Biology subjects

Lau, A. Y.

Publications and source records attributed to Lau, A. Y..

10 recordsLinked to original sources

bioRxiv2026

Directional information flow as a tool for analyzing protein allostery

The ability to tune protein function through the binding of modulatory ligands enables the development of therapeutics that steer a biological system away from dysfunctional states underlying disease. Understanding the dynamic mechanisms by which allosteric ligands alter protein function remains an important open question. Dynamical network models allow us to quantify information flow between protein functional sites. However, existing network models use time-symmetric metrics for computing information from correlated residue motions extracted from molecular dynamics (MD) simulations, failing to fully capture directional information flow between sites. Here, we developed a Python library, TEntroPy, and analysis workflow using transfer entropy to generate a directional protein network from equilibrium MD trajectories. Applying this workflow to proteins with known allosteric ligands, we identified residues in both allosteric and orthosteric (primary) binding sites acting as broadcasters and receivers of information. We then computed optimal paths of directional information flow between binding sites. The presence of temporal asymmetry in residue coupling identified from simulations of the unbound (apo) state suggests that directional information flow is encoded in the intrinsic dynamics of the protein. To test this, we perturbed key binding-site residues and demonstrated that our TE-weighted network captures perturbation-induced changes in dynamics along communication routes between binding sites. Identifying residue pairs with high temporal asymmetry provides an additional tool for understanding the dynamic mechanisms of allosteric communication.

biophysics↗

bioRxiv2026

Substrate binding and activation mechanism of the essential bacterial septal cell wall synthase FtsW

Septal peptidoglycan (sPG) synthesis in most bacteria is driven by the essential, highly conserved FtsWIOLB synthase complex, comprising the SEDS glycosyltransferase FtsW, the monofunctional transpeptidase FtsI, and the scaffolding subcomplex FtsOLB. Although apo structures of FtsWIOLB are known, no substrate-bound structures exist, likely due to the intrinsic flexibility of lipid II (L2) substrates. Here, we combined all-atom molecular dynamics simulations of E. coli FtsWIOLB with cysteine-based mutagenesis and cell-based assays to define donor and acceptor L2 binding sites in FtsW and to elucidate the complexs activation mechanism. We show that conserved arginine residues adjacent to membrane-accessible cavities in FtsW coordinate donor and acceptor L2 pyrophosphate groups, stabilizing substrate binding. The FtsWIOLB complex appears to adopt a self-inhibitory architecture in which gating elements and a periplasmic loop prevent the donor and acceptor from approaching FtsWs catalytic residue D297. Activation by FtsN or a superfission FtsW variant relieves these constraints, stabilizes acceptor-site binding, and reorients both substrates into a catalytically primed state. Long donor glycan chains further stabilize this activated conformation at the acceptor site, promoting processive sPG polymerization. Comparative modeling of ESKAPE pathogen homologs reveals conserved and divergent features of binding-site engagement and activation. We corroborate these computational findings with cysteine-based mutagenesis and crosslinking experiments. These results establish a mechanistic framework for FtsW substrate recognition and functional activation, highlighting tractable sites for structure and mechanism-based antibiotic discovery targeting SEDS-class cell wall synthases.

biophysics↗

bioRxiv2025

Mechanisms of Ion Permeation in the AMPA Receptor Ion Channel

Excitatory synaptic transmission in the human nervous system is mediated by -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), tetrameric ligand-gated ion channels localized in the excitatory post-synaptic membrane. AMPARs are activated by the binding of the neurotransmitter glutamate (Glu), which opens the ion channel and allows the influx of Na+ and Ca2+ ions into the post-synaptic neuron, initiating signal transduction. Despite many efforts, a bona fide ion permeation pathway of both monovalent and divalent cations in AMPARs remains elusive. From analyzing our cryo-electron microscopy (cryo-EM) map of an open calcium-permeable AMPAR (CP-AMPAR) ion channel, we identified potential sites vital to permeation of cations through the channel. To delineate mechanisms of permeation, we studied the channel with all-atom molecular dynamics (MD) simulations. Both Na+ and Ca2+ ions are coordinated by an entry site at the top of the channel prior to entering the selectivity filter. A mutation at the filter (Q607E), implicated in a neurodevelopmental disorder, makes the channel more susceptible to Zn2+ block but also creates a more energetically favorable environment for Na+ and Ca2+ permeation through the ion channel. These findings describe a biophysical basis for ion permeation in CP-AMPARs and how disease mutations alter the channel, which will inform therapeutic design against disease mutations in AMPARs that alter the ion channel.

