Biology subjects

Voth, G. A.

Publications and source records attributed to Voth, G. A..

At least 19 recordsLinked to original sources

bioRxiv2026

HIV-1 Assembly is Assisted by IRSp53 Through an Interaction that Extends Gag Conformation

Budding of the immature virion is an essential part of the HIV-1 life cycle. However, understanding and inhibiting this process has been challenging, as both simulations and theoretical studies suggest that the energy required to bend the membrane is too high for the complete assembly of a spherical viral particle at the plasma membrane without the assistance of cellular host factors. Recent results suggest that the I-BAR protein IRSp53 may be one such host factor, but the molecular mechanism by which the Gag-IRSp53 interaction promotes HIV-1 assembly and budding remains poorly understood. We employ molecular dynamics simulations, starting from AlphaFold models, to show that the SH3 domain of IRSp53 interacts directly with the MACANTD domain of Gag and we experimentally validate this interaction. Our simulations show that the specificity of the interaction with IRSp53 arises from its ability to extend the MACANTD Gag conformation in a manner similar to RNA binding, which is known to activate Gag assembly. We then computationally design HIV-1 Gag mutants that, as confirmed by simulations, do not undergo conformational changes upon interaction with theIRSp53 SH3 domain. Experimentally, these Gag mutants are tested and shown to accumulate in cells. In addition, for these mutants we observe large quantities of Gag assembling at the plasma membrane but exhibiting large budding defects. These results validate the proposed mechanism by which the Gag-IRSp53 interaction promotes completion of HIV-1 budding and suggest new routes for inhibiting viral assembly or budding.

molecular biology↗

bioRxiv2026

HIV-1 Ribonucleoprotein Complex Organization Facilitates Assembly of the Mature HIV-1 Capsid

The encapsulation of the viral RNA condensate inside the capsid is a crucial step in the late stages of the HIV-1 replication cycle. While it is agreed that RNA encapsulation depends on RNA-bound integrase (IN), that capsid-bound IN-RNA filaments are present within the capsid, and that the ribonucleoprotein (RNP) complex takes up only a fraction of the capsid volume, the timing of filament formation and ribonucleoprotein (RNP) encapsulation are unclear. Here, we ran virion-scale, coarse grained molecular dynamics simulations to identify how the organization of the RNP and IN in particular affects mature capsid assembly and genome encapsulation. These virion-scale simulations allow for direct comparison between experimental capsid morphologies and those self-assembled in the simulations. It was found, when relatively few tetramers are on the RNP surface, that IN tetramers could facilitate RNA encapsulation without filling a significant fraction of the capsid volume, but do not form a capsid-templated filament. When octameric IN-RNA filaments are modeled, it was found that WT-like capsid morphologies form only when the IN is organized as a single long filament, in agreement with recent experimental suggestions. These capsids are more conical and elongated than the capsids assembled around the RNP with tetrameric IN. Thus, we suggest the IN-RNA filament assembles ahead of the capsid and directs it to its final conical morphology, consistent with the experimental observation of less elongated capsids when IN oligomerization is disrupted.

molecular biology↗

bioRxiv2026

Zyxin and LPP differ in their sensing of strained actin filaments at tricellular junctions

The actomyosin cytoskeleton plays important roles in cell-cell adhesion by generating and responding to forces. Actin-binding proteins support actomyosin networks by reinforcing actin filaments, promoting actin remodeling, and transmitting forces to transmembrane adhesion proteins. One family of actin-binding proteins, LIM domain-containing proteins, is recruited to strained actin filaments. Here, we investigate two members of the LIM domain family, Zyxin and Lipoma-Preferred Partner (LPP), using the embryonic epithelium of Xenopus laevis, where actin-associated cell-cell junctions connect cells to promote tissue integrity and barrier function. Specifically, we compare Zyxin and LPP's response to increased tension at tricellular junctions (TCJs), sites of heightened mechanical strain within the tissue. Upon increased tension, Zyxin and LPP mechanoaccumulate to different extents relative to F-actin, suggesting distinct mechanisms. Our results demonstrate that Zyxin's and LPP's LIM domain-containing regions (LCRs) are sufficient for mechanoaccumulation as well as responsible for the difference in mechanoaccumulation, while their N-termini play a regulatory role in their mechanosensitive responses. Docking simulations show that a hydrophobic interaction between the LCR and the "crack site" that forms in F-actin filaments under high force may underlie the LCR's sensing of strained F-actin. Additionally, the docking simulations reveal that previously-identified conserved residues are present at the binding interface. Mutations of conserved residues in Zyxin or LPP reduce the extent of mechanoaccumulation at TCJs, supporting the docking prediction. Together, this study advances our understanding of Zyxin and LPP's sensing of strained actin at TCJs under mechanical challenge.

