Biology subjects

Haloi, N.

Publications and source records attributed to Haloi, N..

13 recordsLinked to original sources

bioRxiv2026

CryoLigATE: enhancing the resolvability of cryo-EM maps in protein-ligand complexes using deep learning

Cryo-electron microscopy (cryo-EM) has become a central tool for structure-based drug discovery, yet ligand-binding sites often remain substantially less well resolved than the surrounding protein, limiting reliable atomic interpretation. Although deep-learning methods have substantially improved overall cryo-EM map quality, their predominantly protein-focused training limits their ability to recover ligand density. Here we present CryoLigATE, a deep learning framework specifically designed to enhance densities associated with protein-bound ligands in cryo-EM maps. We curated a chemically and structurally diverse dataset of more than 6,000 protein-ligand complexes from the EMDB and PDB, encompassing drug-like molecules, lipids, steroids, carbohydrates and other ligand classes, and trained a hybrid convolutional-transformer network to enhance local density around binding pockets. During inference, CryoLigATE automatically extracts the target region from a preliminary atomic model, requiring no manual map preparation and completing localized refinement in seconds on a desktop GPU. Evaluation on an independent test set of 649 complexes demonstrates substantial improvements in ligand resolvability, particularly for maps with poorly resolved binding sites, while preserving high-quality experimental densities. The enhanced maps recover chemically meaningful features, including ligand functional groups and topological continuity, enabling more confident atomic modeling. By learning the structural diversity of ligand features, CryoLigATE addresses a longstanding limitation of cryo-EM map enhancement and provides a useful framework for improving structural interpretation and structure-guided drug discovery.

biophysics↗

bioRxiv2026

An atomic interaction conserved for over 600 million years gates inhibitory neurotransmission

Pentameric ligand-gated ion channels (pLGICs) mediate fast inhibitory neurotransmission critical for neuronal network stability. A tyrosine residue in the M2-M3 linker of inhibitory pLGICs, conserved for over 600 million years, is positioned where it could hydrogen bond (H-bond) to the backbone of the neighboring Cys-loop. Given the pathogenic effects of variants of this tyrosine, we hypothesized that this H-bond stabilizes extracellular-to-transmembrane domain coupling essential for channel gating. To test this hypothesis, we used site-directed mutagenesis, noncanonical amino acid incorporation, and electrophysiological recordings in Xenopus laevis oocytes to disrupt this hydrogen bond in GABAA and glycine receptors. Loss of this interaction via tyrosine substitutions or backbone amide modifications that ablate the acceptor or donor, respectively, markedly decrease agonist sensitivity and maximal channel activation, with effects localized to specific subunits. Molecular dynamics simulations support a role for this H-bond in channel gating. These findings reveal a critical atomic interaction underlying a shared mechanism for inhibitory receptor gating and provide a mechanistic explanation for disease-associated mutations linked to epilepsy, neurodevelopmental disability, and hyperekplexia.

biophysics↗

bioRxiv2025

Lipid-facilitated opening of the ADAM10 sheddase revealed by enhanced sampling simulations

ADAM10 is a crucial membrane-bound metalloprotease that regulates cellular physiology by cleaving and releasing membrane-anchored proteins, including adhesion molecules and growth factor precursors, thereby modulating cell signaling, adhesion, and migration. Despite its central role, its activation mechanisms are not fully understood. Here, we model how phosphatidylserine (PS) exposure during apoptosis triggers ADAM10 activation. We confirmed that PS externalization leads to shedding of CD43 from the surface of T cells via ADAM10 activity. Intriguingly, ADAM10 activation correlated with a loss of monoclonal ADAM10 antibody binding, suggesting a PS-induced conformational change that alters epitope accessibility. To explore this lipid-mediated conformational change of ADAM10, we employed molecular dynamics (MD) simulations to map the conformational landscape of ADAM10. Our simulations revealed that in the absence of PS, ADAM10 samples predominantly closed and intermediate states. By contrast, the presence of PS destabilizes the closed conformation, thereby favoring open states. We provide a mechanistic explanation for this PS-induced conformational change which drives ADAM10 activation and loss of mAb binding through conformational change. These findings offer new insights into the lipid-mediated regulation of ADAM10 and its conformational dynamics.

