methionine residues. An evolutionarily conserved region (ECR) of 20 residues within this domain can adopt either of two forms of labile secondary structure. Under normal conditions wherein methionine residues are reduced, the ECR forms a labile cross-{beta} structure that enables RNA granule condensation. Upon methionine oxidation, the ECR undergoes a conformational switch to become an -helix incompatible with self-association and granule integrity. Oxidation of the TDP-43 low complexity domain is hypothesized to occur proximal to mitochondria, thus facilitating dissolution of RNA granules and activation of localized translation.

biochemistry↗