cell biology↗

bioRxiv2026

Mechanistic Insights into HIV-1 Capsid Interactions with CPSF6

A crucial stage of the HIV-1 life cycle is the docking of the viral capsid at, and its translocation through, the nuclear pore complex (NPC). During this process, the capsid interacts with a series of cellular host factors that regulate efficient nuclear entry. One such host factor is cleavage and polyadenylation specificity factor 6 (CPSF6), which plays a critical role in efficient HIV-1 nuclear entry and integration. Experimental evidence suggests that nucleoporins (NUPs), such as NUP153, initially engage the capsid, followed by CPSF6 binding and oligomerization during subsequent stages of nuclear import. However, the mechanistic basis by which CPSF6 binds to the capsid and subsequently oligomerizes remains poorly understood. To address this question, we have developed bottom-up coarse-grained (CG) models that explicitly simulate the process of CPSF6 binding and oligomerization on the HIV-1 capsid. Our simulations show that CPSF6 assembles into a mesh-like coating surrounding the capsid, consistent with experimental observations. Furthermore, we then demonstrate that CPSF6 binding is dependent on the intrinsic curvature of the capsid lattice. Finally, we investigate how perturbing CPSF6-CPSF6 interactions alters binding, oligomerization dynamics, and curvature dependence. Together, these findings provide new mechanistic insights into how CPSF6 regulates HIV-1 nuclear entry.

biophysics↗

bioRxiv2026

Psi RNA-specific Binding Promotes HIV-1 Gag Conformational Change Critical for Immature Viral Particle Assembly

The immature HIV-1 virion is assembled by the Gag polyprotein using inositol hexakisphosphate (IP6) as an essential assembly co-factor. Gag binds the genomic RNA Psi packaging signal via the nucleocapsid (NC) domain and associates with the plasma membrane via the matrix (MA) domain. Previous studies revealed that Gag exists in both compact (C) and extended (E) conformational states in solution. Only E-Gag formed virus-like particles with the correct size and IP6 shifted the equilibrium of DNA-bound Gag to the E state. The influence of specific RNA elements on this conformational change is unknown. In this work, a dual dye-labeled Gag was prepared for probing the effect of RNA binding on Gag conformation using Forster resonance energy transfer (FRET). In low salt and in the absence of other factors, Gag was primarily in the C state. Psi RNA binding induced a more significant FRET decrease than binding to non-Psi RNAs, consistent with a shift to E-Gag. IP6 alone also promoted the E-Gag state in the absence and presence of RNA. Atomistic molecular dynamics simulations are consistent with and provide detail into the role of NC-Psi RNA binding in the conformational switch of C-Gag to assembly-competent E-Gag. Simulations also showed that this switch is driven by capsid (CA) linker domain orientational flexibility and MA-CA unbinding dynamics. Thus, the highly flexible multi-domain Gag polyprotein leverages both viral and host cell factors to sample and stabilize distinct conformations, thereby orchestrating the viral assembly process.

biophysics↗

bioRxiv2026

Structure and Dynamics of the HIV-1 Envelope Protein on the Virion Envelope

HIV-1 buds from infected cells as immature virion particles with a scattered envelope glycoprotein (Env) distribution on their envelope. It then undergoes maturation, during which the viral protease cleaves the Gag polyprotein at multiple sites, leading to structural reorganization of the viral particle and lateral redistribution of Env proteins, ultimately rendering the virion infectious. However, the underlying mechanism of maturation-induced Env reorganization remains elusive. In this study, we combine microsecond-long all-atom (AA), bottom-up coarse-grained (CG) molecular dynamics simulations, and diffusion model-based backmapping to investigate the structural organization and key interactions of Env in viral membranes. AA simulations of fully glycosylated Env embedded in HIV-1 mimetic asymmetric bilayers were first performed to characterize its conformational dynamics and Env-lipid interactions. We then developed a bottom-up CG model of glycosylated Env from that AA data and simulated the mature HIV-1 virion envelope containing multiple Env proteins. The CG simulations predict that Env proteins form clusters through interactions mediated by the cytoplasmic tail domain (CTD) and adopt diverse tilted conformations within these clusters. These CG simulations were then backmapped to AA resolution and further AA simulations were carried out to identify, in detail, the specific interacting residues in the Env clusters. Additionally, analysis of epitope accessibility shows that broadly neutralizing antibodies (bnAbs) targeting the V1/V2 and V3 loops may efficiently interact with Env clusters on the mature virion surface. Together, these results provide a molecular mechanism for Env oligomerization during viral maturation and offer new insights into the accessibility of bnAb epitopes on Env clusters.

