Biology subjects

Do, H. N.

Publications and source records attributed to Do, H. N..

13 recordsLinked to original sources

bioRxiv2026

Reconstructing a Missing Link of HIV-1 Assembly: HIV-1 Envelope-Matrix Interactions in a Native Viral Context

The envelope surface glycoprotein (Env) on HIV-1 drives cell entry and genome delivery through its receptor binding and membrane fusion activities. Incorporation of Env onto assembling virions is governed by its cytoplasmic tail (Env-CT) and the matrix (MA) domain of the PR55Gag (Gag) polyprotein. To better understand how Gag recruits Env onto virions while reducing its antigenic profile by restricting Env copy number to low levels, we used cryo-electron tomography (cryo-ET) and subtomogram averaging combined with molecular dynamics simulations to investigate Env-MA association in intact viral particles. Full-length Env-CT was resolved directly over individual MA trimers in regions of MA lattice discontinuity. We observed that the conserved but enigmatic Kennedy-sequence motif in Env-CT forms a key linkage between Env and MA. Mutational analysis confirmed the impact of specific CT and MA interactions on Env incorporation. Gag maturation released MA lattice restraints on Env, facilitating its clustering and promoting fusion activity.

biochemistry↗

bioRxiv2026

Structure of HIV-1 Env glycoprotein on virions reveals an alternative fusion subunit organization and native membrane coupling

An effective vaccine for Human Immunodeficiency Virus type-1 (HIV-1) has yet to be developed, and detailed characterization of functional Env glycoprotein, the primary antigenic target on virions, has remained elusive. While engineered Env trimers recapitulate many aspects of functional Env, key differences in antigenicity and dynamic behavior have been reported. Here, cryo-electron tomography and subtomogram averaging of HIV-1 virus-like particles (VLPs) revealed conformational differences in critical membrane-proximal regions compared to soluble Envs. Hydrogen/Deuterium-Exchange Mass Spectrometry and Molecular Dynamics captured dynamic profiles of membrane-bound Env and identified critical interactions with membrane. We show that disruption of the viral membrane results in relaxation of Env to a form that resembles engineered, soluble trimers. Additionally, Env from mature and immature VLPs exhibit only minor conformational differences, while surface clustering on virions changes significantly. These studies provide new insights into the essential role the membrane plays in maintaining Env in its native conformational form.

biochemistry↗

bioRxiv2025

Structure-Based Optimization of Pathogen Signal Sequences for Enhanced Antigen Expression in Humans for Vaccine Designs

Signal peptides (SPs) are short amino acid sequences found at the N-terminus of nascent polypeptides, which serve critical roles in trafficking, folding, and post-translational processing of mature proteins. Many sequence-based computational methods have been developed to predict and design optimal SPs for protein secretion in human cells. In this work, we introduce a structure-based workflow for identifying SPs and their regions, aiming to optimize SPs for protein secretion in human cells. Structural modeling of the SPs in complex with human signal recognition particle 54 kDa (SRP54), combined with hydrophobicity plots of the SPs and identification of the cleavage motif, can be used to detect SPs and SP regions. Afterwards, LigandMPNN is applied to redesign the SPs based on their structural complexes with SRP54. We employ our workflow to optimize the bacterial Yersinia pestis F1 SP, Lassa virus glycoprotein (GP) SP, and Venezuelan equine encephalitis virus (VEEV) E3 envelope protein for protein secretion in human cells to serve as vaccine candidates. By comparing the redesigned SPs with the original SPs, we propose that the binding affinity of SPs to SRP54 serve as the most important molecular determinant in the activity of the SPs. Notably, our experiments confirm that the structurally optimized SPs can express the Y. pestis F1 protein, Lassa GP, and VEEV GP in human cells. Overall, we demonstrate that structural modeling can serve as a valuable tool to predict the SPs and SP regions for vaccine antigens, predict whether the SPs can express mature proteins in humans, and optimize the SPs for the expression of mature proteins in humans.

biophysics↗

bioRxiv2025

SignalGen: A Protein Language Model Based AI Agent For Optimal Signal Peptide Prediction