biophysics↗

bioRxiv2025

Modeling ligands in cryo-EM with generative AI and density-guided simulations

Resolving protein-ligand interactions in atomic detail is key to understanding how small molecules regulate macromolecular function. Although recent break-throughs in cryogenic electron microscopy (cryo-EM) have enabled high-quality reconstruction of numerous complex biomolecules, the resolution of bound ligands is often relatively poor. Furthermore, automated methods for building and refining molecular models into cryo-EM maps have largely focused on proteins and may not be optimized for the diverse properties of small-molecule ligands. Here, we present an approach that integrates generative artificial intelligence (AI) with cryo-EM density-guided simulations to fit ligands into experimental maps. Using three inputs: 1) a protein amino acid sequence, 2) a ligand specification, and 3) an experimental cryo-EM map, we validated our approach on a set of biomedically relevant protein-ligand complexes including kinases, GPCRs, and solute transporters, none of which were present in the AI training data. In cases for which generative AI was not sufficient to predict experimental poses outright, integration of flexible fitting into molecular dynamics simulations improved ligand model-to-map cross-correlation relative to the deposited structure from 40-71% to 82-95%. This work offers a straightforward template for integrating generative AI and density-guided simulations to automate model building in cryo-EM maps of ligand-protein complexes, with potential applications for characterization and design of novel modulators and drugs.

biophysics↗

bioRxiv2025

Modeling cryo-EM structures in alternative states with generative AI and density-guided simulations

Modeling atomic coordinates into a target cryo-electron microscopy map is a crucial step in structure determination. Despite recent advances, proteins with multiple functional states remain a challenge - particularly when suitable molecular templates are unavailable for certain states, and the map resolution is not high enough to build de novo models. This is a common scenario, for example, among pharmacologically relevant membrane-bound receptors and transporters. Here, we introduce a refinement approach in which i) several initial models are generated by stochastic subsampling of the multiple sequence alignment (MSA) space in AlphaFold2, ii) the resulting models are subjected to structure-based clustering, iii) density-guided molecular dynamics simulations are performed from the centroid structures, and iv) a final model is selected on the basis of both map fit and model quality. This results in improved fitting accuracy compared to single starting point scenarios for three membrane proteins (the calcitonin receptor-like receptor, L-type amino acid transporter and alanine-serine-cysteine transporter which undergo substantial conformational transitions between functional states. Our results indicate that ensemble construction using generative AI combined with simulation-based refinement facilitates building of alternative states in several families of membrane proteins.

biophysics↗

bioRxiv2024

Adaptive sampling-based structural prediction reveals opening of a GABAA receptor through the αβ interface

GABAA receptors are ligand-gated ion channels in the central nervous system with largely inhibitory function. Despite being a target for drugs including general anesthetics and benzodiazepines, experimental structures have yet to capture an open state of canonical 1{beta}2{gamma}2 GABAA receptors. Here, we use a goal-oriented adaptive sampling strategy in molecular dynamics simulations followed by Markov state modeling to capture an energetically stable putative open state of the receptor. The model conducts chloride ions with comparable conductance as in electrophysiology measurements. The channel desensitizes by narrowing at both the cytoplasmic (-2') and central (9') gates, a motion primarily mediated by transmembrane {beta} subunit interface. Consistent with previous experiments, targeted substitutions disrupting interactions at this interface slowed the open-to-desensitized transition rate. This work demonstrates the capacity of advanced simulation techniques to investigate a computationally and experimentally plausible functionally critical of a complex membrane protein, yet to be resolved by experimental methods.

biophysics↗

bioRxiv2023

Discovering cryptic pocket opening and ligandbinding in a vestibular site of the 5-HT3A receptor

Ligand-gated ion channels propagate electrochemical signals in the nervous system. A diverse set of modulators including stimulants, anesthetics, and lipids regulate their function; however, structures of ligand-bound complexes can be difficult to capture by experimental methods, particularly when binding is dynamic or transient. Here, we used computational methods and electrophysiology to identify a possible bound state of a modulatory stimulant derivative in a cryptic vestibular pocket, distinct from the orthosteric neurotransmitter binding site, of a mammalian serotonin-3A receptor. Starting from a closed-pocket experimental structure, we first applied a molecular dynamics simulations-based goal-oriented adaptive sampling method to identify possible open-pocket conformations. To find plausible ligand-binding poses, we performed Boltzmann docking, which combines traditional docking with Markov state modeling, of the newly sampled conformations. Clustering and analysis of stability and accessibility of docked poses supported a preferred binding site; we further validated this site by mutagenesis and electrophysiology, suggesting a mechanism of potentiation by stabilizing intersubunit contacts. Given the pharmaceutical relevance of serotonin-3 receptors in emesis, psychiatric and gastrointestinal diseases, characterizing relatively unexplored modulatory sites such as these could open valuable avenues to understanding conformational cycling and designing state-dependent drugs. Significance5-HT3A receptors receive the chemical signals of excitatory neurotransmission across the synapse in the central and peripheral nervous systems, and are involved in conditions including emesis, pain, psychiatric disorders, drug abuse, and irritable bowel syndrome. Given their pharmaceutical importance, there is great interest in understanding how and where ligands interact with these receptors. A pocket facing the extracellular vestibule of this membrane protein has been proposed as a modulatory site, but it remains largely uncharacterized in the context of structural modeling or pharmacologically relevant ligands. Here, we are able to identify and investigate binding of a stimulant derivative, 4-bromoamphetamine, in this site by using an integrative computational and experimental approach that is able to account for conformational flexibility.