biophysics↗

bioRxiv2026

Mechanism of HIV-1 Capsid Rupture and Uncoating by Reverse Transcription

One of the key events in the HIV-1 life cycle is reverse transcription, during which single-stranded viral RNA (ssRNA) is converted into double-stranded DNA (dsDNA). This process occurs inside the mature virus capsid and, once it reaches a critical threshold, drives capsid rupture. This uncoating is essential for infection because it releases viral genetic material into the host cell nucleus. Despite its importance, many mechanistic details of this process remain to be fully understood. To address this gap, we develop a multiscale computational method for simulating reverse transcription inside the capsid, termed Coarse-Grained Kinetic Monte Carlo (CG-KMC). CG-KMC stochastically adds deoxynucleotide triphosphates (dNTPs) to the coarse-grained RNA model, enabling stepwise growth of DNA inside the HIV-1 capsid. We implement this method within an integrative coarse-grained framework that combines a "bottom-up" capsid model with a "top-down" representation of the viral RNA/DNA genome. Our simulations phenomenologically capture and predict diverse capsid rupture pathways during reverse transcription. The resulting ruptured structures closely match previously identified cryo-ET images. We further perform an extensive analysis of the rupture process, examining its mechanistic and kinetic aspects as well as the role of capsid-DNA interactions. Our findings illuminate how different capsid-DNA conditions give rise to distinct rupture pathways, which differ from ruptures due to simple outward pressure expansion models from within the capsid. Significance StatementReverse transcription (RT) is a critical step in the life cycle of HIV-1, the causative agent of the AIDS pandemic. During RT, single-stranded RNA (ssRNA), encapsulated inside a mature capsid, is converted into double-stranded DNA (dsDNA). The rigidity of dsDNA generates increased internal pressure inside the capsid which, beyond a critical threshold, drives capsid uncoating, releasing the viral genome into the infected host cell. However, key mechanistic and kinetic aspects of this process remain unresolved. Here, using coarse-grained simulations, we elucidate the mechanistic basis of RT-induced capsid uncoating and quantify the role of capsid-genome interactions. Our results demonstrate that tuning these interactions may provide a potential antiviral strategy to inhibit infectivity by targeting RT.

biophysics↗

bioRxiv2026

Physical Confinement Modulates the Rate-Limiting Transition in the Release of Phosphate from Actin Filaments

The nucleotide state and rates of transitions between states regulate the dynamics of ATPases. Slow inorganic phosphate (Pi) release following ATP hydrolysis is often rate-limiting and associated with key conformational changes. Actin filaments offer a unique opportunity to understand the fundamentals of Pi release, because identical subunits at filament ends and the interior release Pi at markedly different rates. The molecular origin of this difference is debated, so we employed extensive all-atom molecular dynamics simulations to characterize Pi release from different subunits within an actin filament. The calculated dissociation rates of Pi from ADP-Mg2+ in the active site correlate with experimentally measured Pi release rates and scale inversely with the numbers of water molecules in the cavity surrounding the {gamma}-phosphate. Simulations show that egress of Pi through the protein channels, including through the N111-R177 backdoor, is not rate-limiting and, importantly, that subunits at the filament ends use alternative egress pathways. TeaserMolecular dynamics simulations show that dissociation of phosphate from Mg2+ limits release from all parts of actin filaments

biophysics↗

bioRxiv2026

Subdomains of Endophilin-NBAR Can Synergistically Drive Membrane Remodeling and Facilitate Controlled Membrane Scission

