Biology subjects

Sari, L.

Publications and source records attributed to Sari, L..

7 recordsLinked to original sources

bioRxiv2025

Saturation mutagenesis map of generalist versus specialist adaptations of β-lactamase to novel antibiotics

The evolution of {beta}-lactamase proteins is shaped by the need to maintain enzymatic activity against previously prevalent {beta}-lactam antibiotics while expanding substrate range against new classes of antibiotics. Using saturation mutagenesis and sequence-barcoding-based quantification, we comprehensively mapped the response of the fitness landscape of TEM-1 {beta}-lactamase, which evolved against penicillin-class antibiotics, to mutational perturbations against six diverse {beta}-lactam antibiotics. This systematic panel of antibiotic substrates, including representatives from penicillin, cephalosporin, and monobactam classes, allowed us to classify resistance mutations into two categories. Generalist mutations conferred resistance to multiple antibiotics and were consistently restricted to three positions critical for substrate recognition and catalytic function R164, G238, and E240. These substitutions produced broad spectrum resistance through mechanisms such as expansion of the active site and improved substrate accommodation. In contrast, specialist mutations conferred resistance to only a single antibiotic and exhibited much wider positional diversity. Ceftazidime selection yielded the greatest number of distinct specialist mutations, which were frequently found in flexible or peripheral regions including the omega loop. One especially unexpected finding was the identification of the E166P variant. E166 is a catalytic residue required for deacylation during hydrolysis, and substitutions at this site are generally assumed to abolish function. However, E166P conferred a significant increase in ceftazidime resistance despite eliminating activity against penicillins. Molecular dynamics simulations and mutational analysis revealed that the E166P mutant employs an alternative catalytic mechanism, involving residue N132, rather than the canonical pathway. Together, our findings reveal, at the molecular level, how specialist mutations open up a wide range of diverse and idiosyncratic solutions at the expense of generalizability. These insights may inform strategic design of antibiotic administration protocols to systematically lower pathogenic evolvability.

evolutionary biology↗

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↗

bioRxiv2021

All-atom molecular dynamics simulations of synaptic vesicle fusion I: a glimpse at the primed state

Synaptic vesicles are primed into a state that is ready for fast neurotransmitter release upon Ca2+-binding to synaptotagmin-1. This state likely includes trans-SNARE complexes between the vesicle and plasma membranes that are bound to synaptotagmin-1 and complexins. However, the nature of this state and the steps leading to membrane fusion are unclear, in part because of the difficulty of studying this dynamic process experimentally. To shed light into these questions, we performed all-atom molecular dynamics simulations of systems containing trans-SNARE complexes between two flat bilayers or a vesicle and a flat bilayer with or without fragments of synaptotagmin-1 and/or complexin-1. Our results help visualize potential states of the release machinery en route to fusion, and suggest mechanistic features that may control the speed of release. In particular, the simulations suggest that the primed state contains almost fully assembled trans-SNARE complexes bound to the synaptotagmin-1 C2B domain and complexin-1 in a spring-loaded configuration where interactions of the C2B domain with the plasma membrane orient complexin-1 toward the vesicle, avoiding premature membrane merger but keeping the system ready for fast fusion upon Ca2+ influx.

biophysics↗

bioRxiv2021

Control of neurotransmitter release and presynaptic plasticity by re-orientation of membrane-bound Munc13-1

Munc13-1 plays a central role in neurotransmitter release through its conserved C-terminal region, which includes a diacyglycerol (DAG)-binding C1 domain, a Ca2+/PIP2-binding C2B domain, a MUN domain and a C2C domain. Munc13-1 was proposed to bridge synaptic vesicles to the plasma membrane in two different orientations mediated by distinct interactions of the C1C2B region with the plasma membrane: i) one involving a polybasic face that yields a perpendicular orientation of Munc13-1 and hinders release; and ii) another involving the DAG-Ca2+-PIP2-binding face that induces a slanted orientation and facilitates release. Here we have tested this model and investigated the role of the C1C2B region in neurotransmitter release. We find that K603E or R769E point mutations in the polybasic face severely impair synaptic vesicle priming in primary murine hippocampal cultures, and Ca2+-independent liposome bridging and fusion in in vitro reconstitution assays. A K720E mutation in the polybasic face and a K706E mutation in the C2B domain Ca2+-binding loops have milder effects in reconstitution assays and do not affect vesicle priming, but enhance or impair Ca2+-evoked release, respectively. The phenotypes caused by combining these mutations are dominated by the K603E and R769E mutations. Our results show that the C1-C2B region of Munc13-1 plays a central role in vesicle priming and support the notion that re-orientation of Munc13-1 controls neurotransmitter release and short-term presynaptic plasticity.

neuroscience↗

bioRxiv2020

All protein aggregates will thermodynamically order

Proteins can aggregate into disordered liquid droplets or ordered assemblies such as amyloid fibrils. These two distinct phases determine the spatial organization within cells and serve differing roles in a wide range of biological functions including gene regulation, organelle and synapse formation, and memory consolidation. The ordered phase can also give rise to diseases such as Alzheimers. However, how the protein sequence determines aggregation fate is an open question. Here we establish a general statistical mechanical theory of the disordered-to-ordered transition for polymer aggregates, including proteins, thereby completing the phase diagram for this general class of matter. The theory produces a simple universal equation determining the favored phase as a function of the temperature, polymer length, and inter-residue interaction energy variance. We show that the sequence-dependent energy variance can be efficiently calculated from all-atom molecular dynamics simulations, so that the theory has no adjustable parameters. The equation accurately predicts the experimental length-dependent crystallization temperature of synthetic polymers. The theory shows that all protein aggregates, regardless of sequence, will thermodynamically order, even in the most extreme thermophiles. Therefore, energy must be expended to maintain the disordered protein aggregate at steady state. More broadly, the theory establishes a lower bound on the ordering transition temperature for polymers. This bound indicates that condensates of any organic polymer will spontaneously order on all habitable planets.

biophysics↗

bioRxiv2018

Native tau structure is disrupted by disease-associated mutations that promote aggregation

Tauopathies are neurodegenerative diseases characterized by intracellular amyloid deposits of tau protein. Missense mutations in the tau gene (MAPT) correlate with aggregation propensity and cause dominantly inherited tauopathies, but their biophysical mechanism driving amyloid formation is poorly understood. Many disease-associated mutations localize within taus repeat domain at inter-repeat interfaces proximal to amyloidogenic sequences, such as 306VQIVYK311. Using cross-linking mass spectrometry, intramolecular FRET, recombinant protein and synthetic peptide systems, in silico modeling, and cell models, we conclude that the aggregation prone 306VQIVYK311 motif forms metastable compact structures with the upstream sequence that modulates aggregation propensity. Disease-associated mutations, isomerization of a critical proline, or alternative splicing are all sufficient to destabilize this local structure and trigger spontaneous aggregation. These findings provide a biophysical framework to explain the basis of early conformational changes that may underlie genetic and sporadic tau pathogenesis.

biophysics↗