Publications by authors named "Mark S P Sansom"

360 Publications

PyLipID: A Python Package for Analysis of Protein-Lipid Interactions from Molecular Dynamics Simulations.

J Chem Theory Comput 2022 Jan 12. Epub 2022 Jan 12.

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.

Lipids play important modulatory and structural roles for membrane proteins. Molecular dynamics simulations are frequently used to provide insights into the nature of these protein-lipid 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 while 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 data sets of multiple species of membrane proteins.
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http://dx.doi.org/10.1021/acs.jctc.1c00708DOI Listing
January 2022

Structure, mechanism, and inhibition of Hedgehog acyltransferase.

Mol Cell 2021 Dec 9;81(24):5025-5038.e10. Epub 2021 Dec 9.

Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK. Electronic address:

The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.
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http://dx.doi.org/10.1016/j.molcel.2021.11.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8693861PMC
December 2021

Water Nanoconfined in a Hydrophobic Pore: Molecular Dynamics Simulations of Transmembrane Protein 175 and the Influence of Water Models.

ACS Nano 2021 12 16;15(12):19098-19108. Epub 2021 Nov 16.

Department of Biochemistry, University of Oxford, South Parks Road, Oxford, United Kingdom OX1 3QU.

Water molecules within biological ion channels are in a nanoconfined environment and therefore exhibit behaviors which differ from that of bulk water. Here, we investigate the phenomenon of hydrophobic gating, the process by which a nanopore may spontaneously dewet to form a "vapor lock" if the pore is sufficiently hydrophobic and/or narrow. This occurs without steric occlusion of the pore. Using molecular dynamics simulations with both rigid fixed-charge and polarizable (AMOEBA) force fields, we investigate this wetting/dewetting behavior in the transmembrane protein 175 ion channel. We examine how a range of rigid fixed-charge and polarizable water models affect wetting/dewetting 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/dewetting behaviors, but that the polarizable water model resulted in an increased wettability of the hydrophobic gating region of the pore. This has significant implications for molecular simulations of nanoconfined water, as it implies that polarizability may need to be included if we are to gain detailed mechanistic insights into wetting/dewetting processes. These findings are of importance for the design of functionalized biomimetic nanopores (., sensing or desalination) as well as for furthering our understanding of the mechanistic processes underlying biological ion channel function.
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http://dx.doi.org/10.1021/acsnano.1c06443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7612143PMC
December 2021

Influence of water models on water movement through AQP1.

J Chem Phys 2021 Oct;155(15):154502

Universidad Complutense de Madrid, Facultad de Ciencias Fícias, Departamento de Estructura de la Materia, Física Térmica y Electrónica, 28040 Madrid, Spain.

Water diffusion through membrane proteins is a key aspect of cellular function. Essential processes of cellular metabolism are driven by osmotic pressure, which depends on water channels. Membrane proteins such as aquaporins (AQPs) are responsible for enabling water permeation through the cell membrane. AQPs are highly selective, allowing only water and relatively small polar molecules to cross the membrane. Experimentally, estimation of water flux through membrane proteins is still a challenge, and hence, accurate simulations of water permeation are of particular importance. We present a numerical study of water diffusion through AQP1 comparing three water models: TIP3P, OPC, and TIP4P/2005. Bulk diffusion, diffusion permeability, and osmotic permeability are computed and compared among all models. The results show that there are significant differences between TIP3P (a particularly widespread model for simulations of biological systems) and the more recently developed TIP4P/2005 and OPC models. We demonstrate that OPC and TIP4P/2005 reproduce protein-water interactions and dynamics in very good agreement with experimental data. From this study, we find that the choice of the water model has a significant effect on the computed water dynamics as well as its molecular behavior within a biological nanopore.
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http://dx.doi.org/10.1063/5.0063986DOI Listing
October 2021

Membrane-binding mechanism of the EEA1 FYVE domain revealed by multi-scale molecular dynamics simulations.

PLoS Comput Biol 2021 09 23;17(9):e1008807. Epub 2021 Sep 23.

Department of Biochemistry, University of Oxford, Oxford, United Kingdom.