biophysics↗

bioRxiv2023

Structure and dynamics of differential ligand binding in the human {rho}-type GABA(A) receptor

The neurotransmitter {gamma}-aminobutyric acid (GABA) drives critical inhibitory processes in and beyond the nervous system, partly via ionotropic type-A receptors (GABAARs). Pharmacological properties of {rho}-type GABAARs are particularly distinctive, yet the structural basis for their specialization remains unclear. Here we present cryo-EM structures of a lipid-embedded human {rho}1 GABAAR, including a partial intracellular domain, under apo, inhibited, and desensitized conditions. An apparent resting state, determined first in the absence of modulators, was recapitulated with the specific inhibitor (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid and blocker picrotoxin, and provided a rationale for bicuculline insensitivity. Comparative structures, mutant recordings, and molecular simulations with and without GABA further explained the sensitized but slower activation of {rho}1 relative to canonical subtypes. Combining GABA with picrotoxin also captured an apparent uncoupled intermediate state. This work reveals structural mechanisms of gating and modulation with applications to {rho}-specific pharmaceutical design, and to our biophysical understanding of ligand-gated ion channels.

biophysics↗

bioRxiv2023

Interactive computational and experimental approaches improve the sensitivity of periplasmic binding protein-based nicotine biosensors for measurements in biofluids

We developed fluorescent protein sensors for nicotine with improved sensitivity. For iNicSnFR12 at pH 7.4, the proportionality constant for {Delta}F/F0 vs [nicotine] ({delta}-slope, 2.7 M-1) is 6.1-fold higher than the previously reported iNicSnFR3a. The activated state of iNicSnFR12 has a fluorescence quantum yield of at least 0.6. We measured similar dose-response relations for the nicotine-induced absorbance increase and fluorescence increase, suggesting that the absorbance increase leads to the fluorescence increase via the previously described nicotine-induced conformational change, the "candle snuffer" mechanism. Molecular dynamics (MD) simulations identified a binding pose for nicotine, previously indeterminate from experimental data. MD simulations also showed that Helix 4 of the periplasmic binding protein (PBP) domain appears tilted in iNicSnFR12 relative to iNicSnFR3a, likely altering allosteric network(s) that link the ligand binding site to the fluorophore. In thermal melt experiments, nicotine stabilized the PBP of the tested iNicSnFR variants. iNicSnFR12 resolved nicotine in diluted mouse and human serum at 100 nM, the peak [nicotine] that occurs during smoking or vaping, and possibly at the decreasing levels during intervals between sessions. NicSnFR12 was also partially activated by unidentified endogenous ligand(s) in biofluids. Improved iNicSnFR12 variants could become the molecular sensors in continuous nicotine monitors for animal and human biofluids.

bioengineering↗

bioRxiv2021

Investigation of gating in outer membrane porins provides new perspectives on antibiotic resistance mechanisms

Gram-negative bacteria pose a serious public health concern, primarily due to a higher frequency of antibiotic resistance conferred to them as a result of low permeability of their outer membrane (OM). Antibiotics capable of traversing the OM typically permeate through OM porins; thus, understanding the permeation properties of these porins is instrumental to the development of new antibiotics. A common macroscopic feature of many OM porins is their ability to transition between functionally distinct open and closed states that regulate transport properties and rate. To obtain a molecular basis for these processes, we performed tens of microseconds of molecular dynamics simulations of E. coli OM porin, OmpF. We observed that large-scale motion of the internal loop, L3, leads to widening and narrowing of the pore, suggesting its potential role in gating. Furthermore, Markov state analysis revealed multiple energetically stable conformations of L3 corresponding to open and closed states of the porin. Dynamics between these functional states occurs on the time scale of tens of microseconds and are mediated by the movement of highly conserved acidic residues of L3 to form H-bonds with opposing sides of the barrel wall of the pore. To validate our mechanism, we mutated key residues involved in the gating process that alter the H-bond pattern in the open/closed states and performed additional simulations. These mutations shifted the dynamic equilibrium of the pore towards open or closed states. Complementarily, the mutations favoring the open/closed states lead to increased/decreased accumulation of multiple antibiotics in our whole-cell accumulation assays. Notably, porins containing one of the mutations favoring the closed state has previously been found in antibiotic resistant bacterial strains. Overall, our 180 {micro}s of simulation data (wild type and mutants) with concerted experiments suggests that regulation of the dynamic equilibrium between open and closed states of OM porins could be a mechanism by which Gram-negative bacteria acquire antibiotic resistance.

