Biology subjects

Sansom, M.

Publications and source records attributed to Sansom, M..

At least 19 recordsLinked to original sources

bioRxiv2024

The Role of Cholesterol in M2 Clustering and Viral Budding Explained

The proton-conducting domain of the influenza A M2 homotetrameric channel (M2TM-AH; residues 22-62), consisting of four transmembrane (TM; residues 22-46) and four amphipathic helices (AHs; residues 47-62), promotes the release of viral RNA via acidification. Previous studies have also proposed the formation of clusters of M2 channels in the budding neck areas in raft-like domains of the plasma membrane, 1,2 which are rich in cholesterol, resulting in cell membrane scission and viral release. Experiments showed that cholesterol has a significant contribution to lipid bilayer undulations in viral buds suggesting a significant role for cholesterol in the budding process. However, a clear explanation of membrane curvature effect based on the distribution of cholesterol around M2TM-AH clusters is lacking. Using coarse-grained molecular dynamics simulations of M2TM-AH in bilayers, we observed that M2 channels form specific clusters with conical shapes, driven by attraction of their amphipathic helices (AHs). We showed that cholesterol stabilized the formation of M2 channel clusters, by filling and bridging the conical gap between M2 channels at specific sites in the N-terminals of adjacent channels or via the C-terminal region of TM and AHs, the latter sites displaying longer interaction time and higher stability. Potential of mean force calculations showed that when cholesterols occupy the identified interfacial binding sites between two M2 channels, the dimer is stabilized by 11 kJ/mol. This translates to the cholesterol-bound dimer being populated by almost two orders of magnitude compared to a dimer lacking cholesterol. We demonstrated that the cholesterol bridged M2 channels can exert lateral force on the surrounding membrane to induce the necessary negative Gaussian curvature profile which permits the spontaneous scission of the catenoid membrane neck and leads to viral buds and scission. Significance StatementA key role of influenza A M2 channel, a prototype viroporin, is the generation of curvature in the host cell membrane to form budding necks leading to scission and release of the virions. It has been shown experimentally that this process is mediated through the amphipathic helices of M2 and that cholesterol enhanced lipid bilayer undulations in viral budding. Here, using coarse-grained molecular dynamics simulations we revealed that M2 channels form clusters in membranes, induced in an additive manner by both the AHs and cholesterol, which in turn increase local membrane curvature. We showed that both cluster formation and the induced membrane curvature are enhanced by cholesterol by bridging the conical area between M2 channels.

biophysics↗

bioRxiv2023

Electronic Polarizability Tunes the Function of the Human Bestrophin 1 Cl- Channel

Mechanisms of anion permeation within ion channels and nanopores remain poorly understood. Recent cryo-electron microscopy structures of the human bestrophin 1 Cl- channel (hBest1) provide an opportunity to evaluate ion interactions predicted by molecular dynamics (MD) simulations against experimental observations. Here, we implement the fully polarizable forcefield AMOEBA in MD simulations on different conformations of hBest1. This forcefield models multipole moments up to the quadrupole; therefore, it captures induced dipole and anion-{pi} interactions. We show that key biophysical properties of the channel can only be simulated when electronic polarization is included in the molecular models and that Cl- permeation through the neck of the pore is achieved through hydrophobic solvation concomitant with partial ion dehydration. Furthermore, we demonstrate how such polarizable simulations can help determine the identity of ion-like densities within high-resolution cryo-EM structures and that neglecting polarization places Cl- at positions that do not correspond with their experimentally resolved location. Overall, our results demonstrate the importance of including electronic polarization in realistic and physically accurate models of biological systems, especially channels and pores that selectively permeate anions. Statement of SignificanceIon channels are nanoscale protein pores that enable the selective passage of charged ions across cell membranes. Understanding the underlying mechanisms for selective anion permeation through such pores remains a challenge. To simulate their behavior efficiently in silico, fixed charge models are typically employed. However, this approach is insufficient for the study of anions. Here, we use simulations with explicit treatment of electrostatics to investigate the interactions of chloride ions in the human bestrophin 1 channel. We find that electronic polarization tunes the state of the channel and affects the interactions of chloride ions thereby revealing a mechanism for permeation. Furthermore, these simulations can be used to resolve experimental ambiguity in ion-like densities from cryo-EM structures.