Early Endosomal Antigen 1 (EEA1) is a key protein in endosomal trafficking and is implicated in both autoimmune and neurological diseases. The C-terminal FYVE domain of EEA1 binds endosomal membranes, which contain phosphatidylinositol-3-phosphate (PI(3)P). Although it is known that FYVE binds PI(3)P specifically, it has not previously been described of how FYVE attaches and binds to endosomal membranes. In this study, we employed both coarse-grained (CG) and atomistic (AT) molecular dynamics (MD) simulations to determine how FYVE binds to PI(3)P-containing membranes. CG-MD showed that the dominant membrane binding mode resembles the crystal structure of EEA1 FYVE domain in complex with inositol-1,3-diphospate (PDB ID 1JOC). FYVE, which is a homodimer, binds the membrane via a hinge mechanism, where the C-terminus of one monomer first attaches to the membrane, followed by the C-terminus of the other monomer. The estimated total binding energy is ~70 kJ/mol, of which 50-60 kJ/mol stems from specific PI(3)P-interactions. By AT-MD, we could partition the binding mode into two types: (i) adhesion by electrostatic FYVE-PI(3)P interaction, and (ii) insertion of amphipathic loops. The AT simulations also demonstrated flexibility within the FYVE homodimer between the C-terminal heads and coiled-coil stem. This leads to a dynamic model whereby the 200 nm long coiled coil attached to the FYVE domain dimer can amplify local hinge-bending motions such that the Rab5-binding domain at the other end of the coiled coil can explore an area of 0.1 μm2 in the search for a second endosome with which to interact.
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http://dx.doi.org/10.1371/journal.pcbi.1008807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8491906PMC
September 2021

Relative Affinities of Protein-Cholesterol Interactions from Equilibrium Molecular Dynamics Simulations.

J Chem Theory Comput 2021 Oct 15;17(10):6548-6558. Epub 2021 Sep 15.

Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, U.K.

Specific interactions of lipids with membrane proteins contribute to protein stability and function. Multiple lipid interactions surrounding a membrane protein are often identified in molecular dynamics (MD) simulations and are, increasingly, resolved in cryo-electron microscopy (cryo-EM) densities. Determining the relative importance of specific interaction sites is aided by determination of lipid binding affinities using experimental or simulation methods. Here, we develop a method for determining protein-lipid binding affinities from equilibrium coarse-grained MD simulations using binding saturation curves, designed to mimic experimental protocols. We apply this method to directly obtain affinities for cholesterol binding to multiple sites on a range of membrane proteins and compare our results with free energies obtained from density-based equilibrium methods and with potential of mean force calculations, getting good agreement with respect to the ranking of affinities for different sites. Thus, our binding saturation method provides a robust, high-throughput alternative for determining the relative consequence of individual sites seen in, e.g., cryo-EM derived membrane protein structures surrounded by an array of ancillary lipid densities.
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http://dx.doi.org/10.1021/acs.jctc.1c00547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8515805PMC
October 2021

Identification and assessment of cardiolipin interactions with inner membrane proteins.

Sci Adv 2021 Aug 20;7(34). Epub 2021 Aug 20.

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.

Integral membrane proteins are localized and/or regulated by lipids present in the surrounding bilayer. While bacteria have relatively simple membranes, there is ample evidence that many bacterial proteins bind to specific lipids, especially the anionic lipid cardiolipin. Here, we apply molecular dynamics simulations to assess lipid binding to 42 different inner membrane proteins. Our data reveal an asymmetry between the membrane leaflets, with increased anionic lipid binding to the inner leaflet regions of the proteins, particularly for cardiolipin. From our simulations, we identify >700 independent cardiolipin binding sites, allowing us to identify the molecular basis of a prototypical cardiolipin binding site, which we validate against structures of bacterial proteins bound to cardiolipin. This allows us to construct a set of metrics for defining a high-affinity cardiolipin binding site on bacterial membrane proteins, paving the way for a heuristic approach to defining other protein-lipid interactions.
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http://dx.doi.org/10.1126/sciadv.abh2217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8378812PMC
August 2021

Identification and Characterization of Specific Protein-Lipid Interactions Using Molecular Simulation.

Methods Mol Biol 2021 ;2315:121-139

School of Life Sciences and Department of Chemistry, University of Warwick, Coventry, UK.

Interactions with lipids can dramatically shape and define the activity of membrane proteins. Here, we describe tools that allow the identification of these interactions using molecular dynamics simulation. Additionally, we provide the details of how to use different methods to probe the affinity of these interactions.
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http://dx.doi.org/10.1007/978-1-0716-1468-6_8DOI Listing
August 2021

Modulation of adenosine A2a receptor oligomerization by receptor activation and PIP interactions.

Structure 2021 11 15;29(11):1312-1325.e3. Epub 2021 Jul 15.

Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK. Electronic address:

GPCRs have been shown to form oligomers, which generate distinctive signaling outcomes. However, the structural nature of the oligomerization process remains uncertain. We have characterized oligomeric configurations of the adenosine A2a receptor (A2aR) by combining large-scale molecular dynamics simulations with Markov state models. These oligomeric structures may also serve as templates for studying oligomerization of other class A GPCRs. Our simulation data revealed that receptor activation results in enhanced oligomerization, more diverse oligomer populations, and a more connected oligomerization 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 PIP to A2aR stabilizes protein-protein interactions via PIP-mediated association interfaces. These results indicate that A2aR oligomerization is responsive to the local membrane lipid environment. This, in turn, suggests a modulatory effect on A2aR whereby a given oligomerization profile favors the dynamic formation of specific supramolecular signaling complexes.
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http://dx.doi.org/10.1016/j.str.2021.06.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8581623PMC
November 2021

Allosteric activation of T cell antigen receptor signaling by quaternary structure relaxation.