Signal peptides are short amino acid sequences attached to the N-termini of mature proteins. They play determinant roles in protein expression as well as localization of mature proteins. While sequence-based machine learning (ML) models have been developed to identify the signal peptide sequences given the full or mature protein sequences, no model has been created to design optimal signal peptides with the localization of the mature proteins taken into account. Here, we develop a ML model that considers the mature protein sequence, organism, and localization as inputs, encodes and processes them through a Latent Residual Transformer (LRT), and outputs the optimal signal peptide sequences for enhanced expression of the mature proteins, regardless of whether the proteins are non-native to the organism or de novo. The model is trained using the latest data from the UniProt database up until July 2025. Benchmarking of our ML model shows good performance in predicting the signal peptides for both human and non-human proteins from the UniProt database. Furthermore, our ML model is implemented with an artificial intelligence (AI) agent to enhance accessibility for the general scientific community. Findings from this study provide a framework for predicting optimal signal peptides for non-native protein expression of viral and bacterial vaccine candidates in human cells and for enhanced expression of de novo proteins.

bioinformatics↗

bioRxiv2025

Iterative Multiscale Molecular Dynamics: Accelerating Conformational Sampling of Biomolecular Systems by Iterating All-Atom and Coarse-Grained Molecular Dynamics Simulations

All-atom molecular dynamics (AA MD) simulations remain the gold standard for simulating biomolecular dynamics at an atomistic level for decades. However, they often cannot attain the time scales required to sufficiently sample the many biological processes of interest, especially those that involve large biomolecular systems with high energy barriers. Here, we combine the strengths of all-atom (AA) and coarse-grained (CG) MD simulations to develop the iterative multiscale MD (iMMD) simulation workflow in OpenMM by iterating between AA and CG MD simulations to enhance the sampling of biomolecular conformations. The AA-CG-AA iterations are repeated over multiple cycles, facilitating the accelerated sampling of biomolecular conformations at a CG level, while the finer atomistic interactions are refined with AA simulators. We demonstrate the enhanced sampling ability of iMMD over multiple AA-CG-AA iterations on four representative systems, including two globular proteins (fast-folding variant of Trpcage and Z-matrix protein of Mammarenavirus lassaense (LASV)) and two membrane proteins (Torpedo nicotinic acetylcholine receptor (nAChR) and REGN7663 Fab binding to the CXCR4 receptors in a heterogeneous cholesterol/phosphatidylcholine membrane lipid bilayer). In particular, iMMD captures the folding of the fast-folding Trpcage and Z-protein of LASV starting from their extended conformations as well as the binding of the REGN7663 Fab binding to the CXCR4s and dimerization of the CXCR4 receptors within the heterogenous membrane, within few AA-CG-AA iterations. The development of iMMD should allow for unprecedented simulations of biomolecules at much longer biological timescales to explore the biological processes of interest.

biophysics↗

bioRxiv2024

Anti-HIV-1 B cell antigen receptor signaling and structure

The B cell antigen receptor (BCR) complex, comprised of antigen recognition and signaling components, functions in initiating B cell activation. While structural studies have described BCR domain organization, gaps remain in our understanding of its antigen binding domain (Fab, fragment antigen-binding) disposition, and how antigen binding is sensed to initiate signaling. Here, we report antigen affinity and signaling of the immunoglobulin (Ig) class IgM and IgG BCRs and define conformational states of full-length BCRs of two human broadly neutralizing antibodies, the glycan-specific, heavy chain domain-swapped, I-shaped 2G12, and a canonical Y-shaped antibody, CH31, that recognizes the CD4-binding site on the HIV-1 Envelope protein (Env). The BCRs adopted the shapes (I or Y) of their respective soluble antibodies, and both Ig class of BCRs of the same specificity bound Env trimers with similar affinities. We observed antigen-valenc y dependent differential signaling by the 2G12 IgM and IgG BCRs with trimeric Envs. Cryo-electron microscopy of the 2G12 IgG and CH31 IgM BCRs revealed varied Fab orientations. Structural comparisons revealed hinge points and regions of flexibility in the BCRs suggesting a highly dynamic structure of the BCR complex. Taken together, our results provide an integrated understanding of BCR structure, conformation, antigen recognition and signaling, and provide the basis for understanding antigen induced BCR signal transmission. One Sentence SummaryCryo-electron microscopy structures of full-length B cell antigen receptor complex provide a novel dynamic BCR model and a basis for understanding antigen binding induced signal transmission.