biophysics↗

bioRxiv2023

The Energetics and Ion Coupling of Cholesterol Transport Through Patched1

Patched1 (PTCH1) is the principal tumour suppressor protein of the mammalian Hedgehog (HH) signalling pathway, implicated in embryogenesis and tissue homeostasis. PTCH1 inhibits the Class F G protein-coupled receptor Smoothened (SMO) via a debated mechanism involving modulating accessible cholesterol levels within ciliary membranes. Using extensive molecular dynamics (MD) simulations and free energy calculations to evaluate cholesterol transport through PTCH1, we find an energetic barrier of ~15-20 kJ mol-1 for cholesterol export. In simulations we identify cation binding sites within the PTCH1 transmembrane domain (TMD) which may provide the energetic impetus for cholesterol transport. In silico data are coupled to in vivo biochemical assays of PTCH1 mutants to probe coupling between transmembrane motions and PTCH1 activity. Using complementary simulations of Dispatched1 (DISP1) we find that transition between inward-open and solvent occluded states is accompanied by Na+ induced pinching of intracellular helical segments. Thus, our findings illuminate the energetics and ion-coupling stoichiometries of PTCH1 transport mechanisms, whereby 1-3 Na+ or 2-3 K+ couple to cholesterol export, and provide the first molecular description of transitions between distinct transport states.

biophysics↗

bioRxiv2023

Structure of the Native Chemotaxis Core Signalling Unit from E-gene lysed E. coli cells

Motile bacteria detect ambient chemical gradients and control their locomotion via conserved chemotaxis signaling networks, enabling cells to locate nutrients, potential hosts and other important biological niches. The sensory apparatus of the chemotaxis pathway is an array of core-signaling units (CSU) composed of transmembrane chemoreceptors, the histidine kinase CheA and an adaptor protein CheW. Although chemotaxis pathways represent the best understood signaling systems, a detailed mechanistic understanding of signal transduction has been hindered by the lack of a complete structural picture of the CSU and extended array. In this study, we present the structure of the complete CSU from phage E-gene lysed E. coli cells, determined using cryo-electron tomography and sub-tomogram averaging to 12 [A] resolution. Using AlphaFold2, we further predict the atomic structures of the CSUs constituent proteins as well as key protein-protein interfaces, enabling the assembly an all-atom CSU model, which we conformationally refine using our cryoET map. Molecular dynamics simulations of the resulting model provide new insight into the periplasmic organization of the complex, including novel interactions between neighboring receptor ligand binding domains. Our results further elucidate previously unresolved interactions between individual CheA domains, including an anti-parallel P1 dimer and non-productive binding mode between P1 and P4, enhancing our understanding of the structural mechanisms underlying CheA signaling and regulation.

microbiology↗

bioRxiv2023

Influence of Electronic Polarization on the Binding of Anions to a Chloride-Pumping Rhodopsin

The functional properties of some biological ion channels and membrane transport proteins are proposed to exploit anion-hydrophobic interactions. Here, we investigate a chloride-pumping rhodopsin (ClR) as an example of a membrane protein known to contain a defined anion binding site composed predominantly of hydrophobic residues. Using molecular dynamics simulations, we explore Cl- binding to this hydrophobic site and compare the dynamics arising when electronic polarization is neglected (CHARMM36 (c36) fixed-charge force field), included implicitly (via the prosECCo force field), or included explicitly (through the polarizable force field, AMOEBA). Free energy landscapes of Cl- moving out of the binding site and into bulk solution demonstrate that the inclusion of polarization results in stronger ion binding and a second metastable binding site in ClR. Simulations focused on this hydrophobic binding site also indicate longer binding durations and closer ion proximity when polarization is included. Furthermore, simulations reveal that Cl- within this binding site interacts with an adjacent loop to facilitate rebinding events that are not observed when polarization is neglected. These results demonstrate how the inclusion of polarization can influence the behavior of anions within protein binding sites and thereby reveal novel mechanisms. Statement of SignificanceMolecular simulations based on classical (Newtonian) mechanics represent the most common method of visualizing the behavior of water and ions within channels and nanopores. Although computationally efficient, many of the approximations required mean that these simulations often do not fully capture the complex and dynamic interactions involved. Here, we use the prosECCo force field that offers an improved electronic description whilst maintaining computational efficiency. We show that using this method to include the effects of polarization greatly influences the binding dynamics of anions to a protein binding site and yields results similar to more accurate but computationally demanding methods.

biophysics↗

bioRxiv2022

LipIDens: Simulation assisted interpretation of lipid densities in cryo-EM structures of membrane proteins.

