Biology subjects

Kulshrestha, A.

Publications and source records attributed to Kulshrestha, A..

10 recordsLinked to original sources

bioRxiv2026

Cardiolipin regulates free energy of folding and vesicle leakage of CM15 antimicrobial peptides in bacterial inner membranes

Globally rising antimicrobial resistance has led to antimicrobial peptides (AMPs) being explored as alternative therapeutics for bacterial infections. A better mechanistic understanding of the interaction of the peptide with membrane compositions can aid in targeting specific bacterial strains more effectively and lead to more resilient treatments. Using all-atom molecular dynamics simulations, we obtain the free-energy landscape of the cecropin-melittin hybrid peptide CM15 using a path-based method and investigate the effect of cardiolipin (CL), a four - tailed lipid found in bacterial membranes. Our analysis reveals that the transition of CM15 from an unfolded state in solution to a membrane-bound helical state proceeds through three main steps: membrane binding, insertion in an unfolded state, and subsequent folding beneath the lipid headgroups into an -helical conformation. In a phosphatidylethanolamine (PE)/phosphatidylglycerol (PG) membrane lacking CL, folding occurs spontaneously without appreciable free energy barriers. However, the addition of only 5% CL substantially alters the landscape, giving rise to two distinct transition pathways with barriers ranging from 2.5 to 8 k BT. This reflects the presence of a more rugged free-energy landscape associated with local heterogeneity in lipid composition. The emergence of these barriers is driven by strong interactions between CL and CM15, which promote CL sequestration around the peptide in regions of negative membrane curvature. Vesicle leakage experiments reveal the inhibitory influence of CL at lower peptide-to-lipid ratios with delayed kinetics as the CL content is increased. We attribute the reduced activity at low CM15 concentrations to the free-energy barriers associated with peptide folding and to strong CM15-CL interactions that stabilize a membrane bound peptide state, thereby hindering membrane disruption, pore formation, and leakage. Our study illustrates the putative role of CL in pore formation, providing molecular insights that can potentially aid the rational design of strain-specific synthetic AMPs.

biophysics↗

bioRxiv2026

The transmembrane domain regulates the kinetics of the SARS-CoV-2 spike conformational transition

ABSTRACTThe homotrimeric SARS-CoV-2 spike glycoprotein comprises two subunits: S1, which recognizes host-receptors through its receptor-binding domains (RBDs), and S2, anchored to the viral membrane through its transmembrane domain (TMD), which facilitates the fusion of the viral envelope with the host cell membrane. Upon host-receptor engagement and proteolytic activation, S1 dissociates and triggers a large conformational transition in S2, involving structural rearrangements in the S2 ectomembrane-domains and the TMD. While studies have focused on the ectomembrane-domain dynamics, the TMD has typically been modeled as being in a trimeric state. Here, we use molecular dynamics simulations of a coarse-grained structure-based model (SBM) with an implicit membrane to investigate the role of TMD dynamics in modulating S2 conformational conversion. We first recapitulate previous results from an all-atom SBM with a trimeric TMD and re-emphasize that the extended pre-hairpin intermediate state of S2, which brings the two membranes into apposition, is a byproduct of the prefusion-to-postfusion transition. Next, by introducing dynamics into the TMD, we find a late fusion intermediate structurally similar to a recent cryo-EM structure. A dynamic TMD also makes the conformational transition faster. Simulations including the S1/S2 complex reveal coupled RBD-TMD dynamics: when all three RBDs are in the closed state, they can stabilize the TMD in a trimeric configuration, whereas the opening of a single RBD can trigger a transition to a dynamic TMD. So, the dynamics and the conformational preferences of the TMD can be tuned by the presence and conformation of S1. There is some evidence that the TMDs of class I viral fusion proteins, such as spike, contribute to viral fusion by modulating membrane properties. Our simulations indicate an expanded role for the function of the TMD, where it can directly regulate the kinetics of S2 conformational transitions. SIGNIFICANCE STATEMENTThe SARS-CoV-2 fusion protein, spike, undergoes a large conformational transition, which facilitates the fusion of the viral and host-cell membranes and the delivery of the viral genome into the host cell. Despite extensive studies of the spike conformational conversion, its transmembrane domain (TMD) has largely been viewed as a viral membrane anchor. Using coarse-grained structure-based model simulations, we show that TMD dynamics can modulate the timing of the spike-S2 (fusion subunit) prefusion-to-postfusion conformational conversion. The presence of spike-S1 (receptor-recognition subunit) can suppress TMD dynamics, potentially reducing the rate of spike conformational conversion and viral fusion. Thus, the spike TMD regulates the kinetics of spike-mediated membrane fusion, and TMD-targeting strategies can be an additional avenue for antiviral intervention.

