Biology subjects

Weinstein, H.

Publications and source records attributed to Weinstein, H..

11 recordsLinked to original sources

bioRxiv2023

Allosterically coupled conformational dynamics in solution prepare the sterol transfer protein StarD4 to release its cargo upon interaction with target membranes

Complex mechanisms regulate the cellular distribution of cholesterol, a critical component of eukaryote membranes involved in regulation of membrane protein functions directly and through the physiochemical properties of membranes. StarD4, a member of the steroidogenic acute regulator-related lipid-transfer (StART) domain (StARD)-containing protein family, is a highly efficient sterol-specific transfer protein involved in cholesterol homeostasis. Its mechanism of cargo loading and release remains unknown despite recent insights into the key role of phosphatidylinositol phosphates in modulating its interactions with target membranes. We have used large-scale atomistic Molecular dynamics (MD) simulations to study how the dynamics of cholesterol bound to the StarD4 protein can affect interaction with target membranes, and cargo delivery. We identify the two major cholesterol (CHL) binding modes in the hydrophobic pocket of StarD4, one near S136&S147 (the Ser-mode), and another closer to the putative release gate located near W171, R92&Y117 (the Trp-mode). We show that conformational changes of StarD4 associated directly with the transition between these binding modes facilitate the opening of the gate. To understand the dynamics of this connection we apply a machine-learning algorithm for the detection of rare events in MD trajectories (RED), which reveals the structural motifs involved in the opening of a front gate and a back corridor in the StarD4 structure occurring together with the spontaneous transition of CHL from the Ser-mode of binding to the Trp-mode. Further analysis of MD trajectory data with the information-theory based NbIT method reveals the allosteric network connecting the CHL binding site to the functionally important structural components of the gate and corridor. Mutations of residues in the allosteric network are shown to affect the performance of the allosteric connection. These findings outline an allosteric mechanism which prepares the CHL-bound StarD4 to release and deliver the cargo when it is bound to the target membrane.

biophysics↗

bioRxiv2023

Analysis of the molecular determinants for furin cleavage of the spike protein S1/S2 site in defined strains of the prototype coronavirus murine hepatitis virus (MHV)

We have analyzed the spike protein S1/S2 cleavage site of selected strains of MHV by the cellular protease furin, in order to understand the structural requirements underlying the sequence selectivity of the scissile segment. The probability of cleavage of the various MHV strains was first evaluated from furin cleavage scores predicted by the ProP computer software, and then cleavage was measured experimentally with a fluorogenic peptide cleavage assay consisting of S1/S2 peptide mimics and purified furin. We found that in vitro cleavability varied across MHV strains in line with predicted results--but with the notable exception of MHV-A59, which was not cleaved despite a high score predicted for its sequence. Using the known X-Ray structure of furin in complex with a substrate-like inhibitor as an initial structural reference, we carried out molecular dynamics (MD) simulations to learn the modes of binding of the peptides in the furin active site, and the suitability of the complex for initiation of the enzymatic cleavage. We thus identified the 3D structural requirements of the furin active site configuration that enable bound peptides to undergo cleavage, and the way in which the various strains tested experimentally are fulfilling these requirements. We find that despite some flexibility in the organization of the peptide bound to the active site of the enzyme, the presence of a histidine at P2 of MHV-A59 fails to properly orient the sidechain of His194 of the furin catalytic triad and therefore produces a distortion that renders the peptide/complex structural configuration in the active site incompatible with requirements for cleavage initiation. The Ser/Thr in P1 of MHV-2 and MHV-S has a similar effect of distorting the conformation of the furin active site residues produced by the elimination of the canonical salt-bridge formed by arginine in P1 position. This work informs a study of coronavirus infection and pathogenesis with respect to the function of the viral spike protein, and suggests an important process of viral adaptation and evolution within the spike S1/S2 structural loop.

microbiology↗

bioRxiv2022

The allosteric mechanism leading to an open-groove lipid conductive state of the TMEM16F scramblase