Cell Rep 2021 07;36(2):109375

T-cell signalling laboratory, Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK. Electronic address:

The mechanism of T cell antigen receptor (TCR-CD3) signaling remains elusive. Here, we identify mutations in the transmembrane region of TCRβ or CD3ζ that augment peptide T cell antigen receptor (pMHC)-induced signaling not explicable by enhanced ligand binding, lateral diffusion, clustering, or co-receptor function. Using a biochemical assay and molecular dynamics simulation, we demonstrate that the gain-of-function mutations loosen the interaction between TCRαβ and CD3ζ. Similar to the activating mutations, pMHC binding reduces TCRαβ cohesion with CD3ζ. This event occurs prior to CD3ζ phosphorylation and at 0°C. Moreover, we demonstrate that soluble monovalent pMHC alone induces signaling and reduces TCRαβ cohesion with CD3ζ in membrane-bound or solubilised TCR-CD3. Our data provide compelling evidence that pMHC binding suffices to activate allosteric changes propagating from TCRαβ to the CD3 subunits, reconfiguring interchain transmembrane region interactions. These dynamic modifications could change the arrangement of TCR-CD3 boundary lipids to license CD3ζ phosphorylation and initiate signal propagation.
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http://dx.doi.org/10.1016/j.celrep.2021.109375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8293630PMC
July 2021

Binding of Ca-independent C2 domains to lipid membranes: A multi-scale molecular dynamics study.

Structure 2021 10 2;29(10):1200-1213.e2. Epub 2021 Jun 2.

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Electronic address:

C2 domains facilitate protein interactions with lipid bilayers in either a Ca-dependent or -independent manner. We used molecular dynamics (MD) simulations to explore six Ca-independent C2 domains, from KIBRA, PI3KC2α, RIM2, PTEN, SHIP2, and Smurf2. In coarse-grained MD simulations these C2 domains formed transient interactions with zwitterionic bilayers, compared with longer-lived interactions with anionic bilayers containing phosphatidylinositol bisphosphate (PIP). Type I C2 domains bound non-canonically via the front, back, or side of the β sandwich, whereas type II C2 domains bound canonically, via the top loops. C2 domains interacted strongly with membranes containing PIP, causing bound anionic lipids to cluster around the protein. Binding modes were refined via atomistic simulations. For PTEN and SHIP2, CG simulations of their phosphatase plus C2 domains with PIP-containing bilayers were also performed, and the roles of the two domains in membrane localization compared. These studies establish a simulation protocol for membrane-recognition proteins.
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http://dx.doi.org/10.1016/j.str.2021.05.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8507603PMC
October 2021

Switching Cytolytic Nanopores into Antimicrobial Fractal Ruptures by a Single Side Chain Mutation.

ACS Nano 2021 06 22;15(6):9679-9689. Epub 2021 Apr 22.

National Physical Laboratory, Hampton Road, Teddington, TW11 0LW, U.K.

Disruption of cell membranes is a fundamental host defense response found in virtually all forms of life. The molecular mechanisms vary but generally lead to energetically favored circular nanopores. Here, we report an elaborate fractal rupture pattern induced by a single side-chain mutation in ultrashort (8-11-mers) helical peptides, which otherwise form transmembrane pores. In contrast to known mechanisms, this mode of membrane disruption is restricted to the upper leaflet of the bilayer where it exhibits propagating fronts of peptide-lipid interfaces that are strikingly similar to viscous instabilities in fluid flow. The two distinct disruption modes, pores and fractal patterns, are both strongly antimicrobial, but only the fractal rupture is nonhemolytic. The results offer wide implications for elucidating differential membrane targeting phenomena defined at the nanoscale.
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http://dx.doi.org/10.1021/acsnano.1c00218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8219408PMC
June 2021

Large scale model lipid membrane movement induced by a cation switch.

J Colloid Interface Sci 2021 Aug 18;596:297-311. Epub 2021 Mar 18.