The NBAR-domain containing protein endophilin, as a major player in many endocytic pathways, has offered considerable insight into BAR-domain driven membrane remodeling. However, understanding the interaction of the different subdomains of endophilin and their abilities to sense and generate negative Gaussian curvature are yet unanswered questions, with significant implications for the mechanisms and regulation of unconventional endocytic pathways. Using coarse-grained molecular dynamics simulation, we demonstrate the synergistic remodeling capabilities of the NBAR remodeling unit, as well as its ability to sort to and generate membrane regions with negative Gaussian curvature. We find that the assembly of NBAR scaffolds at regions of negative Gaussian curvature facilitate membrane hemifission in dynamic bud formation. These insights provide an additional role for endophilin scaffolds in endocytosis, as well as emphasizing the importance of developing new ways to study negative Gaussian curvature. STATEMENT OF SIGNIFICANCEThis work provides deeper insight into the composite membrane remodeling abilities of NBAR domains in peripheral membrane proteins and their sorting to negative Gaussian curvature. Theis work also explicitly models at the molecular level the {Omega}-shaped membrane geometry, connecting endophilin mechanics to its physiological function in endocytosis.

biophysics↗

bioRxiv2026

Hydration-Controlled Proton Transport in Respiratory Complex I

Proton pumping by respiratory Complex I is one essential element for generating the proton motive force that drives ATP synthesis in mitochondria. Although it is understood that electrons from NADH reduce ubiquinone at the peripheral arm and that four protons are transferred in the membrane domain, the mechanism by which this redox reaction initiates proton translocation remains unclear. A lateral pathway linking the quinone binding site to the membrane domain via ND1, ND3, and ND4L subunits has been proposed as the initial path of an excess proton. However, in experimental structures this region lacks a continuous water network between D66ND3 and E34ND4L, resulting in a hydration bottleneck that may regulate proton transfer. Using multiscale reactive molecular dynamics (MS-RMD) and a water wire connectivity metric, we directly simulate proton transport through this region as coupled the the hydration by water molecules. Our results reveal that proton transfer is thermodynamically feasible when transient hydration aligns with the presence of an excess proton, revealing the strong coupling between hydration and proton (PT) in this region of Complex I. These findings support a model where proton injection enhances local hydration, dynamically opening the pathway for proton transfer and regulating the onset of proton pumping in Complex I.

biophysics↗

bioRxiv2025

Mechanism of Phosphatidylserine Lipid Scrambling by Human SERINC3, an HIV-1Restriction Factor

The HIV-1 restriction factor, hSERINC3, functions as a lipid scramblase, translocating lipids across the bilayer in reconstituted proteoliposomes and the viral envelope. Phosphatidylserine(PS) scrambling and exposure at the outer leaflet are recognized to play important roles in several biological processes. To understand the mechanistic basis for hSERINC3-mediated PS lipid scrambling at atomistic resolution, we implemented the transition-tempered metadynamics (TTMetaD) enhanced sampling method. Our simulations sampled close-to-open hSERINC3 conformational transition during PS scrambling and demonstrated that while other non-ATP-dependent lipid transporters with similar architecture transport lipid following a "trap-and-flip" mechanism, hSERINC3 adopts a "credit card" mechanism of lipid scrambling and does not follow the classical "alternating access" mechanism. Notably, we observe unfolding of the H8 NTD, consistent with the cryo-EM density map of WT-hSERINC3, mediates PS scrambling. A cluster of hydrophilic residues in the hSERINC3 central cavity, forming central gates and interacting with the PS headgroup, stabilizes the intermediate state of inner-groove scrambling and is also observed in the AlphaFold2 model of hSERINC5 that exhibits the highest viral restriction activity. Surprisingly, our simulations reveal distinct pathways for lipid translocation and pathway-dependent alterations of hSERINC3 central cavity, providing direct evidence for a non-canonical, closed-state out-of-groove PS scrambling in a complex membrane environment.

biophysics↗

bioRxiv2025

Mutation and ACE2-induced Allosteric Network Rewiring in Delta and Omicron SARS-CoV-2 Spike Proteins