TMEM16F is a Ca2+-activated phospholipid scramblase in the TMEM16 family of membrane proteins. Unlike other TMEM16s exhibiting a membrane-exposed hydrophilic groove that serves as a translocation pathway for lipids, the experimentally determined structures of TMEM16F shows the groove in a closed conformation even under conditions of maximal scramblase activity. Here we describe the analysis of [~]400 {micro}s all-atom ensemble molecular dynamics (MD) simulations of the TMEM16F dimer that revealed an allosteric mechanism leading to an open-groove state of the protein that is competent for lipid scrambling. The grove opens into a continuous hydrophilic conduit that is highly similar in structure to that seen in other activated scramblases. The allosteric pathway connects this opening to an observed destabilization of the Ca2+ ion bound at the distal site near the dimer interface, to the dynamics of specific protein regions that produces the open-groove state seen in our MD simulations to scramble phospholipids.

biophysics↗

bioRxiv2022

The interaction of calcium ions with specific residues in the SARS-CoV fusion peptide and the regulation of viral infectivity

The SARS-CoV-1 spike glycoprotein contains a fusion peptide (FP) segment that mediates fusion of the viral and host cell membranes. Calcium ions are thought to position the FP optimally for membrane insertion by interacting with negatively charged residues in this segment (E801, D802, D812, E821, D825, and D830); however, which residues bind to calcium and in what combinations supportive of membrane insertion are unknown. Using biological assays and molecular dynamics studies, we have determined the functional configurations of FP-Ca+2 binding which promote membrane insertion. We first mutated the negatively charged residues in the SARS CoV-1 FP to assay their role in cell entry and syncytia formation, finding that charge loss in the D802A or D830A mutants reduced syncytia formation and pseudoparticle transduction. Interestingly, the D812A mutation led to increased pseudoparticle transduction, indicating the Ca2+ effect depends on binding at specific FP sites. To interpret mechanistically these results and learn how specific modes of FP-Ca2+ binding modulate membrane insertion, we performed molecular dynamics simulations. Preferred residue pairs for Ca2+ binding were identified (E801/D802; E801/D830; D812/E821) which promote FP membrane insertion. In contrast, binding to residues E821/D825 inhibited FP membrane insertion, which is also supported by our biological assays. Our findings show that Ca2+ binding to SARS-CoV-1 FP residue pairs E801/D802 and D812/E821 facilitates membrane insertion, whereas binding to the E801/D802 and D821/D825 pairs is detrimental. These conclusions provide an improved and nuanced mechanistic understanding of calcium binding modes to FP residues and their dynamic effects on host cell entry.

microbiology↗

bioRxiv2020

Ca2+-dependent mechanism of membrane insertion and destabilization by the SARS-CoV-2 fusion peptide

Cell penetration after recognition of the SARS-CoV-2 virus by the ACE2 receptor, and the fusion of its viral envelope membrane with cellular membranes, are the early steps of infectivity. A region of the Spike protein (S) of the virus, identified as the "fusion peptide" (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca2+ ions to the FPs of corona viruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca2+ binding to the SARS-CoV-2-FP, the role of Ca2+ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca2+-bound SARS-CoV-2-FP and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics (MD) simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca2+-bound peptide models with lipid membranes SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca2+ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca2+ ions. These findings provide novel mechanistic insights regarding the role of Ca2+ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry. STATEMENT OF SIGNIFICANCESARS-CoV-2, the cause of the COVID-19 pandemic, penetrates host cell membranes and uses viral-to-cellular membrane fusion to release its genetic material for replication. Experiments had identified a region termed "fusion peptide" (FP) in the Spike proteins of coronaviruses, as the spearhead in these initial processes, and suggested that Ca2+ is needed to support both functions. Absent structure and dynamics-based mechanistic information these FP functions could not be targeted for therapeutic interventions. We describe the development and determination of the missing information from analysis of extensive MD simulation trajectories, and propose specific Ca2+-dependent mechanisms of SARS-CoV-2-FP membrane insertion and destabilization. These results offer a structure-specific platform to aid the ongoing efforts to use this target for the discovery and/or of inhibitors.

biophysics↗

bioRxiv2020

An equilibrium constant pH molecular dynamics method for accurate prediction of pH-dependence in protein systems: Theory and application