biophysics↗

bioRxiv2026

Sphingomyelin-Cholesterol Synergy as a Lipid Chaperone Driving Cytolysin A Pore Formation

Bacterial pathogens rely on pore-forming toxins (PFTs) to breach host barriers, a feat that requires water-soluble monomers to spontaneously metamorphose into membrane-inserted pores. In the extracellular milieu, these toxins must navigate complex folding landscapes without ATP-dependent chaperones. Here, we show that the synergistic interplay of host sphingomyelin (SM) and cholesterol (CHOL) acts as an intrinsic "lipid chaperone" that drives the conformational maturation of the bacterial toxin Cytolysin A (ClyA). Using leakage assays on vesicles and suspended lipid bilayer (SuLBs) arrays, atomistic molecular dynamics (MD) simulations, cell membrane permeabilization assays and biophysical measurements, we demonstrate that SM-CHOL synergy accelerates the rate-limiting unfolding of the toxins membrane bound {beta}-tongue motif into a reactive "molten globule" intermediate and guides its subsequent refolding into functional -helical conformation crucial for pore formation. We identify conserved lysine residues (K175 and K206) as critical molecular sensors that detect this specific lipid signature to drive productive transformation. This assembly process reciprocally remodels the host membrane and dismantling liquid-ordered domains, suggesting a mechanical coupling between toxin folding and the disruption of host lipid homeostasis. Our findings establish a paradigm of lipid-mediated chaperoning, revealing how pathogens co-evolve to exploit host lipid complexity to overcome the energetic barriers of membrane insertion.

biophysics↗

bioRxiv2025

Multiscale simulations elucidate the mechanism of polyglutamine aggregation and the role of flanking domains in fibril polymorphism

Protein aggregation, which is implicated in aging and neurodegenerative diseases, typically involves a transition from soluble monomers and oligomers to insoluble fibrils. Polyglutamine (polyQ) tracts in proteins can form amyloid fibrils, which are linked to polyQ diseases, including Huntingtons disease (HD), where the length of the polyQ tract inversely correlates with the age of onset. Despite significant research on the mechanisms of Httex1 aggregation, atomistic information regarding the intermediate stages of its fibrillation and the morphological characteristics of the end-state amyloid fibrils remains limited. Recently, molecular dynamics (MD) simulations based on a hybrid multistate structure-based model, Multi-eGO, have shown promise in capturing the kinetics and mechanism of amyloid fibrillation with high computational efficiency while achieving qualitative agreement with experiments. Here, we utilize the multi-eGO simulation methodology to study the mechanism and kinetics of polyQ fibrillation and the effect of the N17 flanking domain of Huntingtin protein. Aggregation simulations of polyQ produced highly heterogeneous amyloid fibrils with variable-width branched morphologies by incorporating combinations of {beta}-turn, {beta}-arc, and {beta}-strand structures, while the presence of the N17 flanking domain reduces amyloid fibril heterogeneity by favoring {beta}-strand conformations. Our simulations reveal that the presence of N17 domain enhanced aggregation kinetics by promoting the formation of large, structurally stable oligomers. Furthermore, the early-stage aggregation process involves two distinct mechanisms: backbone interactions driving {beta}-sheet formation and side-chain interdigitation. Overall, our study provides detailed insights into fibrillation kinetics, mechanisms, and end-state polymorphism associated with Httex1 amyloid aggregation. SIGNIFICANCE STATEMENTPolyglutamine (polyQ) aggregation is central to Huntingtons disease and related neurodegenerative disorders. Despite extensive experimental efforts, a complete molecular understanding of this process--from early aggregation events to the origins of fibril polymorphism--has remained elusive, with varied interpretations of complex fibril architectures. Through multiscale simulations, we reveal how polyQ fibrils adopt diverse tertiary and quaternary structures and demonstrate how the N-terminal flanking domain (N17) modulates fibril architecture and accelerates aggregation. Our hybrid multi-eGO simulations capture early-stage fibrillation kinetics and identify distinct structural polymorphs that align with experimental observations. This work provides a molecular framework for understanding amyloid polymorphism and illuminates the role of flanking domains in shaping aggregation pathways-- offering valuable insights for therapeutic strategies targeting early toxic intermediates.

