Biology subjects

Biggin, P. C.

Publications and source records attributed to Biggin, P. C..

14 recordsLinked to original sources

bioRxiv2026

Structural mechanism of TRPC3 inhibition by a potent and selective antagonist

The transient receptor channel canonical family member 3 (TRPC3) is a non-selective, calcium-permeable cation channel within the larger TRP superfamily. TRPC3 is expressed in both excitable and non-excitable cells, where it integrates multiple signaling pathways and is directly activated by binding of diacylglycerol following activation of G-protein coupled receptors. Increased TRPC3 signaling is implicated in several diseases, highlighting TRPC3 as a potential therapeutic target. However, the development of TRPC3 inhibitors with suitable potency and selectivity has been challenging due to the channels high structural and sequence homology with related family members, particularly TRPC6. Here, we present a novel, selective and highly potent TRPC3 antagonist (GSK2820986A) featuring an anilino-thiazole pharmacophore. Through a combination of molecular dynamic simulations and functional assays, we identify a putative binding site for GSK2820986A in the S4-S5 pocket and propose a potential inhibitory mechanism. Our findings provide a powerful tool compound and mechanistic framework to advance the investigation and therapeutic targeting of TRPC3 in disease.

biochemistry↗

bioRxiv2025

Structural determinants for GqPCR-mediated inhibition of TASK K2P K+ channels and their dysfunction in disease

Two-Pore Domain K+(K2P) channels are crucial determinants of cellular electrical excitability. TASK-1 and TASK-3 K2P channels regulate the resting membrane potential in many different cell types where their activity is also coupled to GqPCR signalling pathways via direct inhibition by diacylglycerol (DAG) generated as a result of phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis. This regulation is defective in two different TASK channelopathies, but the molecular mechanisms underlying this inhibition remain unclear. Here, we demonstrate that DAG inhibition of TASK channel activity is state-dependent. Single channel recordings show that the sensitivity to GqPCR inhibition inversely correlates with channel open probability and that DAG destabilises the open state of TASK-1 to promote channel closure. Combining Molecular Dynamics simulations with mutagenesis studies, we also identify a binding site for DAG in a groove between the M2, M3 and M4 domains, and highlight the crucial role of a specific residue within on M4 (T230) in mediating this inhibitory effect as well as defining the difference in GPCR sensitivity between TASK-1 and TASK-3. Together, these results provide a better understanding of the molecular mechanisms underlying GqPCR regulation of TASK channels and the pathogenic effect of K2P channelopathies linked to TASK channel dysregulation.

biophysics↗

bioRxiv2025

Steric control of signaling bias in the immunometabolic receptor GPR84

Biased signaling in G protein-coupled receptors offers therapeutic promise, yet rational design of biased ligands remains challenging due to limited mechanistic understanding. Here, we report a molecular framework for controlling signaling bias at the immunometabolic receptor GPR84. We identified three structurally-matched ligands (OX04529, OX04954, and OX04539) with varying steric profiles that exhibit comparable Gi protein activation but dramatically different {beta}-arrestin recruitment capacities. A high- resolution cryo-EM structure of GPR84-Gi in complex with OX04529, complemented by molecular dynamics simulations and targeted mutagenesis, revealed that steric interactions between ligand substituents and Leu3366.52 and Phe1875.47 indirectly disrupt a critical polar network involving Tyr3326.48, Asn1043.36 and Asn3627.45 essential for {beta}-arrestin recruitment. Based on these insights, we developed a steric-dependent model that enabled rational design of G protein-biased agonists with predictable {beta}-arrestin recruitment profiles. This mechanistic framework provides a blueprint for designing biased agonists with customized signaling profiles at GPR84 and potentially other class A GPCRs.