Cryo-electron microscopy (cryo-EM) enables the determination of membrane protein structures in native-like environments. Characterising how membrane proteins interact with the surrounding membrane lipid environment is assisted by resolution of lipid-like densities visible in cryo-EM maps. Nevertheless, establishing the molecular identity of putative lipid and/or detergent densities remains challenging. Here we present LipIDens, a pipeline for molecular dynamics (MD) simulation-assisted interpretation of lipid and lipid-like densities in cryo-EM structures. The pipeline integrates the implementation and analysis of multi-scale MD simulations for identification, ranking and refinement of lipid binding poses which superpose onto cryo-EM map densities. Thus, LipIDens enables direct integration of experimental and computational structural approaches to facilitate the interpretation of lipid-like cryo-EM densities and to reveal the molecular identities of protein-lipid interactions within a bilayer environment. The LipIDens code is open-source and embedded within a notebook format to assist automation and usability.

biophysics↗

bioRxiv2021

Influence of effective polarization on ion and water interactions within a biomimetic nanopore

Interactions between ions and water at hydrophobic interfaces within ion channels and nanopores are suggested to play a key role in the movement of ions across biological membranes. Previous molecular dynamics (MD) simulations have shown that the affinity of polarizable anions to aqueous/hydrophobic interfaces can be markedly influenced by including polarization effects through an electronic continuum correction (ECC). Here, we designed a model biomimetic nanopore to imitate the polar pore openings and hydrophobic gating regions found in pentameric ligand-gated ion channels. MD simulations were then performed using both a non-polarizable force field and the ECC method to investigate the behavior of water, Na+ and Cl- ions confined within the hydrophobic region of the nanopore. Number density distributions revealed preferential Cl- adsorption to the hydrophobic pore walls, with this interfacial layer largely devoid of Na+. Free energy profiles for Na+ and Cl- permeating the pore also display an energy barrier reduction associated with the localization of Cl- to this hydrophobic interface, and the hydration number profiles reflect a corresponding reduction in the first hydration shell of Cl-. Crucially, these ion effects were only observed through inclusion of effective polarization which therefore suggests that polarizability may be essential for an accurate description for the behavior of ions and water within hydrophobic nanoscale pores, especially those that conduct Cl-.

biophysics↗

bioRxiv2021

Hepatocyte cholesterol content modulates glucagon receptor signalling

Glucagon decreases liver fat, and non-alcoholic fatty liver disease (NAFLD) is associated with hepatic glucagon resistance. Increasingly it is recognised that the function of G protein-coupled receptors can be regulated by their local plasma membrane lipid environment. The aim of this study was to evaluate the effects of experimentally modulating hepatocyte cholesterol content on the function of the glucagon receptor (GCGR). We found that glucagon-mediated cAMP production is inversely proportional to cholesterol content of human hepatoma and primary mouse hepatocytes after treatment with cholesterol-depleting and loading agents, with ligand internalisation showing the opposite trend. Mice fed a high cholesterol diet had increased hepatic cholesterol and a blunted hyperglycaemic response to glucagon, both of which were partially reversed by simvastatin. Molecular dynamics simulations identified potential membrane-exposed cholesterol binding sites on the GCGR. Overall, our data suggest that increased hepatocyte membrane cholesterol could directly contribute to glucagon resistance in NAFLD.

pharmacology and toxicology↗

bioRxiv2021

Water Nanoconfined in a Hydrophobic Pore: MD Simulations and Water Models

Water molecules within biological ion channels are in a nano-confined environment and therefore exhibit novel behaviours which differ from that of bulk water. Here, we investigate the phenomenon of hydrophobic gating, the process by which a nanopore may spontaneously de-wet to form a vapour lock if the pore is sufficiently hydrophobic and/or narrow. Notably, this occurs without steric occlusion of the pore. Using molecular dynamics simulations with both additive and polarisable (AMOEBA) force fields, we investigate this wetting/de-wetting behaviour in the TMEM175 ion channel. We examine how a range of rigid fixed-charge (i.e. additive) and polarisable water models affect wetting/de-wetting in both the wild-type structure and in mutants chosen to cover a range of nanopore radii and pore-lining hydrophobicities. Crucially, we find that the rigid fixed-charge water models lead to similar wetting/de-wetting behaviours, but that the polarisable water model resulted in an increased wettability of the hydrophobic gating region of the pore. This has significant implications for molecular simulations of nano-confined water, as it implies that polarisability may need to be included if we are to gain detailed mechanistic insights into wetting/de-wetting processes. These findings are of importance for the design of functionalised biomimetic nanopores (for e.g. sensing or desalination), as well as for furthering our understanding of the mechanistic processes underlying biological ion channel function.