biophysics↗

bioRxiv2025

A graphical user interface for wrmXpress 2.0 streamlines helminth phenotypic screening

Image-based phenotypic screening is a fundamental technique used to better understand the basic biology of helminths and advance discovery of new anthelmintics. Miniaturization of screening platforms and automated microscopy have led to a surge in imaging data and necessitated software to organize and analyze these data. Traditionally, these analyses are performed remotely on high-performance computers, often requiring an understanding of a command-line interface (CLI) and the ability to write scripts to control the software or job scheduler. Requiring access to specialized computing equipment and advanced computational skills raises the barrier to entry for these sorts of studies. The development of efficient, performant computer and graphical processing units for personal computers and cheaper imaging solutions has made the requirement of remote servers superfluous for many small to medium-scale screens, but most analytical software still require interaction with a CLI. To democratize the analysis of image-based phenotypic screens, we have developed a graphical user interface (GUI) for wrmXpress, a tool that integrates many popular computational pipelines for analyzing imaging data of parasitic and free-living worms. The GUI operates on any personal computer using the operating systems native web browser, allowing users to configure and run analyses using a point-and-click approach. Containerization of the application eliminates the need to install specialized programming libraries and dependencies, further increasing the ease of use. GUI development required a substantial reorganization of the wrmXpress backend codebase, which allowed for the addition a new pipeline for high-resolution tracking of worm behavior, and we demonstrate its functionality by showing that praziquantel modulates the behavior of Schistosoma mansoni miracidia. These advances make cutting-edge analyses of image-based phenotyping of worms more equitable and accessible.

bioinformatics↗

bioRxiv2023

Purinergic P2Y2 Receptor-Induced Activation of Endothelial TRPV4 Channels Mediates Lung Ischemia-Reperfusion Injury

Lung ischemia-reperfusion injury (IRI), characterized by inflammation, vascular permeability, and lung edema, is the major cause of primary graft dysfunction after lung transplantation. We recently reported that endothelial cell (EC) TRPV4 channels play a central role in lung edema and dysfunction after IR. However, the cellular mechanisms for lung IR-induced activation of endothelial TRPV4 channels are unknown. In a left-lung hilar ligation model of IRI in mice, we found that lung IR increases the efflux of extracellular ATP (eATP) through pannexin 1 (Panx1) channels at the EC membrane. Elevated eATP activated elementary Ca2+ influx signals through endothelial TRPV4 channels through purinergic P2Y2 receptor (P2Y2R) signaling. P2Y2R-dependent activation of TRPV4 channels was also observed in human and mouse pulmonary microvascular endothelium in ex vivo and in vitro surrogate models of lung IR. Endothelium-specific deletion of P2Y2R, TRPV4, and Panx1 in mice had substantial protective effects against lung IR-induced activation of endothelial TRPV4 channels, lung edema, inflammation, and dysfunction. These results identify endothelial P2Y2R as a novel mediator of lung edema, inflammation, and dysfunction after IR, and show that disruption of endothelial Panx1-P2Y2R-TRPV4 signaling pathway could represent a promising therapeutic strategy for preventing lung IRI after transplantation.

physiology↗

bioRxiv2023

Cholesterol catalyzes unfolding in membrane inserted motifs of the pore forming protein cytolysin A