ISIS Pulsed Neutron and Muon Source, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 OQX, UK. Electronic address:

A biomembrane sample system where millimolar changes of cations induce reversible large scale (≥ 200 Å) changes in the membrane-to-surface distance is described. The system composes of a free-floating bilayer, formed adjacent to a self-assembled monolayer (SAM). To examine the membrane movements, differently charged floating bilayers in the presence and absence of Ca and Na, respectively, were examined using neutron reflectivity and quartz crystal microbalance measurements, alongside molecular dynamics simulations. In neutron reflectivity the variation of Ca and Na concentration enabled precision manipulation of the membrane-to-surface distance. Simulations suggest that Ca ions bridge between SAM and bilayer whereas the more diffuse binding of Na, especially to bilayers, is unable to fully overcome the repulsion between anionic floating bilayer and anionic SAM. Reproduced neutron reflectivity results with quartz crystal microbalance demonstrate the potential of this easily producible sample system to become a standard analysis tool for e.g. investigating membrane binding effects, endocytosis and cell signaling.
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http://dx.doi.org/10.1016/j.jcis.2021.03.078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8109235PMC
August 2021

Effect of Water Models on Transmembrane Self-Assembled Cyclic Peptide Nanotubes.

ACS Nano 2021 04 19;15(4):7053-7064. Epub 2021 Mar 19.

Center for Research in Biological Chemistry and Molecular Materials (CIQUS), University of Santiago de Compostela, 15782 Santiago de Compostela, Spain.

Self-assembling cyclic peptide nanotubes can form nanopores when they are inserted in lipid bilayers, acting as ion and/or water permeable channels. In order to improve the versatility of these systems, it is possible to specifically design cyclic peptides with a combination of natural and non-natural amino acids, enabling the control of the nature of the inner cavity of the channels. Here, the behavior of two types of self-assembling peptide motifs, alternating α-amino acids with γ- or δ-aminocycloalkanecarboxylic acids, is studied molecular dynamics (MD) simulations. The behavior of water molecules in nanopores is expected to affect the properties of these channels and therefore merits detailed examination. A number of water models commonly used in MD simulations have been validated by how well they reproduce bulk water properties. However, it is less clear how these water models behave in the nanoconfined condition inside a channel. The behavior of four different water models-TIP3P, TIP4P, TIP4P/2005, and OPC-are evaluated in MD simulations of self-assembled cyclic peptide nanotubes of distinct composition and diameter. The dynamic behavior of the water molecules and ions in these designed artificial channels depends subtly on the water model used. TIP3P water molecules move faster than those of TIP4P, TIP4P/2005, and OPC. This demeanor is clearly observed in the filling of the nanotube, in water diffusion within the pore, and in the number and stability of hydrogen bonds of the peptides with water. It was also shown that the water model influences the simulated ion flux through the nanotubes, with TIP3P producing the greatest ion flux. Additionally, the two more recent models, TIP4P/2005 and OPC, which are known to reproduce the experimental self-diffusion coefficient of bulk water quite well, exhibit very similar results under the nanoconfined conditions studied here. Because none of these models have been parametrized specifically for waters confined in peptide nanotubes, this study provides a point of reference for further validation.
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http://dx.doi.org/10.1021/acsnano.1c00155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8485350PMC
April 2021

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

J Phys Chem B 2021 03 15;125(11):2929-2941. Epub 2021 Mar 15.

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.

α-Synuclein (αS) is a presynaptic protein that binds to cell membranes and is linked to Parkinson's disease (PD). Binding of αS to membranes is a likely first step in the molecular pathophysiology of PD. The αS molecule can adopt multiple conformations, being largely disordered in water, adopting a β-sheet conformation when present in amyloid fibrils, and forming a dynamic multiplicity of α-helical conformations when bound to lipid bilayers and related membrane-mimetic surfaces. Multiscale molecular dynamics simulations in conjunction with nuclear magnetic resonance (NMR) and cross-linking mass spectrometry (XLMS) measurements are used to explore the interactions of αS with an anionic lipid bilayer. The simulations and NMR measurements together reveal a break in the helical structure of the central non-amyloid-β component (NAC) region of αS in the vicinity of residues 65-70, which may facilitate subsequent oligomer formation. Coarse-grained simulations of αS starting from the structure of αS when bound to a detergent micelle reveal the overall pattern of protein contacts to anionic lipid bilayers, while subsequent all-atom simulations provide details of conformational changes upon membrane binding. In particular, simulations and NMR data for liposome-bound αS indicate incipient β-strand formation in the NAC region, which is supported by intramolecular contacts seen XLMS and simulations. Markov state models based on the all-atom simulations suggest a mechanism of conformational change of membrane-bound αS a dynamic helix break in the region of residue 65 in the NAC region. The emergent dynamic model of membrane-interacting αS advances our understanding of the mechanism of PD, potentially aiding the design of novel therapeutic approaches.
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http://dx.doi.org/10.1021/acs.jpcb.1c01281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8006134PMC
March 2021

Molecular Simulations of Hydrophobic Gating of Pentameric Ligand Gated Ion Channels: Insights into Water and Ions.

J Phys Chem B 2021 02 13;125(4):981-994. Epub 2021 Jan 13.

Department of Biochemistry, University of Oxford, Oxford, U.K.