biophysics↗

bioRxiv2021

Rationalizing generation of broad spectrum antibiotics with the addition of a primary amine

Antibiotic resistance of Gram-negative bacteria is largely attributed to the low permeability of their outer membrane (OM). Recently, we disclosed the eNTRy rules, a key lesson of which is that the introduction of a primary amine enhances OM permeation in certain contexts. To understand the molecular basis for this finding, we perform an extensive set of molecular dynamics (MD) simulations and free energy calculations comparing the permeation of aminated and amine-free antibiotic derivatives through the most abundant OM porin of E. coli, OmpF. To improve sampling of conformationally flexible drugs in MD simulations, we developed a novel, Monte Carlo and graph theory based algorithm to probe more efficiently the rotational and translational degrees of freedom visited during the permeation of the antibiotic molecule through OmpF. The resulting pathways were then used for free-energy calculations, revealing a lower barrier against the permeation of the aminated compound, substantiating its greater OM permeability. Further analysis revealed that the amine facilitates permeation by enabling the antibiotic to align its dipole to the luminal electric field of the porin and while forming favorable electrostatic interactions with specific, highly-conserved charged residues. The importance of these interactions in permeation was further validated with experimental mutagenesis and whole cell accumulation assays. Overall, this study provides insights on the importance of the primary amine for antibiotic permeation into Gram-negative pathogens that could help the design of future antibiotics. We also offer a new computational approach for calculating free-energy of processes where relevant molecular conformations cannot be efficiently captured.

biophysics↗

bioRxiv2020

Structural Basis of Complex Formation Between Mitochondrial Anion Channel VDAC1 and Hexokinase-II

Complex formation between hexokinase-II (HKII) and the mitochondrial channel VDAC1 plays a crucial role in regulating cell growth and survival; however, structural details of this complex remain elusive. We hypothesize that a conserved, hydrophobic helix (H-anchor) of HKII first inserts into the outer membrane of mitochondria (OMM) and then interacts with VDAC1 on the cytosolic leaflet of OMM to form a binary complex. To systematically investigate this process, we adopted a hybrid approach: 1) the membrane binding of HKII was first described with molecular dynamics (MD) simulations employing a membrane mimetic model with enhanced lipid diffusion, then 2) the resulting membrane-bound HKII was used to form complex with VDAC1 in millisecond-scale Brownian dynamics (BD) simulations. We show that H-anchor inserts its first 10 residues into the membrane, substantiating previous experimental findings. The insertion depth of the H-anchor was used to derive positional restraints in subsequent BD simulations to preserve the membrane-bound pose of HKII during the formation of the HKII/VDAC1 binary complex. Multiple BD-derived structural models were further refined with MD simulations, resulting in one stable complex. A major feature in the complex is the partial (not complete) blockade of VDAC1s permeation pathway by HKII, a result supported by our comparative electrophysiological measurements of the channel in the presence and absence of HKII. Additionally, we showed how VDAC1 phosphorylation disrupts HKII binding, a feature that is verified by our electrophysiology recordings and have implications in mitochondria-mediated cell death.

biophysics↗

bioRxiv2020

The Structures of Secretory and Dimeric Immunoglobulin A

Secretory (S) Immunoglobulin (I) A is the predominant mucosal antibody, which binds pathogens and commensal microbes. SIgA is a polymeric antibody, typically containing two copies of IgA that assemble with one joining-chain (JC) to form dimeric (d) IgA that is bound by the polymeric Ig-receptor ectodomain, called secretory component (SC). Here we report the cryo-electron microscopy structures of murine SIgA and dIgA. Structures reveal two IgAs conjoined through four heavy-chain tailpieces and the JC that together form a {beta}-sandwich-like fold. The two IgAs are bent and tilted with respect to each other, forming distinct concave and convex surfaces. In SIgA, SC is bound to one face, asymmetrically contacting both IgAs and JC. The bent and tilted arrangement of complex components limits the possible positions of both sets of antigen binding fragments (Fabs) and preserves steric accessibility to receptor binding sites, likely influencing antigen binding and effector functions.

immunology↗