Plasma membrane induced protein folding and conformational transitions play a central role in cellular homeostasis. Several transmembrane proteins are folded in the complex lipid milieu to acquire a specific structure and function. Bacterial pore forming toxins (PFTs) are proteins expressed by a large class of pathogenic bacteria that exploit the plasma membrane environment to efficiently undergo secondary structure changes, oligomerize and form transmembrane pores. Unregulated pore formation causes ion imbalance leading to cell death and infection. Determining the free energy landscape of these membrane driven transitions remains a challenging problem. Although cholesterol recognition is required for lytic activity of several proteins in the PFT family of toxins, the regulatory role of cholesterol for the -PFT, cytolysin A expressed by E. coli is less understood. In a recent free energy computation, we have shown that the {beta}-tongue, a critical membrane inserted motif of the ClyA toxin, has an on-pathway partially unfolded intermediate that refolds into the helix-turn-helix motif of the pore state.1 To understand the molecular role played by cholesterol, we have carried out string method based computations in membranes devoid of cholesterol which reveals an increase of[~] 30 times in the free energy barrier for the loss of {beta}-sheet secondary structure when compared with membranes containing cholesterol. Specifically the tyrosine-cholesterol interaction was found to be critical to stabilizing the unfolded intermediate. In the absence of cholesterol the membrane was found to undergo large curvature deformations in both leaflets of the membrane accompanied by bilayer thinning. Our study with the -toxin, ClyA illustrates that cholesterol is critical to catalyzing and stabilizing the unfolded state of the {beta}-tongue in the membrane, opening up fresh insights into cholesterol assisted unfolding of membrane proteins. SignificanceCholesterol, an integral part of mammalian cell membranes, is necessary for activity of pathogenic toxins. Our understanding of the thermodynamic and molecular underpinnings of cholesterol-protein interactions during different stages of toxin activity is unclear. Using path based all atom molecular dynamics simulations, we illustrate lowered free energy barriers and enhanced stability of the membrane unfolded intermediate of an -pore forming toxin (PFT) ClyA providing insights into the increased pore formation kinetics with cholesterol. Thus, membrane cholesterol generally believed to play a passive receptor function for PFT activity is involved in a more complex regulatory role in assisting secondary structure transitions critical to PFT lytic activity. Our findings could aid in drug development strategies for mitigating PFT mediated bacterial infections.

biophysics↗

bioRxiv2022

Computing the mechanism of α-helix to β-sheet transition in proteins using the finitetemperature string method

The transition of an -helix to a {beta}-sheet in proteins is among the most complex conformational changes seen in bio-molecular systems. Currently, it is difficult to study such protein conformational changes in a direct molecular dynamics simulation. This limitation is typically overcome using an indirect approach wherein one computes the free energy landscape associated with the transition. Computation of free energy landscapes, however, requires a suitable set of collective variables that describe the transition. In this work we demonstrate the use of path collective variables [J. Chem. Phys. 126, 054103 (2007)] and combine it with the finite temperature string (FTS) method [J. Phys. Chem. B 109, 6688-6693 (2005)] to determine the molecular mechanisms involved during the structural transition of the mini G-protein from an -helix to a {beta}-hairpin. The transition from the -helix proceeds via unfolding of the terminal residues, giving rise to a {beta}-turn unfolded intermediate to eventually form the {beta}-hairpin. Our proposed algorithm uses umbrella sampling simulations to simulate images along the string and the weighted histogram analysis to compute the free energy along the computed transition path. This work demonstrates that the string method in combination with path collective variables can be exploited to study complex protein conformational changes such as a complete change in the secondary structure.

biophysics↗

bioRxiv2021

Conformational flexibility is a key determinant of the lytic activity of the pore forming protein, Cytolysin A