Ion channels are proteins which form gated nanopores in biological membranes. Many channels exhibit hydrophobic gating, whereby functional closure of a pore occurs by local dewetting. The pentameric ligand gated ion channels (pLGICs) provide a biologically important example of hydrophobic gating. Molecular simulation studies comparing additive vs polarizable models indicate predictions of hydrophobic gating are robust to the model employed. However, polarizable models suggest favorable interactions of hydrophobic pore-lining regions with chloride ions, of relevance to both synthetic carriers and channel proteins. Electrowetting of a closed pLGIC hydrophobic gate requires too high a voltage to occur physiologically but may inform designs for switchable nanopores. Global analysis of ∼200 channels yields a simple heuristic for structure-based prediction of (closed) hydrophobic gates. Simulation-based analysis is shown to provide an aid to interpretation of functional states of new channel structures. These studies indicate the importance of understanding the behavior of water and ions within the nanoconfined environment presented by ion channels.
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http://dx.doi.org/10.1021/acs.jpcb.0c09285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7869105PMC
February 2021

Membrane Binding of Antimicrobial Peptides Is Modulated by Lipid Charge Modification.

J Chem Theory Comput 2021 Feb 4;17(2):1218-1228. Epub 2021 Jan 4.

Department of Biochemistry, University of Oxford, Oxford OX1 3QU, U.K.

Peptide interactions with lipid bilayers play a key role in a range of biological processes and depend on electrostatic interactions between charged amino acids and lipid headgroups. Antimicrobial peptides (AMPs) initiate the killing of bacteria by binding to and destabilizing their membranes. The multiple peptide resistance factor (MprF) provides a defense mechanism for bacteria against a broad range of AMPs. MprF reduces the negative charge of bacterial membranes through enzymatic conversion of the anionic lipid phosphatidyl glycerol (PG) to either zwitterionic alanyl-phosphatidyl glycerol (Ala-PG) or cationic lysyl-phosphatidyl glycerol (Lys-PG). The resulting change in the membrane charge is suggested to reduce the binding of AMPs to membranes, thus impeding downstream AMP activity. Using coarse-grained 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. More than 5000 simulations in total are used to define the relationship between lipid bilayer composition, peptide sequence (using five different membrane-active peptides), and peptide binding to membranes. The degree of interaction of a peptide with a membrane correlates with the membrane surface charge density. Free energy profile (potential of mean force) calculations reveal that the lipid modifications due to MprF alter the energy barrier to peptide helix penetration of the bilayer. These results will offer a guide to the design of novel peptides, which addresses the issue of resistance via MprF-mediated membrane modification.
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http://dx.doi.org/10.1021/acs.jctc.0c01025DOI Listing
February 2021

Coarse-Grained Simulations Suggest the Epsin N-Terminal Homology Domain Can Sense Membrane Curvature without Its Terminal Amphipathic Helix.

ACS Nano 2020 Dec 10. Epub 2020 Dec 10.

Department of Materials, Imperial College London, London SW7 2AZ, United Kingdom.

Nanoscale membrane curvature is a common feature in cell biology required for functions such as endocytosis, exocytosis and cell migration. These processes require the cytoskeleton to exert forces on the membrane to deform it. Cytosolic proteins contain specific motifs which bind to the membrane, connecting it to the internal cytoskeletal machinery. These motifs often bind charged phosphatidylinositol phosphate lipids present in the cell membrane which play significant roles in signaling. These lipids are important for membrane deforming processes, such as endocytosis, but much remains unknown about their role in the sensing of membrane nanocurvature by protein domains. Using coarse-grained molecular dynamics simulations, we investigated the interaction of a model curvature active protein domain, the epsin N-terminal homology domain (ENTH), with curved lipid membranes. The combination of anionic lipids (phosphatidylinositol 4,5-bisphosphate and phosphatidylserine) within the membrane, protein backbone flexibility, and structural changes within the domain were found to affect the domain's ability to sense, bind, and localize with nanoscale precision at curved membrane regions. The findings suggest that the ENTH domain can sense membrane curvature without the presence of its terminal amphipathic α helix another structural region we have denoted as H3, re-emphasizing the critical relationship between nanoscale membrane curvature and protein function.
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http://dx.doi.org/10.1021/acsnano.0c05960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760104PMC
December 2020

The MscS-like channel YnaI has a gating mechanism based on flexible pore helices.

Proc Natl Acad Sci U S A 2020 11 4;117(46):28754-28762. Epub 2020 Nov 4.