biophysics↗

bioRxiv2024

SARS-CoV-2 3CLPro Dihedral Angles Reveal Allosteric Signaling

In allosteric proteins, identifying the pathways that signals take from allosteric ligand-binding sites to enzyme active sites or binding pockets and interfaces remains challenging. This avenue of research is motivated by the goals of understanding particular macromolecular systems of interest and creating general methods for their study. An especially important protein that is the subject of many investigations in allostery is the SARS-CoV-2 main protease (Mpro), which is necessary for coronaviral replication. It is both an attractive drug target and, due to intense interest in it for the development of pharmaceutical compounds, a gauge of the state-of-the-art approaches in studying protein inhibition. Here we develop a computational method for characterizing protein allostery and use it to study Mpro. We propose a role of the proteins C-terminal tail in allosteric modulation and warn of unintuitive traps that can plague studies of the role of protein dihedrals angles in transmitting allosteric signals.

biophysics↗

bioRxiv2023

Allosteric Competition and Inhibition in AMPA Receptors

Excitatory neurotransmission is principally mediated by AMPA-subtype ionotropic glutamate receptors (AMPARs). Dysregulation of AMPARs is the cause of many neurological disorders and how therapeutic candidates such as negative allosteric modulators inhibit AMPARs is unclear. Here, we show that non-competitive inhibition desensitizes AMPARs to activation and prevents positive allosteric modulation. We dissected the noncompetitive inhibition mechanism of action by capturing AMPARs bound to glutamate and the prototypical negative allosteric modulator, GYKI-52466, with cryo-electron microscopy. Noncompetitive inhibition by GYKI-52466, which binds in the transmembrane collar region surrounding the ion channel, negatively modulates AMPARs by decoupling glutamate binding in the ligand binding domain from the ion channel. Furthermore, during allosteric competition between negative and positive modulators, negative allosteric modulation by GKYI-52466 outcompetes positive allosteric modulators to control AMPAR function. Our data provide a new framework for understanding allostery of AMPARs and foundations for rational design of therapeutics targeting AMPARs in neurological diseases.

biophysics↗

bioRxiv2022

Conformational changes in the essential E. coli septal cell wall synthesis complex suggest an activation mechanism

The bacterial divisome, a macromolecular machine that is composed of more than thirty proteins in E. coli, orchestrates the essential process of cell wall constriction during cell division. Novel antimicrobial strategies can target protein-protein interactions within the divisome and will benefit from insights into divisome structure and dynamics. In this work, we combined structure prediction, molecular dynamics simulation, single-molecule imaging, and mutagenesis to construct a model of the core complex of the E. coli divisome composed of the essential septal cell wall synthase complex formed by FtsW and FtsI, and its regulators FtsQ, FtsL, FtsB, and FtsN. We observed extensive interactions in four key regions in the periplasmic domains of the complex. FtsQ, FtsL, and FtsB scaffold FtsI in an extended conformation with the FtsI transpeptidase domain lifted away from the membrane through interactions among the C-terminal domains. FtsN binds between FtsI and FtsL in a region rich in residues with superfission (activating) and dominant negative (inhibitory) mutations. Mutagenesis experiments in cellulo and in silico revealed that the essential domain of FtsN functions as a tether to tie FtsI and FtsL together, impacting interactions between the anchor-loop of FtsI and the putative catalytic region of FtsW, suggesting a mechanism of how FtsN activates the cell wall synthesis activities of FtsW and FtsI.

biophysics↗

bioRxiv2022

NIS metastable intermediates provide insights into conformational transition between principal thermodynamic states