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates viral entry by binding its receptor-binding domain (RBD) to the host receptor ACE2. Spike mutations in different variants have been experimentally shown to influence the rate of conformational transitions and alter viral infectivity. In parallel, both experimental and computational studies have reported the presence of long-range allosteric communication within the spike protein, suggesting that such mutations may also affect allosteric signaling pathways involved in viral function. A detailed understanding of the allosteric residue network is essential for rational antiviral drug design. In this study, we performed extensive atomistic molecular dynamics (MD) simulations of the spike proteins from the Delta and Omicron variants in both ACE2-bound and unbound states. By integrating linear mutual information (LMI) calculations and graph theory-based analysis, we delineated the long-range allosteric communication networks embedded within the spike protein. Betweenness centrality metrics enabled the identification of residues that act as key mediators of information flow. Notably, ACE2 binding markedly enhances allosteric coupling throughout the spike. We identified three key linkers, Link1 (NTD-RBD), Link2 (RBD-SD1), and Link3 (SD2-FP), as primary mediators of allosteric communication. Delta exhibits stronger signaling through Link1 and Link2, whereas Omicron redirects communication via Link3. While Delta maintains localized connectivity within the S1 domain but loses long-range contact with the S2 core, Omicron forms a broader yet weaker S1 network and establishes long-range coupling. We propose that the N856K and T547K mutations reshape the conformational landscape, reconfiguring allosteric communication pathways in Omicron. Furthermore, our analysis reveals distinct domain-level allosteric couplings in Delta and Omicron, pointing to variant-specific differences in fusogenicity and immune evasion. By mapping key allosteric sites and mutation-induced conformational shifts, our study may provide a framework for developing robust antiviral strategies resilient to future emerging SARS-CoV-2 variants. SignificanceReceptor engagement at the RBD rewires long range allostery in the SARS CoV 2 spike. Using LMI and graph theory-based analyses, we map communication paths and pinpoint residues that govern spike opening and infection. ACE2 binding globally strengthens coupling, but energy and signals propagate along variant specific routes: Delta biases toward openness, channeling binding energy into RBD opening; Omicron remains less open, routing energy to the S2 core to prime fusion. Information flows through three linkers - Link1 (NTD to RBD), Link2 (RBD to SD1), and Link3 (SD2 to FP), with Delta emphasizing Link1/Link2 and Omicron shifting to Link3 and CD connections. We suggest that the N856K and T547K mutations reshape this landscape in Omicron.

biophysics↗

bioRxiv2025

On the Mechanism of Ezrin Activation

Ezrin is a peripheral membrane protein that contributes to the organization and stability of cellular membrane structures by reversibly linking the plasma membrane to actin filaments. The formation of this membrane-actin linkage has been experimentally shown to require ezrin N-terminal (FERM) domain binding to PI(4,5)P2 phospholipid-enriched membrane sites and the phosphorylation of the ezrin C-terminal domain (CTD) at residue T567. Collectively, membrane association and T567 phosphorylation are believed to promote separation of the FERM and CTD domains; however, the underlying molecular mechanism remains less clear. In this study, we investigate the mechanistic steps of ezrin activation and the thermodynamic free energy landscape of FERM-CTD dissociation using enhanced sampling molecular dynamics (MD). We find that upon ezrin attachment to a lipid membrane, PI(4,5)P2 molecules outcompete other phospholipids at the surface of the FERM F1 and F3 subdomains. This interaction triggers a major conformational rearrangement within the FERM domain that destabilizes the FERM F2-CTD interface and initiates dissociation between the FERM and CTD. By employing well-tempered metadynamics (WTMetaD) with a contact-map collective variable, we determine that the principal barrier to FERM-CTD dissociation comes from F3-CTD interactions and that this dissociation can happen spontaneously with a moderate free energy barrier. We also show that the FERM-CTD reassociation after ezrin T567 phosphorylation is impeded due to reduced dissociation energy barrier. The free energy profile of dissociation between FERM and the CTD-replacing EBP50 protein is similar to that of the FERM-CTD system with nonphosphorylated T567, which agrees well with an in vivo experimental observation that EBP50 competes with the CTD for F2-F3 binding after CTD is dissociated. Together, our results help establish a revised view on the ezrin activation mechanism where FERM binding to PI(4,5)P2 enables spontaneous dissociation of the nonphosphorylated CTD. SIGNIFICANCEEzrin and related ERM proteins control how cells link their plasma membrane to the actin cytoskeleton, a process fundamental to cell shape, signaling and motility. Despite decades of study, the molecular basis of ezrin activation - how it transitions from a self-inhibited to an active membrane-bound state - has remained unresolved. Using atomistic and enhanced sampling molecular dynamics together with biochemical validation, we show that binding of the FERM domain to PI(4,5)P2-enriched membranes alone is sufficient to trigger spontaneous dissociation of the nonphosphorylated C-terminal domain. Phosphorylation of T567 subsequently stabilizes the open conformation and prevents domain reassociation, enabling actin engagement and binding of FERM partners such as EBP50. Collectively, these findings advance a more integrated view of ezrin activation, highlighting how membrane interactions, conformational flexibility and phosphorylation act in concert to regulate membrane-cytoskeleton coupling.