Biocenter, Julius-Maximilians-Universität Würzburg, 97080 Würzburg, Germany;

The mechanosensitive channel of small conductance (MscS) is the prototype of an evolutionarily diversified large family that fine-tunes osmoregulation but is likely to fulfill additional functions. has six osmoprotective paralogs with different numbers of transmembrane helices. These helices are important for gating and sensing in MscS but the role of the additional helices in the paralogs is not understood. The medium-sized channel YnaI was extracted and delivered in native nanodiscs in closed-like and open-like conformations using the copolymer diisobutylene/maleic acid (DIBMA) for structural studies. Here we show by electron cryomicroscopy that YnaI has an extended sensor paddle that during gating relocates relative to the pore concomitant with bending of a GGxGG motif in the pore helices. YnaI is the only one of the six paralogs that has this GGxGG motif allowing the sensor paddle to move outward. Access to the pore is through a vestibule on the cytosolic side that is fenestrated by side portals. In YnaI, these portals are obstructed by aromatic side chains but are still fully hydrated and thus support conductance. For comparison with large-sized channels, we determined the structure of YbiO, which showed larger portals and a wider pore with no GGxGG motif. Further in silico comparison of MscS, YnaI, and YbiO highlighted differences in the hydrophobicity and wettability of their pores and vestibule interiors. Thus, MscS-like channels of different sizes have a common core architecture but show different gating mechanisms and fine-tuned conductive properties.
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http://dx.doi.org/10.1073/pnas.2005641117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682570PMC
November 2020

Water in Nanopores and Biological Channels: A Molecular Simulation Perspective.

Chem Rev 2020 09 25;120(18):10298-10335. Epub 2020 Aug 25.

Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, U.K.

This Review explores the dynamic behavior of water within nanopores and biological channels in lipid bilayer membranes. We focus on molecular simulation studies, alongside selected structural and other experimental investigations. Structures of biological nanopores and channels are reviewed, emphasizing those high-resolution crystal structures, which reveal water molecules within the transmembrane pores, which can be used to aid the interpretation of simulation studies. Different levels of molecular simulations of water within nanopores are described, with a focus on molecular dynamics (MD). In particular, models of water for MD simulations are discussed in detail to provide an evaluation of their use in simulations of water in nanopores. Simulation studies of the behavior of water in idealized models of nanopores have revealed aspects of the organization and dynamics of nanoconfined water, including wetting/dewetting in narrow hydrophobic nanopores. A survey of simulation studies in a range of nonbiological nanopores is presented, including carbon nanotubes, synthetic nanopores, model peptide nanopores, track-etched nanopores in polymer membranes, and hydroxylated and functionalized nanoporous silica. These reveal a complex relationship between pore size/geometry, the nature of the pore lining, and rates of water transport. Wider nanopores with hydrophobic linings favor water flow whereas narrower hydrophobic pores may show dewetting. Simulation studies over the past decade of the behavior of water in a range of biological nanopores are described, including porins and β-barrel protein nanopores, aquaporins and related polar solute pores, and a number of different classes of ion channels. Water is shown to play a key role in proton transport in biological channels and in hydrophobic gating of ion channels. An overall picture emerges, whereby the behavior of water in a nanopore may be predicted as a function of its hydrophobicity and radius. This informs our understanding of the functions of diverse channel structures and will aid the design of novel nanopores. Thus, our current level of understanding allows for the design of a nanopore which promotes wetting over dewetting or vice versa. However, to design a novel nanopore, which enables fast, selective, and gated flow of water de novo would remain challenging, suggesting a need for further detailed simulations alongside experimental evaluation of more complex nanopore systems.
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http://dx.doi.org/10.1021/acs.chemrev.9b00830DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7517714PMC
September 2020

Mechanisms of activation and desensitization of full-length glycine receptor in lipid nanodiscs.

Nat Commun 2020 07 27;11(1):3752. Epub 2020 Jul 27.

Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, 44106-4970, USA.

Glycinergic synapses play a central role in motor control and pain processing in the central nervous system. Glycine receptors (GlyRs) are key players in mediating fast inhibitory neurotransmission at these synapses. While previous high-resolution structures have provided insights into the molecular architecture of GlyR, several mechanistic questions pertaining to channel function are still unanswered. Here, we present Cryo-EM structures of the full-length GlyR protein complex reconstituted into lipid nanodiscs that are captured in the unliganded (closed), glycine-bound (open and desensitized), and allosteric modulator-bound conformations. A comparison of these states reveals global conformational changes underlying GlyR channel gating and modulation. The functional state assignments were validated by molecular dynamics simulations, and the observed permeation events are in agreement with the anion selectivity and conductance of GlyR. These studies provide the structural basis for gating, ion selectivity, and single-channel conductance properties of GlyR in a lipid environment.
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http://dx.doi.org/10.1038/s41467-020-17364-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7385131PMC
July 2020

Electric Field Induced Wetting of a Hydrophobic Gate in a Model Nanopore Based on the 5-HT Receptor Channel.