immunology↗

bioRxiv2024

Insertion and Anchoring of HIV-1 Fusion Peptide into Complex Membrane Mimicking Human T-cell

A fundamental understanding of how HIV-1 envelope (Env) protein facilitates fusion is still lacking. The HIV-1 fusion peptide, consisting of 15 to 22 residues, is the N-terminus of the gp41 subunit of the Env protein. Further, this peptide, a promising vaccine candidate, initiates viral entry into target cells by inserting and anchoring into human immune cells. The influence of membrane lipid reorganization and the conformational changes of the fusion peptide during the membrane insertion and anchoring processes, which can significantly affect HIV-1 cell entry, remains largely unexplored due to the limitations of experimental measurements. In this work, we investigate the insertion of the fusion peptide into an immune cell membrane mimic through multiscale molecular dynamics simulations. We mimic the native T-cell by constructing a 9-lipid asymmetric membrane, along with geometrical restraints accounting for insertion in the context of gp41. To account for the slow timescale of lipid mixing while enabling conformational changes, we implement a protocol to go back and forth between atomistic and coarse-grained simulations. Our study provides a molecular understanding of the interactions between the HIV-1 fusion peptide and the T-cell membrane, highlighting the importance of conformational flexibility of fusion peptides and local lipid reorganization in stabilizing the anchoring of gp41 into the targeted host membrane during the early events of HIV-1 cell entry. Importantly, we identify a motif within the fusion peptide critical for fusion that can be further manipulated in future immunological studies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/606381v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@d80eedorg.highwire.dtl.DTLVardef@bc2482org.highwire.dtl.DTLVardef@f715org.highwire.dtl.DTLVardef@15dba11_HPS_FORMAT_FIGEXP M_FIG Table of Content. C_FIG

biophysics↗

bioRxiv2024

Dynamics and Activation of Membrane-Bound B Cell Receptor Assembly

B-cell receptor complexes (BCR) are expressed on the surface of a B-cell and are critical in antigen recognition and modulating the adaptive immune response. Even though the relevance of antibodies has been known for almost a hundred years, the antigen-dependent activation mechanism of B-cells has remained elusive. Several models have been proposed for BCR activation, including cross-linking, conformation-induced oligomerization, and dissociation activation models. Recently, the first cryo-EM structures of the human B-cell antigen receptor of the IgM and IgG isotypes were published that validated the asymmetric organization of the BCR complex. Here, we have carried out extensive molecular dynamics simulations to probe the conformational changes upon antigen binding and the influence of the membrane lipids. We identified two critical dynamical events that could be associated with antigen-dependent activation of BCR. First, antigen binding caused increased flexibility in regions distal to the antigen binding site. Second, antigen binding altered the rearrangement of IgM transmembrane helices, including the relative interaction of Ig/Ig{beta} that mediates intracellular signaling. Furthermore, these transmembrane rearrangements led to changes in localized lipid composition.

biophysics↗

bioRxiv2024

Diverse toxins exhibit a common binding mode to the Nicotinic Acetylcholine Receptors revealing a new molecular determinant