biophysics↗

bioRxiv2025

Lattice Instability Drives Formation of Protofilament Clusters at the Microtubule Plus End Tips

Microtubules (MTs) are dynamic cytoskeletal filaments composed of - and {beta}-tubulin protein dimers. They are crucial for maintaining cell structure, facilitating intracellular transport, and ensuring proper chromosome segregation among other things. These biological functions are influenced by the dynamic instability of the MT plus-end tip. Recent simulations have discovered formation of protofilament (PF) clusters at the MT plus-end tip, but reliable extrapolation of PF cluster dynamics and detailed microscopic mechanism are yet to be developed to understand their behavior thoroughly. In this work, we have constructed, from "bottom up", a relatively high resolution coarse-grained (CG) molecular dynamics (MD) model for tubulins with 20 CG sites per tubulin monomer, performed extensive CG MD simulations on MT lattices with 8 and 40 layers of heterodimers, and conducted comprehensive atomistic-level analysis. Our findings demonstrate that, in both GTP and GDP states, PF clusters are stable up to tens of microseconds of CG MD simulation time during spontaneous outward bending relaxation. PF clustering is initiated by longitudinal relaxation, stabilized by residual lateral interaction in the PF clusters. This process is thermodynamically driven by intrinsic lattice instability. In longer microtubules, this instability accumulates and further facilitates PF bending and clustering at the plus-end tip, but it can also be released via lattice curvature and supertwist. GDP-MTs form more PF clusters than GTP-MT on average and undergo more lateral cleavage and faster bending relaxation due to weaker lateral interactions, which facilitates MT catastrophe. GTP-MT forms flatter and more rigid PF clusters that favor nucleotide addition. Our findings highlight the critical role of lattice instability in microtubule dynamics and offer new insights on the conformational variability of MT plus-end tips. SIGNIFICANCEThe dynamical structure and behavior of microtubule (MT) plus-end tips remains an important topic in structural and computational biophysics. For the recently discovered protofilament clusters at MT plus-end tips, this study provides a mechanism for their formation through comprehensive analyses of tubulin-tubulin interaction interfaces in all-atom simulations of complete MT lattices. This study also makes long-time predictions for the dynamics of PF clusters through a coarse-grained model for the tubulin heterodimers to extend the effective simulation time and system size, while preserving key details of the lateral and longitudinal interaction interfaces. The findings deepen understanding of the life cycle of PF clusters, while adding new insight on microtubule dynamics and the role of lattice destabilization.

biophysics↗

bioRxiv2025

Membrane Remodeling by the Collective Action of Caveolin-1

Caveolin-1 proteins scaffold 50-100nm large invaginations in the plasma membrane to mediate critical cellular processes. As revealed recently by cryo-electron microscopy, several caveolin-1 protomers can fold into a disk-like structure that embeds in the cytoplasmic leaflet. This 8S complex represents a basal component to drive membrane curvature via higher-order interactions. The biophysical mechanisms behind the membrane remodeling, however, have remained elusive. To address this shortcoming, we have developed a new bottom-up coarse-grained model to overcome the substantial computational limitations for this large system. During simulations with the coarse-grained model, the complexes increasingly coordinate as partially mediated by attractive electrostatic interactions between scaffolding domains. The coordination of complexes strongly correlates with membrane protrusion, as approaching complexes amplify localized stress in the exoplasmic leaflet. Thus, proximity of two CAV1-8S complexes induces dynamic curvature generation that can facilitate access for signaling partners. This mechanism is further explored in clusters of multiple CAV1-8S complexes that form large-scale membrane invaginations.

biophysics↗

bioRxiv2025

Adaptations in Plasmodium tubulin determine unique microtubule architectures, mechanics and drug susceptibility

Microtubules are ubiquitous yet diverse cytoskeleton filaments. However, tubulin conservation presents challenges in understanding the origins of diverse microtubule architectures. The mechanisms by which microtubule architecture varies through the life cycle of the malaria-causing parasite Plasmodium are not understood and provide a valuable framework for exploring how intrinsic properties of tubulin contribute to architectural variety. Using parasite-purified tubulin, we determined structures of P. falciparum microtubules by cryo-electron microscopy. Parasite-specific sequences change the tubulin dimer structure, thereby modifying drug susceptibility and polymer mechanical properties. Within the P. falciparum microtubule, lateral contacts are smaller but stronger, and the lattice is stiffer than in mammalian microtubules. Non-canonical microtubule architectures found in parasites are highly similar to those observed in vitro, validating the physiological relevance of these properties. Our findings show how evolutionary adaptation of tubulin modulates the material properties of the microtubule cytoskeleton.