pharmacology and toxicology↗

bioRxiv2025

Allosteric Control of Super-Agonism in a Ligand-Gated Ion Channel

Ligand-gated ion channels open in response to the binding of an agonist. The agonist binding site is typically located tens of Angstroms away from the channel gate, which lies within the membrane and thus the gating mechanism is considered a classic example of allostery within proteins. The multi-subunit nature of these proteins also means that modulatory effects on the gating process can also be mediated by several other distinct regions - so called allosteric modulatory sites. One of the most well-studied channels in this regard, is the nicotinic acetylcholine receptor. Super-agonists are compounds that can produce a greater maximal response than the endogenous ligand (acetylcholine in this case). They are able to stabilize the open state of the ion channel and this can have important consequences for neuronal signaling. Some super-agonist effects can be mediated through the orthosteric binding site, but others can be mediated by alternative binding sites. The latter are often much harder to identify and can depend very precisely on the subunit composition of the receptor. In this work we sought to identify the mechanism by which TC-2559, a known super-agonist that acts as such only at one particular combination of neuronal nicotinic acetylcholine receptors - the high sensitivity receptor which is comprised of 2 alpha subunits and 3 beta subunits (as opposed to the low sensitivity receptor which has 3 alpha and 2 beta subunits). By using advanced computational methods, supported by two-voltage electrode clamp experiments, we were able to show that TC-2559 not only binds to the orthosteric but also binds to the unique b2-b2 interface of the HS receptor. The binding of TC-2559 to this interface exerts unique interactions that other agonists are not able to make, but more importantly it induces changes in the interface that support that concept of an allosteric gain in overall efficacy. Our results highlight how allosteric control exists to modulate receptor function.

biochemistry↗

bioRxiv2024

Exploring the Influence of Pore Shape on Conductance and Permeation

There are increasing numbers of ion channel structures featuring heteromeric subunit assembly, exemplified by synaptic 1{beta}B Glycine and 4{beta}2 Nicotinic receptors. These structures exhibit inherent pore asymmetry, but the relevance of this to function is unknown. Furthermore, molecular dynamics simulations performed on symmetrical homomeric channels often leads to thermal distortion whereby conformations of the resulting ensemble are also asymmetrical. When functionally annotating ion channels, researchers often rely on minimal constrictions determined via radius-profile calculations performed with computer programs, such as HOLE or CHAP, coupled with an assessment of pore hydrophobicity. However, such tools typically employ spherical probe particles, limiting their ability to accurately capture pore asymmetry. Here, we introduce an algorithm that employs ellipsoidal probe particles, enabling a more comprehensive representation of the pore geometry. Our analysis reveals that the use of non-spherical ellipsoids for pore characterization, provides a more accurate and easily interpretable depiction of conductance. To quantify the implications of pore asymmetry on conductance, we systematically investigated carbon nanotubes (CNTs) with varying degrees of pore asymmetry as model systems. The conductance through these channels shows surprising effects that would otherwise not be predicted with spherical probes. The results have broad implications not only for the functional annotation of biological ion channels, but also for the design of synthetic channel systems for use in areas such as water filtration. Furthermore, we make use of the more accurate characterization of channel pores to refine a physical conductance model to obtain a heuristic estimate for single channel conductance. The code is freely available, obtainable as pip-installable python package and provided as a webservice.

biophysics↗

bioRxiv2023

Small molecule positive allosteric modulation of homomeric kainate receptors GluK1-3: Development of screening assays and insight into GluK3 structure

The kainate receptors GluK1-3 belong to the family of ionotropic glutamate receptors and are essential for fast excitatory neurotransmission in the brain and associated with neurological and psychiatric diseases. How these receptors can be modulated by small molecule agents is not well-understood, especially for GluK3. We show that the positive allosteric modulator BPAM344 can be used to establish robust calcium-sensitive fluorescence-based assays at GluK1-3 for testing agonists, antagonists, and positive allosteric modulators. The EC50 of BPAM344 for potentiating the response of 100 {micro}M kainate was determined to 26.3 {micro}M at GluK1, 75.4 {micro}M at GluK2, and 639 {micro}M at GluK3. In the presence of 150 {micro}M BPAM344, domoate was found to be a potent agonist at GluK1 and GluK2 with EC50 of 0.77 {micro}M and 1.33 {micro}M, respectively. At GluK3, domoate acts as a very weak agonist or antagonist with IC50 of 14.5 {micro}M, in the presence of 500 {micro}M BPAM344 and 100 {micro}M kainate. Using H523A mutated GluK3, we determined the first dimeric structure of the ligand-binding domain by X-ray crystallography, allowing location of BPAM344, zinc, sodium, and chloride ion binding sites at the dimer interface. Molecular dynamics simulations support the stability of the ion sites as well as the involvement of Asp761, Asp790, and Glu797 in binding of zinc ions. Using electron microscopy, we show that in the presence of glutamate and BPAM344, full-length GluK3 adopts a dimer-of-dimers arrangement. This study may contribute to unravelling the potential of kainate receptors as targets for treatment of brain diseases.