biophysics↗

bioRxiv2021

PyLipID: A Python package for analysis of protein-lipid interactions from MD simulations

Lipids play important modulatory and structural roles for membrane proteins. Molecular dynamics simulations are frequently used to provide insights into the nature of these proteinlipid interactions. Systematic comparative analysis requires tools that provide algorithms for objective assessment of such interactions. We introduce PyLipID, a python package for the identification and characterization of specific lipid interactions and binding sites on membrane proteins from molecular dynamics simulations. PyLipID uses a community analysis approach for binding site detection, calculating lipid residence times for both the individual protein residues and the detected binding sites. To assist structural analysis, PyLipID produces representative bound lipid poses from simulation data, using a density-based scoring function. To estimate residue contacts robustly, PyLipID uses a dual-cutoff scheme to differentiate between lipid conformational rearrangements whilst bound from full dissociation events. In addition to the characterization of protein-lipid interactions, PyLipID is applicable to analysis of the interactions of membrane proteins with other ligands. By combining automated analysis, efficient algorithms, and open-source distribution, PyLipID facilitates the systematic analysis of lipid interactions from large simulation datasets of multiple species of membrane proteins. ToC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/452312v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1f6530corg.highwire.dtl.DTLVardef@c639d5org.highwire.dtl.DTLVardef@1fef967org.highwire.dtl.DTLVardef@a80446_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Allosteric activation of T cell antigen receptor signalling by quaternary structure relaxation

The mechanism of T cell antigen receptor (TCR-CD3) signalling remains elusive. Here, we identified mutations in the transmembrane region of TCR{beta} or CD3{zeta} that augmented pMHC-induced signalling, not explicable by enhanced ligand binding, lateral diffusion, clustering or co-receptor function. Using a novel biochemical assay and molecular dynamics simulation, we demonstrated that the gain-of-function mutations loosened interaction between TCR{beta} and CD3{zeta}. We found that, similar to the activating mutations, pMHC binding reduced TCR{beta} cohesion with CD3{zeta}. This event occurred prior to CD3{zeta} phosphorylation and at 0{degrees}C. Moreover, we demonstrated that soluble monovalent pMHC alone induced signalling and reduced TCR{beta} cohesion with CD3{zeta} in membrane-bound or solubilised TCR-CD3. Our data provide compelling evidence that pMHC binding suffices to activate allosteric changes propagating from TCR{beta} to the CD3 subunits, reconfiguring interchain transmembrane region interactions. These dynamic modifications could change the arrangement of TCR-CD3 boundary lipids to licence CD3{zeta} phosphorylation and initiate signal propagation.

immunology↗

bioRxiv2020

Binding of Ca2+-Independent C2 Domains to Lipid Membranes: a Multi-Scale Molecular Dynamics Study

C2 domains facilitate protein-lipid interaction in cellular recognition and signalling processes. They possess a {beta}-sandwich structure, with either type I or type II topology. C2 domains can interact with anionic lipid bilayers in either a Ca2+-dependent or a Ca2+-independent manner. The mechanism of recognition of anionic lipids by Ca2+-independent C2 domains is incompletely understood. We have used molecular dynamics (MD) simulations to explore the membrane interactions of six Ca2+- independent C2 domains, from KIBRA, PI3KC2, RIM2, PTEN, SHIP2, and Smurf2. In coarse grained MD simulations these C2 domains bound to lipid bilayers, forming transient interactions with zwitterionic (phosphatidylcholine, PC) bilayers compared to long lived interactions with anionic bilayers also containing either phosphatidylserine (PS) or PS and phosphatidylinositol bisphosphate (PIP2). Type I C2 domains bound non-canonically via the front, back or side of the {beta} sandwich, whereas type II C2 domains bound canonically, via the top loops (as is typically the case for Ca2+-dependent C2 domains). C2 domains interacted strongly (up to 120 kJ/mol) with membranes containing PIP2 causing the bound anionic lipids to clustered around the protein. The C2 domains bound less strongly to anionic membranes without PIP2 (<50 kJ/mol), and most weakly to neutral membranes (<33 kJ/mol). Productive binding modes were identified and further analysed in atomistic simulations. For PTEN and SHIP2, CG simulations were also performed of the intact enzymes (i.e. phosphatase domain plus C2 domain) with PIP2-contating bilayers and the roles of the two domains in membrane localization were compared. From a methodological perspective, these studies establish a multiscale simulation protocol for studying membrane binding/recognition proteins, capable of revealing binding modes alongside details of lipid binding affinity and specificity.