Nicotinic acetylcholine receptors (nAChRs) are critical ligand-gated ion channels in the human nervous system. They are targets for various neurotoxins produced by algae, plants, and animals. While there have been many structures of nAChRs bound by neurotoxins published, the binding mechanism of toxins to the nAChRs remains uncleared. In this work, we have performed extensive Gaussian accelerated molecular dynamics simulations on several Aplysia californica (AC) nAChRs in complex with -conotoxins, strychnine, and pinnatoxins, as well as human nAChRs in complex with -bungarotoxin and -conotoxin for a total of 60 s of simulation time to determine the binding and dissociation pathways of the toxins to the nAChRs and the associated effects. We uncovered two common binding and dissociation pathways shared by toxins and nAChRs. In the primary binding pathway, the toxins diffused from the bulk solvent to first bind a region near the extracellular pore before moving downwards along the nAChRs to the nAChR orthosteric pocket. The second binding pathway involved a direct diffusion of the toxins from the bulk solvent into the nAChR orthosteric pocket. The dissociation pathways were the reverse of the observed binding pathways. We also found that the toxins enacted their toxicity upon binding by restricting the necessary movements required by the nAChRs to open their extracellular and intracellular pores for the ions to pass through. Notably, the electrostatically bipolar interactions between nAChR orthosteric pocket and toxins provides a molecular level explanation for the common binding mode shared by diverse toxins and serve as a key determinant for toxicity.

biophysics↗

bioRxiv2023

Molecular Dynamics Activation of γ-Secretase for Cleavage of Notch1 Substrate

{gamma}-Secretase is an intramembrane aspartyl protease complex which cleaves the transmembrane domain of over 150 peptide substrates, including amyloid precursor protein (APP) and the Notch family of receptors, via two conserved aspartates D257 and D385 in the Presenilin-1 (PS1) catalytic subunit. However, while the activation of {gamma}-secretase for cleavage of APP has been widely studied, the cleavage of Notch by {gamma}-secretase remains poorly explored. Here, we combined Gaussian accelerated Molecular Dynamics (GaMD) simulations and mass spectrometry (MS) analysis of proteolytic products to present the first dynamic models for cleavage of Notch by {gamma}-secretase. MS showed that {gamma}-secretase cleaved the WT Notch at Notch residue G34, while cleavage of L36F mutant Notch occurred at Notch residue C33. Initially, we prepared our simulation systems starting from the cryoEM structure of Notch-bound {gamma}-secretase (PDB: 6IDF) and failed to capture the proper cleavages of WT and L36F Notch by {gamma}-secretase. We then discovered an incorrect registry of the Notch substrate in the PS1 active through alignment of the experimental structure of Notch-bound (PDB: 6IDF) and APP-bound {gamma}-secretase (PDB: 6IYC). Every residue of APP substrate was systematically mutated to the corresponding Notch residue to prepare a resolved model of Notch-bound {gamma}-secretase complexes. GaMD simulations of the resolved model successfully captured {gamma}-secretase activation for proper cleavages of both WT and L36F mutant Notch. Our findings here provided mechanistic insights into the structural dynamics and enzyme-substrate interactions required for {gamma}-secretase activation for cleavage of Notch and other substrates.

biophysics↗

bioRxiv2023

Artificial Intelligence Boosted Molecular Dynamics

We have developed a new Deep Boosted Molecular Dynamics (DBMD) method. Probabilistic Bayesian neural network models were implemented to construct boost potentials that exhibit Gaussian distribution with minimized anharmonicity, thereby allowing for accurate energetic reweighting and enhanced sampling of molecular simulations. DBMD was demonstrated on model systems of alanine dipeptide and the fast-folding protein and RNA structures. For alanine dipeptide, 30ns DMBD simulations captured up to 83-125 times more backbone dihedral transitions than 1{micro}s conventional molecular dynamics (cMD) simulations and were able to accurately reproduce the original free energy profiles. Moreover, DBMD sampled multiple folding and unfolding events within 300ns simulations of the chignolin model protein and identified low-energy conformational states comparable to previous simulation findings. Finally, DBMD captured a general folding pathway of three hairpin RNAs with the GCAA, GAAA, and UUCG tetraloops. Based on Deep Learning neural network, DBMD provides a powerful and generally applicable approach to boosting biomolecular simulations. DBMD is available with open source in OpenMM at https://github.com/MiaoLab20/DBMD/.