neuroscience↗

bioRxiv2023

Tackling hysteresis in conformational sampling - how to be forgetful with MEMENTO

The structure of proteins has long been recognised to hold the key to understanding and engineering their function, and rapid advances in structural biology (and protein structure prediction) are now supplying researchers with an ever-increasing wealth of structural information. Most of the time, however, structures can only be determined in free energy minima, one at a time. While conformational flexibility may thus be inferred from static end-state structures, their interconversion mechanisms -- a central ambition of structural biology -- are often beyond the scope of direct experimentation. Given the dynamical nature of the processes in question, many studies have attempted to explore conformational transitions using molecular dynamics (MD). However, ensuring proper convergence and reversibility in the predicted transitions is extremely challenging. In particular, a commonly used technique to map out a path from a starting to a target conformation called targeted MD (tMD) can suffer from starting-state dependence (hysteresis) when combined with techniques such as umbrella sampling (US) to compute the free energy profile of a transition. Here, we study this problem in detail on conformational changes of increasing complexity. We also present a new, history-independent approach that we term "MEMENTO" (Morphing End states by Modelling Ensembles with iNdependent TOpologies) to generate paths that alleviate hysteresis in the construction of conformational free energy profiles. MEMENTO utilises template-based structure modelling to restore physically reasonable protein conformations based on coordinate interpolation (morphing) as an ensemble of plausible intermediates, from which a smooth path is picked. We compare tMD and MEMENTO on well-characterized test cases (the toy peptide deca-alanine and the enzyme adenylate kinase) before discussing its use in more complicated systems (the kinase P38 and the bacterial leucine transporter LeuT). Our work shows that for all but the simplest systems tMD paths should not in general be used to seed umbrella sampling or related techniques, unless the paths are validated by consistent results from biased runs in opposite directions. MEMENTO, on the other hand performs well as a flexible tool to generate intermediate structures for umbrella sampling. We also demonstrate that extended end-state sampling combined with MEMENTO can aid the discovery of collective variables on a case-by-case basis. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/525919v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@a5de3corg.highwire.dtl.DTLVardef@1a17305org.highwire.dtl.DTLVardef@867721org.highwire.dtl.DTLVardef@1f73798_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Binding and activation of serotonergic G-protein coupled receptors by the multimodal antidepressant vortioxetine.

G-protein coupled receptors are important pharmacological targets. Despite substantial progress, important questions still remain concerning the details of activation: how can a ligand act as an agonist in one receptor, but as an antagonist in a homologous receptor, and how can agonists activate a receptor despite lacking polar functional groups able to interact with helix 5? Studying vortioxetine, an important multimodal antidepressant drug, may elucidate both questions. Herein, we present a thorough in silico analysis of vortioxetine binding to 5-HT1A, 5-HT1B, and 5-HT7 receptors and compare to available experimental data. We are able to rationalize the differential mode of action of vortioxetine at different receptors, but also, in the case of the 5-HT1A receptor, we observe the initial steps of activation suggesting that interaction with helix 5 does not necessarily require a hydrogen bond as previously suggested. The results extend our current understanding of agonist and antagonist action at GPCRs.

biophysics↗

bioRxiv2020

The KDEL Trafficking Receptor Exploits pH to Tune the Strength of an Unusual Short Hydrogen Bond.

The endoplasmic reticulum (ER) is the main site of protein synthesis in eukaryotic cells and requires a high concentration of luminal chaperones to function. During protein synthesis, ER luminal chaperones are swept along the secretory pathway and must be retrieved to maintain cell viability. ER protein retrieval is achieved by the KDEL receptor, which recognises a C-terminal Lys-Asp-Glu-Leu (KDEL) sequence. Recognition of ER proteins by the KDEL receptor is pH dependent, with binding occurring under acidic conditions in the Golgi and release under conditions of higher pH in the ER. Recent crystal structures of the KDEL receptor in the apo and peptide bound state suggested that peptide binding drives the formation of a short-hydrogen bond that locks the KDEL sequence in the receptor and activates the receptor for COPI binding in the cytoplasm. Using quantum mechanical calculations we demonstrate that the strength of this short hydrogen bond is reinforced following protonation of a nearby histidine, linking receptor protonation to high affinity peptide binding. Protonation also controls the wetting of a cavity adjacent to the peptide binding site, leading to a conformational change that ultimately allows the complex to be recognized by the COPI system.