biophysics↗

bioRxiv2020

The guidance and adhesion protein FLRT2 dimerizes in cis via dual Small-X3-Small transmembrane motifs

Fibronectin Leucine-rich Repeat Transmembrane (FLRT 1-3) proteins are a family of broadly expressed single-spanning transmembrane receptors that play key roles in development. Their extracellular domains mediate homotypic cell-cell adhesion and heterotypic protein interactions with other receptors to regulate cell adhesion and guidance. These in trans FLRT interactions determine the formation of signaling complexes of varying complexity and function. Whether FLRTs also interact at the surface of the same cell, in cis, remains unknown. Here, molecular dynamics simulations reveal two dimerization motifs in the FLRT2 transmembrane helix. Single particle tracking experiments show that these Small-X3-Small motifs synergize with a third dimerization motif encoded in the extracellular domain to permit the cis association and co-diffusion patterns of FLRT2 receptors on cells. These results may point to a competitive switching mechanism between in cis and in trans interactions which suggests that homotypic FLRT interaction mirrors the functionalities of classic adhesion molecules. FieldsStructural Biology and Biophysics / Computational Biology

biophysics↗

bioRxiv2020

GPCR Oligomerisation Modulation by Conformational State and Lipid Interactions Revealed by MD Simulations and Markov Models

G protein-coupled receptors (GPCRs) play key roles in cellular signalling. GPCRs are suggested to form dimers and higher order oligomers in response to activation. However, we do not fully understand GPCR activation at larger scales and in an in vivo context. We have characterised oligomeric configurations of the adenosine 2a receptor (A2aR) by combining large-scale molecular dynamics simulations with Markov state models. Receptor activation results in enhanced oligomerisation, more diverse oligomer populations, and a more connected oligomerisation network. The active state conformation of the A2aR shifts protein-protein association interfaces to those involving intracellular loop ICL3 and transmembrane helix TM6. Binding of PIP2 to A2aR stabilises protein-protein interactions via PIP2-mediated association interfaces. These results indicate that A2aR oligomerisation is responsive to the local membrane lipid environment. This in turn suggests a modulatory effect on A2aR whereby a given oligomerisation profile favours the dynamic formation of specific supra-molecular signalling complexes.

biophysics↗

bioRxiv2020

Membrane Interactions of α-Synuclein Revealed by Multiscale Molecular Dynamics Simulations, Markov State Models, and NMR

-Synuclein is a presynaptic protein that binds to cell membranes and is linked to Parkinsons disease (PD). Whilst the normal function of remains -synuclein remains uncertain, it is thought that oligomerization of the protein on the cell membrane contributes to cell damage. Knowledge of how -synuclein binds to lipid bilayers is therefore of great interest as a likely first step in the molecular pathophysiology of PD, and may provide insight of the phenotype of PD-promoting mutations. We use coarse-grained and atomistic simulations in conjunction with NMR and cross-linking mass spectrometry studies of -synuclein bound to anionic lipid bilayers to reveal a break in the helical structure of the NAC region, which may give rise to subsequent oligomer formation. Coarse-grained simulations of -synuclein show that the interhelical region leads recognition and binding to both POPG and mixed composition bilayers and identifies important protein-lipid contacts, including those in the region between the two helices in the folded structure. We extend these simulations with all-atom simulations of the initial binding event to reveal details of the time-progression of lipid binding. We present secondary structure analysis that reveals points of possible {beta}-strand formation in the structure, and investigate intramolecular contacts with simulations and mass-spectrometry crosslinking. Additionally we show how Markov state models can be used to investigate possible conformational changes of membrane bound -synuclein in the NAC region, and we extract representative structures. These structural insights will aid the design and development of novel therapeutic approaches.

biophysics↗

bioRxiv2020

Electric field induced wetting of a hydrophobic gate in a model nanopore based on the 5-HT3 receptor channel