Computational modeling and simulation of biomolecular systems at their functional pH ranges requires an accurate approach to exploring the pH dependence of conformations and interactions. Here we present a new approach - the Equilibrium Constant pH (ECpH) method - to perform conformational sampling of protein systems in the framework of molecular dynamics simulations in an N, P, T-thermodynamic ensemble. The performance of ECpH is illustrated for two proteins with experimentally determined conformational responses to pH change: the small globular water-soluble bovine b-lactoglobulin (BBL), and the dimer transmembrane antiporter CLC-ec1 Cl-/H+. We show that with computational speeds comparable to equivalent canonical MD simulations we performed, the ECpH trajectories reproduce accurately the pH-dependent conformational changes observed experimentally in these two protein systems, some of which were not seen in the corresponding canonical MD simulations. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=153 SRC="FIGDIR/small/394015v1_ufig1.gif" ALT="Figure 1"> View larger version (79K): org.highwire.dtl.DTLVardef@b9c6bdorg.highwire.dtl.DTLVardef@a6d0d3org.highwire.dtl.DTLVardef@1d7fb23org.highwire.dtl.DTLVardef@a8f760_HPS_FORMAT_FIGEXP M_FIG Table of Contents artwork C_FIG

biophysics↗

bioRxiv2019

Monitoring Dynamics of Large Membrane Proteins by 19F Paramagnetic Longitudinal Relaxation: Domain Movement in a Glutamate Transporter Homolog

In proteins where conformational changes are functionally important, the number of accessible states and their dynamics are often difficult to establish. Here we describe a novel 19F-NMR spectroscopy approach to probe dynamics of large membrane proteins. We labeled a glutamate transporter homologue with a 19F probe via cysteine chemistry and with a Ni2+ ion via chelation by a di-histidine motif. We used distance-dependent enhancement of the longitudinal relaxation of 19F nuclei by the paramagnetic metal to assign the observed resonances. We identified two outward- and one inward-facing states of the transporter, in which the substrate-binding site is near the extracellular and intracellular solutions, respectively. We then resolved the structure of the unanticipated second outward-facing state by Cryo-EM. Finally, we showed that the rates of the conformational exchange are accessible from measurements of the metal-enhanced longitudinal relaxation of 19F nuclei.

biophysics↗

bioRxiv2019

The allosteric mechanism of substrate-specific transport in SLC6 is mediated by a volumetric sensor

Neurotransmitter:sodium symporters (NSS) in the SLC6 family terminate neurotransmission by coupling the thermodynamically favorable transport of ions to the thermodynamically unfavorable transport of neurotransmitter back into presynaptic neurons. While a combination of structural, functional, and computational studies on LeuT, a bacterial NSS homolog, has provided critical insight into the mechanism of sodium-coupled transport, the mechanism underlying substrate-specific transport rates is still not understood. We present a combination of MD simulations, single-molecule FRET imaging, and measurements of Na+ binding and substrate transport that reveal an allosteric mechanism in which residues F259 and I359 in the substrate binding pocket couple substrate binding to Na+ release from the Na2 site through allosteric modulation of the stability of a partially-open, inward-facing state. We propose a new model for transport selectivity in which the two residues act as a volumetric sensor that inhibits the transport of bulky amino acids.