ACS Nano 2020 08 23;14(8):10480-10491. Epub 2020 Jul 23.

Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.

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-HT receptor in its closed state, with a field of at least ∼100 mV nm (corresponding to a supra-physiological potential difference of ∼0.85 V across the membrane) 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 characterizing the phase transitions of water confined within the nanopore, revealing liquid-vapor 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.
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http://dx.doi.org/10.1021/acsnano.0c04387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7450702PMC
August 2020

The Glycosphingolipid GM3 Modulates Conformational Dynamics of the Glucagon Receptor.

Biophys J 2020 07 17;119(2):300-313. Epub 2020 Jun 17.

Department of Biochemistry, University of Oxford, Oxford, United Kingdom. Electronic address:

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. Although recent studies suggest a role for membrane lipids in the modulation of class A and class F GPCR signaling properties, little is known about the effect of lipids on class B1 receptors. In this study, we employed multiscale molecular dynamics 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 transmembrane domain. ECD movements were modulated by binding of the glycosphingolipid GM3. These large-scale fluctuations in ECD conformation may affect the ligand binding and receptor activation properties. We also identify a unique phosphatidylinositol (4,5)-bisphosphate (PIP) interaction profile near intracellular loop (ICL) 2/TM3 at the G-protein-coupling interface, suggesting a mechanism of engaging G-proteins that may have a distinct dependence on PIP compared with class A GPCRs. Given the structural conservation of class B1 GPCRs, the modulatory effects of GM3 and PIP on GCGR may be conserved across these receptors, offering new insights into potential therapeutic targeting.
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http://dx.doi.org/10.1016/j.bpj.2020.06.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376093PMC
July 2020

Atomistic mechanism of transmembrane helix association.

PLoS Comput Biol 2020 06 4;16(6):e1007919. Epub 2020 Jun 4.

Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

Transmembrane helix association is a fundamental step in the folding of helical membrane proteins. The prototypical example of this association is formation of the glycophorin dimer. While its structure and stability have been well-characterized experimentally, the detailed assembly mechanism is harder to obtain. Here, we use all-atom simulations within phospholipid membrane to study glycophorin association. We find that initial association results in the formation of a non-native intermediate, separated by a significant free energy barrier from the dimer with a native binding interface. We have used transition-path sampling to determine the association mechanism. We find that the mechanism of the initial bimolecular association to form the intermediate state can be mediated by many possible contacts, but seems to be particularly favoured by formation of non-native contacts between the C-termini of the two helices. On the other hand, the contacts which are key to determining progression from the intermediate to the native state are those which define the native binding interface, reminiscent of the role played by native contacts in determining folding of globular proteins. As a check on the simulations, we have computed association and dissociation rates from the transition-path sampling. We obtain results in reasonable accord with available experimental data, after correcting for differences in native state stability. Our results yield an atomistic description of the mechanism for a simple prototype of helical membrane protein folding.
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http://dx.doi.org/10.1371/journal.pcbi.1007919DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272003PMC
June 2020

Induced Polarization in Molecular Dynamics Simulations of the 5-HT Receptor Channel.

J Am Chem Soc 2020 05 8;142(20):9415-9427. Epub 2020 May 8.

Department of Biochemistry, University of Oxford, Oxford OX1 3QU, U.K.

Ion channel proteins form water-filled nanoscale pores within lipid bilayers, and their properties are dependent on the complex behavior of water in a nanoconfined environment. Using a simplified model of the pore of the 5-HT receptor (5HT3R) which restrains the backbone structure to that of the parent channel protein from which it is derived, we compare additive with polarizable models in describing the behavior of water in nanopores. Molecular dynamics simulations were performed with four conformations of the channel: two closed state structures, an intermediate state, and an open state, each embedded in a phosphatidylcholine bilayer. Water density profiles revealed that for all water models, the closed and intermediate states exhibited strong dewetting within the central hydrophobic gate region of the pore. However, the open state conformation exhibited varying degrees of hydration, ranging from partial wetting for the TIP4P/2005 water model to complete wetting for the polarizable AMOEBA14 model. Water dipole moments calculated using polarizable force fields also revealed that water molecules remaining within dewetted sections of the pore resemble gas phase water. Free energy profiles for Na and for Cl ions within the open state pore revealed more rugged energy landscapes using polarizable force fields, and the hydration number profiles of these ions were also sensitive to induced polarization resulting in a substantive reduction of the number of waters within the first hydration shell of Cl while it permeates the pore. These results demonstrate that induced polarization can influence the complex behavior of water and ions within nanoscale pores and provides important new insights into their chemical properties.
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http://dx.doi.org/10.1021/jacs.0c02394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7243253PMC
May 2020

Defining how multiple lipid species interact with inward rectifier potassium (Kir2) channels.