biophysics↗

bioRxiv2025

Structural Heterogeneity of the Membrane-Interacting Region of the HIV-1 Envelope Glycoprotein

The HIV-1 envelope glycoprotein (Env) trimer (gp120/gp41)3 forms the key functional envelope spike and is the target of neutralizing antibodies. The glycoprotein gp41 component mediates the fusion of viral and host cell membranes. In addition to its ectodomain, the membrane-interacting C-terminal domain of gp41 plays a crucial role in maintaining the fusogenicity and antigenic characteristics of Env. The membrane-interacting domain of gp41 consists of the highly conserved membrane proximal external region (MPER), which contains epitopes for broadly neutralizing antibodies, the transmembrane domain (TMD), which anchors Env in the membrane and mediates trimer formation, and the cytoplasmic tail (CT) domain, which plays an important role in Env trafficking to HIV-1 assembly sites. Previous experimental studies have extensively characterized the structure of the C-terminal domain of gp41; however, they reported different conformational states of the MPER and TMD. In this study, we used all-atom molecular dynamics simulations to investigate the structure and function of the membrane-interacting domain of gp41 in an HIV-1 mimetic membrane bilayer. The basic residues in the CT domain were found to interact favorably with PIP2, leading to lateral demixing of lipids and the accumulation of PIP2 in the cytofacial leaflet around the CT baseplate. Additionally, analysis based on an artificial intelligence (AI) machine learning based protocol revealed a diverse conformational ensemble of MPER-TMD, consistent with previous experimental observations. The MPER-TMD adopts both helix-turn-helix and extended helical conformations. We propose that the inherent flexibility of the MPER and the N-terminal region of TMD can play an important role in facilitating the late stages of membrane fusion. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/676619v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1ca7fc0org.highwire.dtl.DTLVardef@1894c82org.highwire.dtl.DTLVardef@fe22fcorg.highwire.dtl.DTLVardef@aaf7ee_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Reaching the full potential of cryo-EM reconstructions with molecular dynamics simulations at 310 K: Actin filaments as an example

Cryo-electron microscopy (cryo-EM) structures of multi-protein complexes such as actin filaments help explain the mechanisms of assembly and interactions with partner proteins. Yet, rapid cooling during freezing may not preserve the conformations at physiological temperature. All-atom molecular dynamics simulations starting with cryo-EM reconstructions can provide additional insights. For example, at 310 K the states of ADP-actin filaments consistent with higher entropy favor partly twisted subunits and smaller rotations along short-pitch helix than the cryo-EM reconstructions, while cryogenic temperatures favor flattened conformations. In the active site, the positions of Q137 and the catalytic water 1 and activating water 2 optimal for in line attack on the {gamma}-phosphate of ATP are very rare at 310 K, explaining in part the slow rate of ATP hydrolysis in filaments. This favorable arrangement of the waters is not observed in simulations of actin monomers. At 310 K subunits in ADP-Pi-actin filaments have their backdoor gates open 60% of the time for phosphate release, a conformation not observed by cryo-EM. Rare fluctuations open binding sites for cofilin and phalloidin. The twisted conformations of pointed end subunits and interactions of the D-loop of the penultimate subunit explain the slow association of new subunits. The terminal subunit at the barbed end is tethered to its neighbor along the long-pitch helix but transiently dissociates from its lateral neighbor. These effects of subfreezing temperatures on actin filaments are surely not an isolated example, so MD simulations of structures of other frozen proteins will be informative. Significance statementFunctionally important properties of proteins are tightly linked to their conformations at physiological temperatures. While cryo-EM reconstructions and crystal structure of frozen proteins provide a molecular resolution of protein structures, they might differ from the conformations at physiological temperature. Using the actin filament as a case study, we find cryo-EM reconstructed structures correspond to low entropy conformations that differ from the ensemble of structures in molecular dynamics simulations at 310 K. The fluctuations of subunit dihedral angles, short-pitch rotations and some side chains explain functionally important properties of actin filaments, including the slow rates of ATP hydrolysis and phosphate release as well as the slow binding of the protein cofilin and cyclic peptide phalloidin.

biophysics↗