biophysics↗

bioRxiv2018

Gramicidin increases lipid flip-flop in symmetric and asymmetric lipid vesicles

Unlike most transmembrane proteins, phospholipids can migrate from one leaflet of the membrane to the other. Because this spontaneous lipid translocation (flip-flop) tends to be very slow, cells facilitate the process with enzymes that catalyze the transmembrane movement and thereby regulate the transbilayer lipid distribution. Non-enzymatic membrane-spanning proteins with unrelated primary functions have also been found to accelerate lipid flip-flop in a nonspecific manner and by various hypothesized mechanisms. Using deuterated phospholipids, we examined the acceleration of flip-flop by gramicidin channels which have well-defined structures and known function, features that make them ideal candidates for probing the protein-membrane interactions underlying lipid flip-flop. To study compositionally and isotopically asymmetric proteoliposomes containing gramicidin, we expanded a recently developed protocol for the preparation and characterization of lipid-only asymmetric vesicles. Channel incorporation, conformation, and function were examined with small-angle X-ray scattering, circular dichroism and a stopped-flow spectrofluorometric assay, respectively. As a measure of lipid scrambling we used differential scanning calorimetry to monitor the effect of gramicidin on the melting transition temperatures of the two bilayer leaflets. The two calorimetric peaks of the individual leaflets merged into a single peak over time suggestive of scrambling activity, and the effect of the channel on the transbilayer lipid distribution in both symmetric POPC and asymmetric POPC/DMPC vesicles was quantified from proton NMR measurements. Our results show that gramicidin increases lipid flip-flop in a complex, concentration-dependent manner. To determine the molecular mechanism of the process we used molecular dynamics simulations and further computational analysis of the trajectories to estimate the amount of membrane deformation in the samples. Together, the experimental and computational approaches were found to constitute an effective means for studying the effects of transmembrane proteins on lipid distribution in both symmetric and asymmetric model membranes.

biophysics↗

bioRxiv2018

A new computational method for membrane compressibility: Bilayer mechanical thickness revisited

Because lipid bilayers can bend and stretch in ways similar to thin elastic sheets, physical models of bilayer deformation have utilized mechanical constants such as the moduli for bending rigidity ({kappa}C) and area compressibility (KA). However, the use of these models to quantify the energetics of membrane deformation associated with protein-membrane interactions and the membrane response to stress is often hampered by the shortage of experimental data suitable for the estimation of the mechanical constants of various lipid mixtures. While computational tools such as Molecular Dynamics (MD) simulations can provide alternative means to estimate KA values, current approaches suffer significant technical limitations. Here, we present a novel computational framework that allows for a direct estimation of KA values for individual bilayer leaflets. The theory is based on the concept of elasticity and derives KA from real-space analysis of local thickness fluctuations sampled in MD simulations. We explore and validate the model on a large set of single and multicomponent bilayers of different lipid composition and sizes, simulated at different temperatures. The calculated bilayer compressibility moduli agree with values estimated previously from experiments and those obtained from a standard computational method based on a series of constrained tension simulations. We further validate our framework in a comparison with an existing polymer brush model (PBM) and confirm the PBMs predicted linear relationship with proportionality coefficient of 24 using elastic parameters calculated from the simulation trajectories. The robustness of the results that emerge from the new method allows us to revisit the origins of the bilayer mechanical (compressible) thickness and in particular, its dependence on acyl chain unsaturation and the presence of cholesterol.

biophysics↗

bioRxiv2017

How structural elements added in evolution from bacterial transporters serve human SLC6 homologs

Much of the structure-based mechanistic understandings of the function of SLC6A neurotransmitter transporters emerged from the study of their bacterial LeuT-fold homologs. It has become evident, however, that structural differences such as the long N- and C-termini of the eukaryotic neurotransmitter transporters impart an expanded set of functional properties to the eukaryotic transporters, which are not shared by the bacterial homologs that lack the structural elements that appeared later in evolution. However, mechanistic insights into some of the measured functional properties of the eukaryotic transporters, that have been suggested to involve these structural elements, are sparse. To learn how the structural elements added in evolution enable mechanisms of the eukaryotic transporters in ways not shared with their bacterial LeuT-like homologs, we focused on the human dopamine transporter (hDAT) as a prototype. We present the results of a study employing large-scale molecular dynamics simulations and comparative Markov State Model analysis of experimentally determined properties of the wild type and mutant hDAT constructs, which reveal a rich spectrum of interactions of the hDAT N-terminus and the mechanisms by which these contribute to regulation (e.g., by phosphorylation), or to entirely new phenotypes (e.g., reverse uptake - efflux) added in evolution. We reveal separate roles for the distal and proximal segments of the much larger N-terminus shared by the eukaryotic transporters compared to the bacterial ones, consistent with the proposal that the size of this region increased during evolution to enable more, and different, modes of regulation that are not shared with the bacterial homologs.

biophysics↗