bioinformatics↗

bioRxiv2023

Deep Learning Dynamic Allostery of G-Protein-Coupled Receptors

G-protein-coupled receptors (GPCRs) are the largest superfamily of human membrane proteins and represent primary targets of [~]1/3 of currently marketed drugs. Allosteric modulators have emerged as more selective drug candidates compared with orthosteric agonists and antagonists. However, many X-ray and cryo-EM structures of GPCRs resolved so far exhibit negligible differences upon binding of positive and negative allosteric modulators (PAMs and NAMs). Mechanism of dynamic allosteric modulation in GPCRs remains unclear. In this work, we have systematically mapped dynamic changes in free energy landscapes of GPCRs upon binding of allosteric modulators using the Gaussian accelerated molecular dynamics (GaMD), Deep Learning (DL) and free energy prOfiling Workflow (GLOW). A total of 18 available high-resolution experimental structures of allosteric modulator-bound class A and B GPCRs were collected for simulations. A number of 8 computational models were generated to examine selectivity of the modulators by changing their target receptors to different subtypes. All-atom GaMD simulations were performed for a total of 66 {micro}s on 44 GPCR systems in the presence/absence of the modulator. DL and free energy calculations revealed significantly reduced conformational space of GPCRs upon modulator binding. While the modulator-free GPCRs often sampled multiple low-energy conformational states, the NAMs and PAMs confined the inactive and active agonist-G protein-bound GPCRs, respectively, to mostly only one specific conformation for signaling. Such cooperative effects were significantly reduced for binding of the selective modulators to "non-cognate" receptor subtypes in the computational models. Therefore, comprehensive DL of extensive GaMD simulations has revealed a general dynamic mechanism of GPCR allostery, which will greatly facilitate rational design of selective allosteric drugs of GPCRs.

bioinformatics↗

bioRxiv2021

Mechanism of Tripeptide Trimming by γ-Secretase

The membrane-embedded {gamma}-secretase complex processively cleaves within the transmembrane domain of amyloid precursor protein (APP) to produce 37-to-43-residue amyloid {beta}-peptides (A{beta}) of Alzheimers disease (AD). Despite its importance in pathogenesis, the mechanism of processive proteolysis by {gamma}-secretase remains poorly understood. Here, mass spectrometry and western blotting were used to quantify the efficiency of the first tripeptide trimming step (A{beta}49[->]A{beta}46) of wildtype (WT) and familial AD (FAD) mutant APP substrate. In comparison to WT APP, the efficiency of this first trimming step was similar for the I45F, A42T and V46F APP FAD mutants, but substantially diminished for the I45T and T48P mutants. In parallel with biochemical experiments, all-atom simulations using a novel Peptide Gaussian accelerated molecular dynamics (Pep-GaMD) method were applied to investigate tripeptide trimming of A{beta}49 by {gamma}-secretase. The starting structure was active {gamma}-secretase bound to A{beta}49 and APP intracellular domain (AICD), as generated from our previous study that captured activation of {gamma}-secretase for the initial endoproteolytic cleavage of APP (Bhattarai et al., ACS Cent Sci, 2020, 6:969-983). Pep-GaMD simulations captured remarkable structural rearrangements of both the enzyme and substrate, in which hydrogen-bonded catalytic aspartates and water became poised for tripeptide trimming of A{beta}49 to A{beta}46. These structural changes required a positively charged N-terminus of endoproteolytic coproduct AICD, which could dissociate during conformational rearrangements of the protease and A{beta}49. The simulation findings were highly consistent with biochemical experimental data. Taken together, our complementary biochemical experiments and Pep-GaMD simulations have enabled elucidation of the mechanism of tripeptide trimming by {gamma}-secretase. Significance statementProduction of amyloid {beta}-peptide (A{beta}) of Alzheimers disease (AD) from its precursor protein requires a series of proteolytic events carried out by the membrane-embedded {gamma}-secretase complex. Mutations in the substrate and enzyme that produce A{beta} cause hereditary AD and these mutations affect tripeptide trimming of initially formed long A{beta} peptides by {gamma}-secretase. Little is known about the structural mechanism of this trimming process. Here, we have developed a molecular dynamic model for the first step of trimming (A{beta}49 to A{beta}46) by {gamma}-secretase. Conformational changes in the enzyme-substrate complex to set up this trimming step require the presence of N-terminally charged endoproteolytic cleavage co-product. Computational effects of AD-causing mutations in A{beta}49 on the trimming process were validated through biochemical experiments.

biochemistry↗