Bacterial pore-forming toxins (PFTs) bind and oligomerize on mammalian cell membranes forming nanopores, that cause cell lysis to promote a wide range of bacterial infections. Cytolysin A (ClyA), an alpha()-PFT, is known to undergo one of the largest conformational changes during its transition from a water soluble monomeric form to the membrane embedded dodecameric nanopore assembly. Despite extensive work on the structure and assembly of ClyA, a complete molecular picture of the interplay between the protein segments and membrane lipids driving this transformation remains elusive. In this study, we combine experiments and all-atom molecular dynamics (MD) simulations of ClyA and its mutants to unravel the role of the two key membrane interacting motifs, namely, the {beta}-tongue and N-terminus helix, in facilitating this critical transition. Erythrocyte turbidity and vesicle leakage assays reveal a loss of activity for {beta}-tongue mutant (Y178F), and delayed kinetics for the N-terminus mutants (Y27A and Y27F). All atom, thermal unfolding molecular dynamics simulations of the monomer carried out at 310, 350 and 400 K reveal a distinct reduction in the flexibility in both the {beta}-tongue and N-terminal regions of the mutants when compared with the wild type. This decreased loss of conformational flexbility correlates positively with the reduced lytic and leakage activity observed in experiments, indicating that the tendency to lose secondary structure in the {beta}-tongue region is an important step in the conformational transition bistability of the ClyA protein. Simulations with the membrane inserted oligomeric arcs representing the pore state reveal a greater destabilization tendency among the {beta}-tongue mutant as inferred from secondary structure and N-terminal positioning. Our combined experimental and simulation study, reveals that conformational flexibility is indispensable for the outward movement of the {beta}-tongue and the tendency to induce disorder in the {beta}-tongue is an important step in the transition to the membrane mediated helix-turn-helix motif integral to ClyA pore formation. This observed loss of secondary structure is akin to the structural transitions observed in intrinsically disordered proteins (IDPs) to support protein function. Our finding suggest that inherent flexibility in the protein could play a wider and hitherto unrecognized role in the membrane mediated conformational transitions of PFTs in general. Author summaryBacterial pore-forming toxins (PFTs) bind and oligomerize on mammalian cell membranes to form bilayer spanning nanopores. Unregulated pore formation disrupts ionic balance that compromises the permeability of the cell, leading to cell death and infection. PFTs display remarkable structural plasticity that allow these proteins to interconvert between the two physiochemically distinct water soluble and membrane bound forms. However the molecular mechanism for this robust interconversion is poorly understood. For example, upon membrane binding, cytolysin A (ClyA), an -PFT, shows one of the largest conformational changes in protein structure among the PFT family of proteins. In order to understand this transtition we characterized several point mutations in ClyA using experiments and molecular dynamics (MD) simulations to understand the role of two essential ClyA motifs ({beta} tongue and N-terminus) implicated in the conformational changes responsible for oligomerization and conferring stability to the pore state. Our study reveals that the innate conformational flexibility of the {beta} tongue results in a disordered intermediate state that facilitate the complete transition to the pore state. This tendency to disorder is compromised to varying degrees in ClyA mutants, correlating with the loss of lytic activity. Our results suggest that the finely tuned conformational flexibility in the membrane motifs of ClyA are critical to its function, revealing a broader paradigm that could be at play during membrane associated secondary structure transitions of proteins in general.

biophysics↗

bioRxiv2021

Accelerating COVID-19 research with graph mining and transformer-based learning

In 2020, the White House released the, "Call to Action to the Tech Community on New Machine Readable COVID-19 Dataset," wherein artificial intelligence experts are asked to collect data and develop text mining techniques that can help the science community answer high-priority scientific questions related to COVID-19. The Allen Institute for AI and collaborators announced the availability of a rapidly growing open dataset of publications, the COVID-19 Open Research Dataset (CORD-19). As the pace of research accelerates, biomedical scientists struggle to stay current. To expedite their investigations, scientists leverage hypothesis generation systems, which can automatically inspect published papers to discover novel implicit connections. We present an automated general purpose hypothesis generation systems AGATHA-C and AGATHA-GP for COVID-19 research. The systems are based on graph-mining and the transformer model. The systems are massively validated using retrospective information rediscovery and proactive analysis involving human-in-the-loop expert analysis. Both systems achieve high-quality predictions across domains (in some domains up to 0.97% ROC AUC) in fast computational time and are released to the broad scientific community to accelerate biomedical research. In addition, by performing the domain expert curated study, we show that the systems are able to discover on-going research findings such as the relationship between COVID-19 and oxytocin hormone. ReproducibilityAll code, details, and pre-trained models are available at https://github.com/IlyaTyagin/AGATHA-C-GP CCS CONCEPTS* Applied computing [->] Bioinformatics; Document management and text processing; * Computing methodologies [->] Learning latent representations; Neural networks; Information extraction; Semantic networks.

bioinformatics↗