In this study we examined the influence of a transmembrane voltage on the hydrophobic gating of nanopores using molecular dynamics simulations. We observed electric field induced wetting of a hydrophobic gate in a biologically inspired model nanopore based on the 5-HT3 receptor in its closed state, with a field of at least [~]100 mV nm-1 was required to hydrate the pore. We also found an unequal distribution of charged residues can generate an electric field intrinsic to the nanopore which, depending on its orientation, can alter the effect of the external field, thus making the wetting response asymmetric. This wetting response could be described by a simple model based on water surface tension, the volumetric energy contribution of the electric field, and the influence of charged amino acids lining the pore. Finally, the electric field response was used to determine time constants characterising the phase transitions of water confined within the nanopore, revealing liquid-vapour oscillations on a time scale of ~5 ns. This time scale was largely independent of the water model employed and was similar for different sized pores representative of the open and closed states of the pore. Furthermore, our finding that the threshold voltage required for hydrating a hydrophobic gate depends on the orientation of the electric field provides an attractive perspective for the design of rectifying artificial nanopores. ToC/Abstract Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/114157v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@c75d59org.highwire.dtl.DTLVardef@c1a884org.highwire.dtl.DTLVardef@1e81e2org.highwire.dtl.DTLVardef@e13b43_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Modified Bacterial Lipids Which Alter Membrane Surface Charge Reduce Binding of Antimicrobial Peptides

Antimicrobial peptides (AMPs) initiate killing of bacteria by binding to and destabilizing their membranes. The multiple peptide resistance factor (MprF) provides a defence mechanism for bacteria against a broad range of AMPs. MprF reduces the negative charge of both Gram-positive and Gram--negative bacterial membranes through enzymatic conversion of the anionic lipid phosphatidyl glycerol (PG) to either zwitterionic alanyl-phosphatidyl glycerol (Ala-PG) or cationic lysylphosphatidyl glycerol (Lys-PG). The resulting change in membrane charge is suggested to reduce AMP-membrane binding and hinder downstream AMP activity. Using molecular dynamics to investigate the effects of these modified lipids on AMP-binding to model membranes, we show that AMPs have substantially reduced affinity for model membranes containing Ala-PG or Lys-PG. A total of ~7000 simulations are used to define the relationship between bilayer composition and binding for 5 different membrane active peptides. The reduction of degree of interaction of a peptide with the membrane is shown to correlate with the change in membrane surface charge density. Free energy profile (potential of mean force) calculations reveal that these lipid modifications alter the energy barrier to peptide helix penetration of the bilayer. These results will enable us to guide design of novel peptides which address the issue of resistance via MprF-mediated membrane modification.

biophysics↗

bioRxiv2020

The Glycosphingolipid GM3 Modulates Conformational Dynamics of the Glucagon Receptor

The extracellular domain (ECD) of Class B1 G-protein coupled receptors (GPCRs) plays a central role in signal transduction and is uniquely positioned to sense both the extracellular and membrane environments. Whilst recent studies suggest a role for membrane lipids in the modulation of Class A and Class F GPCR signalling properties, little is known about the effect of lipids on Class B1 receptors. In this study, we employed multiscale molecular dynamics (MD) simulations to access the dynamics of the glucagon receptor (GCGR) ECD in the presence of native-like membrane bilayers. Simulations showed that the ECD could move about a hinge region formed by residues Q122-E126 to adopt both closed and open conformations relative to the TMD. ECD movements were modulated by binding of the glycosphingolipid GM3. These large-scale fluctuations in ECD conformation that may affect the ligand binding and receptor activation properties. We also identify a unique PIP2 interaction profile near ICL2/TM3 at the G-protein coupling interface, suggesting a mechanism of engaging G-proteins which may have a distinct dependence on PIP2 compared to Class A GPCRs. Given the structural conservation of Class B1 GPCRs, the modulatory effects of GM3 and PIP2 on GCGR may be conserved across these receptors, offering new insights into potential therapeutic targeting. Statement of SignificanceThe role of lipids in regulation of Class B GPCRs remains elusive, despite recent structural advances. In this study, multi-scale molecular dynamics simulations are used to evaluate lipid interactions with the glucagon receptor, a Class B1 GPCR. We find that the glycosphingolipid GM3 binds to the glucagon receptor extracellular domain (ECD), modulating the dynamics of the ECD and promoting movement away from the transmembrane domain. We also identify a unique PIP2 interaction fingerprint in a region known to be important for bridging G-protein coupling in Class A GPCRs. Thus, this study provides molecular insight into the behaviour of the glucagon receptor in a complex lipid bilayer environment which may aid understanding of glucagon receptor signalling properties.

biophysics↗