Proc Natl Acad Sci U S A 2020 04 25;117(14):7803-7813. Epub 2020 Mar 25.

Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom

Protein-lipid interactions are a key element of the function of many integral membrane proteins. These potential interactions should be considered alongside the complexity and diversity of membrane lipid composition. Inward rectifier potassium channel (Kir) Kir2.2 has multiple interactions with plasma membrane lipids: Phosphatidylinositol (4, 5)-bisphosphate (PIP) activates the channel; a secondary anionic lipid site has been identified, which augments the activation by PIP; and cholesterol inhibits the channel. Molecular dynamics simulations are used to characterize in molecular detail the protein-lipid interactions of Kir2.2 in a model of the complex plasma membrane. Kir2.2 has been simulated with multiple, functionally important lipid species. From our simulations we show that PIP interacts most tightly at the crystallographic interaction sites, outcompeting other lipid species at this site. Phosphatidylserine (PS) interacts at the previously identified secondary anionic lipid interaction site, in a PIP concentration-dependent manner. There is interplay between these anionic lipids: PS interactions are diminished when PIP is not present in the membrane, underlining the need to consider multiple lipid species when investigating protein-lipid interactions.
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http://dx.doi.org/10.1073/pnas.1918387117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7149479PMC
April 2020

Multiple lipid binding sites determine the affinity of PH domains for phosphoinositide-containing membranes.

Sci Adv 2020 02 19;6(8):eaay5736. Epub 2020 Feb 19.

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.

Association of peripheral proteins with lipid bilayers regulates membrane signaling and dynamics. Pleckstrin homology (PH) domains bind to phosphatidylinositol phosphate (PIP) molecules in membranes. The effects of local PIP enrichment on the interaction of PH domains with membranes is unclear. Molecular dynamics simulations allow estimation of the binding energy of GRP1 PH domain to PIP-containing membranes. The free energy of interaction of the PH domain with more than two PIP molecules is comparable to experimental values, suggesting that PH domain binding involves local clustering of PIP molecules within membranes. We describe a mechanism of PH binding proceeding via an encounter state to two bound states which differ in the orientation of the protein relative to the membrane, these orientations depending on the local PIP concentration. These results suggest that nanoscale clustering of PIP molecules can control the strength and orientation of PH domain interaction in a concentration-dependent manner.
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http://dx.doi.org/10.1126/sciadv.aay5736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7030919PMC
February 2020

The energetics of protein-lipid interactions as viewed by molecular simulations.

Biochem Soc Trans 2020 02;48(1):25-37

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.

Membranes are formed from a bilayer containing diverse lipid species with which membrane proteins interact. Integral, membrane proteins are embedded in this bilayer, where they interact with lipids from their surroundings, whilst peripheral membrane proteins bind to lipids at the surface of membranes. Lipid interactions can influence the function of membrane proteins, either directly or allosterically. Both experimental (structural) and computational approaches can reveal lipid binding sites on membrane proteins. It is, therefore, important to understand the free energies of these interactions. This affords a more complete view of the engagement of a particular protein with the biological membrane surrounding it. Here, we describe many computational approaches currently in use for this purpose, including recent advances using both free energy and unbiased simulation methods. In particular, we focus on interactions of integral membrane proteins with cholesterol, and with anionic lipids such as phosphatidylinositol 4,5-bis-phosphate and cardiolipin. Peripheral membrane proteins are exemplified via interactions of PH domains with phosphoinositide-containing membranes. We summarise the current state of the field and provide an outlook on likely future directions of investigation.
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http://dx.doi.org/10.1042/BST20190149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054751PMC
February 2020

Lipid Interactions of a Ciliary Membrane TRP Channel: Simulation and Structural Studies of Polycystin-2.

Structure 2020 02 2;28(2):169-184.e5. Epub 2019 Dec 2.

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Electronic address:

Polycystin-2 (PC2) is a transient receptor potential (TRP) channel present in ciliary membranes of the kidney. PC2 shares a transmembrane fold with other TRP channels, in addition to an extracellular domain found in TRPP and TRPML channels. Using molecular dynamics (MD) simulations and cryoelectron microscopy we identify and characterize PIP and cholesterol interactions with PC2. PC2 is revealed to have a PIP binding site close to the equivalent vanilloid/lipid binding site in the TRPV1 channel. A 3.0-Å structure reveals a binding site for cholesterol on PC2. Cholesterol interactions with the channel at this site are characterized by MD simulations. The two classes of lipid binding sites are compared with sites observed in other TRPs and in Kv channels. These findings suggest PC2, in common with other ion channels, may be modulated by both PIPs and cholesterol, and position PC2 within an emerging model of the roles of lipids in the regulation and organization of ciliary membranes.
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http://dx.doi.org/10.1016/j.str.2019.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001106PMC
February 2020
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