The Sodium/Iodide Symporter (NIS), a thirteen-helix transmembrane protein found in the thyroid and other tissues, transports iodide, a required constituent of thyroid hormones T3 and T4. Despite extensive experimental information and clinical data, structural details of the intermediate microstates comprising the conformational transition of NIS between its inwardly and outwardly open states remain unresolved. We present data from a combination of enhanced sampling and transition path molecular dynamics (MD) simulations that elucidate the nature of the principal intermediate states comprising the transition between the inwardly and outwardly open metastable states of fully bound and unbound NIS under an enforced ionic gradient. Our findings suggest that in both the absence and presence of bound physiological ions, NIS principally occupies a proximally inward to inwardly open state, whereas when fully bound, it also occupies a rare but thermodynamically favorable inward occluded state. The results of this work provide novel insight into the populations of NIS intermediates and the free energy landscape comprising the conformational transition, adding to a mechanistic understanding of NIS ion transport. Moreover, the knowledge gained from this approach can serve as a basis for studies of NIS mutants to target therapeutic interventions. O_FIG O_LINKSMALLFIG WIDTH=192 HEIGHT=200 SRC="FIGDIR/small/512170v1_ufig1.gif" ALT="Figure 1"> View larger version (80K): org.highwire.dtl.DTLVardef@e826e0org.highwire.dtl.DTLVardef@1504d00org.highwire.dtl.DTLVardef@ef7104org.highwire.dtl.DTLVardef@1e4c95f_HPS_FORMAT_FIGEXP M_FIG For Table of Contents Only C_FIG

biophysics↗

bioRxiv2022

Phosphorylation at Ser65 modulates ubiquitin conformational dynamics

Phosphorylation of ubiquitin at Ser65 increases the population of a rare C-terminally retracted (CR) conformation. Transition between the CR and the Major ubiquitin conformations is critical for promoting mitochondrial degradation. The mechanisms by which the Major and CR conformations of Ser65-phosphorylated (pSer65) ubiquitin interconvert, however, have not yet been revealed. Here, we perform all-atom molecular dynamics simulations using the string method with swarms of trajectories to calculate the lowest free-energy path between these two conformers. Our analysis reveals the existence of a Bent intermediate in which the C-terminal residues of the {beta}5 strand shift to resemble the CR conformation, while pSer65 retains contacts resembling the Major conformation. This stable intermediate was reproduced in well-tempered metadynamics calculations, with the exception of a Gln2Ala mutant that disrupts contacts with pSer65. Lastly, dynamical network modelling reveals that the transition from the Major to CR conformations involves a decoupling of residues near pSer65 from the adjacent {beta}1 strand.

biophysics↗

bioRxiv2022

Excitatory and inhibitory D-serine binding to the NMDA receptor

N-methyl-D-aspartate receptors (NMDARs) uniquely require binding of two different neurotransmitter agonists for synaptic transmission. D-serine and glycine bind to one subunit, GluN1, while glutamate binds to the other, GluN2. These agonists bind to the receptors bi-lobed ligand-binding domains (LBDs), which close around the agonist during receptor activation. To better understand the unexplored mechanisms by which D-serine contributes to receptor activation, we performed multi-microsecond molecular dynamics simulations of the GluN1/GluN2A LBD dimer with free D-serine and glutamate agonists. Surprisingly, we observed D-serine binding to both GluN1 and GluN2A LBDs, suggesting that D-serine competes with glutamate for binding to GluN2A. This mechanism is confirmed by our electrophysiology experiments, which show that D-serine is indeed inhibitory at high concentrations. Although free energy calculations indicate that D-serine stabilizes the closed GluN2A LBD, its inhibitory behavior suggests that it either does not remain bound long enough or does not generate sufficient force for ion channel gating. We developed a workflow using pathway similarity analysis to identify groups of residues working together to promote binding. These conformation-dependent pathways were not significantly impacted by the presence of N-linked glycans, which act primarily by interacting with the LBD bottom lobe to stabilize the closed LBD.

biophysics↗

bioRxiv2021

Finding Druggable Sites in Proteins using TACTICS

Structure-based drug discovery efforts require knowledge of where drug-binding sites are located on target proteins. To address the challenge of finding druggable sites, we developed a machine-learning algorithm called TACTICS (Trajectory-based Analysis of Conformations To Identify Cryptic Sites), which uses an ensemble of molecular structures (such as molecular dynamics simulation data) as input. First, TACTICS uses k-means clustering to select a small number of conformations that represent the overall conformational heterogeneity of the data. Then, TACTICS uses a random forest model to identify potentially bindable residues in each selected conformation, based on protein motion and geometry. Lastly, residues in possible binding pockets are scored using fragment docking. As proof-of-principle, TACTICS was applied to the analysis of simulations of the SARS-CoV-2 main protease and methyltransferase and the Yersinia pestis aryl carrier protein. Our approach recapitulates known small-molecule binding sites and predicts the locations of sites not previously observed in experimentally determined structures. The TACTICS code is available at https://github.com/Albert-Lau-Lab/tactics_protein_analysis.

biophysics↗