biophysics↗

bioRxiv2020

Structural correlates of human muscle nicotinic acetylcholine receptor subunit assembly mediated by δ(+) interface residues

Muscle nicotinic acetylcholine receptors are a class of heteropentameric ligand-gated cation channels with constituent subunits adopting a fixed stoichiometric arrangement. The specific amino acid residues that govern subunit ordering are however, only partially understood. By integrating all-atom molecular dynamics simulations, bioinformatics, two-electrode voltage clamp electrophysiology and 125I--bungarotoxin assays of chimeric nAChR subunits, we identify residues across the extracellular, transmembrane and extended M4 helix of the {delta} subunit that make structural signatures that contribute to intransigent assembly rules. Furthermore, functional differences observed in 2{delta}2{beta} receptors can be rationalized by changes in dynamical behavior that manifest themselves at the agonist binding site.

neuroscience↗

bioRxiv2020

State-dependent protein-lipid interactions of a pentameric ligand-gated ion channel in a neuronal membrane

Pentameric ligand-gated ion channels (pLGICs) are receptor proteins that are sensitive to their membrane environment, but the mechanism for how lipids modulate function under physiological conditions in a state dependent manner is not known. The glycine receptor is a pLGIC whose structure has been resolved in different functional states. Using a realistic model of a neuronal membrane coupled with coarse-grained molecular dynamics simulations, we demonstrate that the lipid-protein interactions are dependent on the receptor state, suggesting that lipids may regulate the receptors conformational dynamics. Comparison with existing structural data confirms known lipid binding sites, but we also predict further protein-lipid interactions including a site at the communication interface between the extracellular and transmembrane domain. Moreover, in the active state, cholesterol can bind to the binding site of the positive allosteric modulator ivermectin. These protein-lipid interaction sites could in future be exploited for the rational design of lipid-like allosteric drugs. Author SummaryIon channels are proteins that control the flow of ions into the cell. The family of ion channels known as the pentameric ligand gated ion channels (pLGICS) open in response to the binding of a neurotransmitter, moving the channel from a resting state to an open state. The glycine receptor is a pLGIC whose structure has been resolved in different functional states. It is also known that the response of pLGICs can also be modified by different types of lipid found within the membrane itself but exactly how is unclear. Here, we used a realistic model of a neuronal membrane and performed molecular dynamics simulations to show various lipid-protein interactions that are dependent on the channel state. Our work also reveals previously unconsidered protein-lipid interactions at a key junction of the channel known to be critical for the transmission of the opening process. We also demonstrate that cholesterol interacts with the protein at a site already known to bind to another compound that modulates the channel, called ivermectin. The work should be useful for future drug design.

biophysics↗

bioRxiv2019

A Refined Open State of the Glycine Receptor Obtained Via Molecular Dynamics Simulations

Fast neurotransmission is mediated by pentameric ligand-gated ion channels. Glycine receptors are chloride-selective members of this receptor family that mediate inhibitory synaptic transmission and are implicated in neurological disorders including autism and hyperekplexia. They have been structurally characterized by both X-ray crystallography and cryo electron microscopy studies, with the latter giving rise to what was proposed as a possible open state. However, recent work has questioned the physiological relevance of this open state structure, since it rapidly collapses in molecular dynamics simulations. Here, we show that the collapse can be avoided by a careful equilibration protocol that reconciles the more problematic regions of the original electron-density map and gives a stable open state that shows frequent selective chloride permeation. The protocol developed in this work provides a means to refine open-like structures of the whole pentameric ligand-gated ion channel superfamily and reconciles the previous issues with the cryo-EM structure.

biophysics↗

bioRxiv2019

Proton-control of transitions in an amino-acid transporter

Amino acid transport into the cell is often coupled to the proton electrochemical gradient, as found in the solute carrier (SLC) 36 family of proton coupled amino acid transporters (PATs). Although no structure of a human PAT exists, the crystal structure of a related homolog, GkApcT, from bacteria has recently been solved in an inward occluded state and allows an opportunity to examine how protons are coupled to amino acid transport. Our working hypothesis is that release of the amino acid substrate is facilitated by deprotonation of a key glutamate residue (E115), located at the bottom of the binding pocket and which forms part of the intracellular gate, allowing the protein to transition from an inward-occluded to an inward-open conformation. During unbiased molecular dynamics, we observed a transition from the inward-occluded state captured in the crystal structure, to a much more open state, which we consider likely to be representative of the inward-open substrate release state. To explore this and the role of protons in these transitions, we have used umbrella sampling to demonstrate that the transition from inward-occluded to inward-open is more energetically favourable when E115 is deprotonated. That E115 is likely to be protonated in the inward-occluded state and deprotonated in the inward-open state is further confirmed via the use of absolute binding free energies. Finally, we also show, via the use of absolute binding free energy calculations, that the affinity of the protein for alanine is very similar regardless of either the state or the protonation of E115, presumably reflecting key interactions deep within the binding cavity. Together, our results give a detailed picture of the role of protons in driving one of the major transitions in this transporter.\n\nSignificance StatementFor transporter proteins that utilize the proton gradient to drive the uptake of solutes, the precise mechanistic details of proton-coupling remain poorly understood. Structures can only infer the position of protons. All-atom molecular dynamics simulations however, are the ideal complementary tool. Here, we report extensive MD simulations on GkApcT, a proton-coupled transporter. We observe a spontaneous transition from the crystallographically derived inward-occluded state, to an inward-open state, which we then characterise with umbrella sampling and absolute binding free energy calculations. The results suggest that a conserved glutamate is protonated in the inward-occluded state and subsequent deprotonation of this glutamate allows the transporter to move into the inward-open state, thus facilitating substrate release into the cell.

biophysics↗

bioRxiv2018

Substrate conformational dynamics drive structure-specific recognition of gapped DNA by DNA polymerase

DNA-binding proteins utilise different recognition mechanisms to locate their DNA targets. Some proteins recognise specific nucleotide sequences, while many DNA repair proteins interact with specific (often bent) DNA structures. While sequence-specific DNA binding mechanisms have been studied extensively, structure-specific mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I (Pol) substrates both alone and in Pol-DNA complexes. Using a rigid-body docking approach based on a network of 73 distance restraints collected using single-molecule FRET, we determined a novel solution structure of the singlenucleotide-gapped DNA-Pol binary complex. The structure was highly consistent with previous crystal structures with regards to the downstream primer-template DNA substrate; further, our structure showed a previously unobserved sharp bend (~120{degrees}) in the DNA substrate; we also showed that this pronounced bending of the substrate is present in living bacteria. All-atom molecular dynamics simulations and single-molecule quenching assays revealed that 4-5 nt of downstream gap-proximal DNA are unwound in the binary complex. Coarsegrained simulations on free gapped substrates reproduced our experimental FRET values with remarkable accuracy (<{Delta}FRET> = -0.0025 across 34 independent distances) and revealed that the one-nucleotide-gapped DNA frequently adopted highly bent conformations similar to those in the Pol-bound state ({Delta}G < 4 kT); such conformations were much less accessible to nicked (> 7 kT) or duplex (>> 10 kT) DNA. Our results suggest a mechanism by which Pol and other structure-specific DNA-binding proteins locate their DNA targets through sensing of the conformational dynamics of DNA substrates.\n\nSignificance StatementMost genetic processes, including DNA replication, repair and transcription, rely on DNA-binding proteins locating specific sites on DNA; some sites contain a specific sequence, whereas others present a specific structure. While sequence-specific recognition has a clear physical basis, structure-specific recognition mechanisms remain obscure. Here, we use single-molecule FRET and computer simulations to show that the conformational dynamics of an important repair intermediate (1nt-gapped DNA) act as central recognition signals for structure-specific binding by DNA polymerase I (Pol). Our conclusion is strongly supported by a novel solution structure of the Pol-DNA complex wherein the gapped-DNA is significantly bent. Our iterative approach combining precise single-molecule measurements with molecular modelling is general and can elucidate the structure and dynamics for many large biomachines.

biophysics↗