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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.

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 via XLMS and simulations. Markov state models based on the all-atom simulations suggest a mechanism of conformational change of membrane-bound αS via 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.

Objective To determine whether glucagon receptor (GCGR) actions are modulated by cellular cholesterol levels. Methods We determined the effects of experimental cholesterol depletion and loading on glucagon-mediated cAMP production, ligand internalisation and glucose production in human hepatoma cells, mouse and human hepatocytes. GCGR interactions with lipid bilayers were explored using coarse-grained molecular dynamic simulations. Glucagon responsiveness was measured in mice fed a high cholesterol diet with or without simvastatin to modulate hepatocyte cholesterol content. Results GCGR cAMP signalling was reduced by higher cholesterol levels across different cellular models. Ex vivo glucagon-induced glucose output from mouse hepatocytes was enhanced by simvastatin treatment. 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. Simulations identified likely membrane-exposed cholesterol binding sites on the GCGR, including a site where cholesterol is a putative negative allosteric modulator. Conclusions Our results indicate that cellular cholesterol content influences glucagon sensitivity and indicate a potential molecular basis for this phenomenon. This could be relevant to the pathogenesis of non-alcoholic fatty liver disease, which is associated with both hepatic cholesterol accumulation and glucagon resistance.

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.

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 via another structural region we have denoted as H3, re-emphasizing the critical relationship between nanoscale membrane curvature and protein function.

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. Escherichia coli 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.

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.

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., Glycinergic synapses play a central role in motor control and pain processing in the central nervous system. Here, authors present cryo-EM structures of the full-length glycine receptors (GlyRs) reconstituted into lipid nanodiscs in the unliganded, glycine-bound and allosteric modulator-bound conformations and reveal global conformational changes underlying GlyR channel gating and modulation.

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-HT3 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.

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 (e.g., sensing or desalination) as well as for furthering our understanding of the mechanistic processes underlying biological ion channel function.

The CorA family of proteins regulates the homeostasis of divalent metal ions in many bacteria, archaea, and eukaryotic mitochondria, making it an important target in the investigation of the mechanisms of transport and its functional regulation. Although numerous structures of open and closed channels are now available for the CorA family, the mechanism of the transport regulation remains elusive. Here, we investigated the conformational distribution and associated dynamic behaviour of the pentameric Mg2+ channel CorA at room temperature using small-angle neutron scattering (SANS) in combination with molecular dynamics (MD) simulations and solid-state nuclear magnetic resonance spectroscopy (NMR). We find that neither the Mg2+-bound closed structure nor the Mg2+-free open forms are sufficient to explain the average conformation of CorA. Our data support the presence of conformational equilibria between multiple states, and we further find a variation in the behaviour of the backbone dynamics with and without Mg2+. We propose that CorA must be in a dynamic equilibrium between different non-conducting states, both symmetric and asymmetric, regardless of bound Mg2+ but that conducting states become more populated in Mg2+-free conditions. These properties are regulated by backbone dynamics and are key to understanding the functional regulation of CorA.

Summary 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 PIP2 to A2aR stabilizes protein-protein interactions via PIP2-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., Graphical abstract, Highlights • MD simulation and Markov state models are used to study oligomerization of A2aR • A2aR oligomerization is enhanced by receptor activation and by PIP2 interactions • The population of A2aR oligomers may change in response to environmental cues • Oligomerization may control combinatory allosteric modulation of GPCR signaling, Song et al. use molecular dynamics simulation and Markov state models to show that oligomerization of the adenosine 2a receptor (a class A GPCR) is enhanced by both receptor activation and by PIP2 interactions. Oligomerization may influence allosteric modulation of GPCRs.

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., Author summary Peripheral proteins bind to the surfaces of specific membranes within eukaryotic cells and thereby coordinate dynamic interactions between sub-cellular compartments. The FYVE domain of EEA1 recognizes the membranes of endosomes by binding to the anionic headgroup of a specific lipid, phosphatidylinositol-3-phosphate (PI(3)P), which is present in those membranes. We use molecular dynamics simulations at two different resolutions (coarse-grained and all atom) to define the mechanism whereby a dimeric FYVE domain binds to model membranes containing PI(3)P. Hinge-bending flexibility between the bound FYVE domain and the coiled coil stalk to which it is attached is observed. In the intact EEA1 molecule the coiled coil is 200 nm long. This ‘amplifies’ the flexibility observed at the FYVE hinge end such that the other end of the EAA1 protein can sweep out an area of about 0.1 μm2 in its search for a second endosome with which to interact. In this way EAA1 acts as a long stalk with two ‘sticky’ ends to help draw together two specific membrane surfaces.

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 Escherichia coli 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.

Summary 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., Graphical abstract, Highlights • Mutations in TCRβ and CD3ζ TMRs that reduce their interaction augment signaling • pMHC and anti-CD3 binding to TCR-CD3 induce similar quaternary structure relaxation • Soluble monovalent pMHC alone signals and reduces TCRαβ cohesion with CD3ζ • Allosteric changes in TCR-CD3 dynamics instigate T cell activation, T cell antigen receptor is central in adaptive immunity; however, the mechanism that couples ligand binding and intracellular signaling is still controversial. Here, Lanz et al. have taken an interdisciplinary approach demonstrating that binding of soluble monovalent pMHC to TCR-CD3 allosterically reduces TCRαβ cohesion with CD3ζ and initiates signal transduction.

Cinnamycin is a lantibiotic peptide, which selectively binds to and permeabilizes membranes containing phosphatidylethanolamine (PE) lipids. As PE is a major component of many bacterial cell membranes, cinnamycin has considerable potential for destroying these. In this study, molecular dynamics simulations are used to elucidate the structure of a lipid-cinnamycin complex and the origin of selective lipid binding. The simulations reveal that cinnamycin selectively binds to PE by forming an extensive hydrogen-bonding network involving all three hydrogen atoms of the primary ammonium group of the PE head group. The substitution of a single hydrogen atom with a methyl group on the ammonium nitrogen destabilizes this hydrogen-bonding network. In addition to binding the primary ammonium group, cinnamycin also interacts with the phosphate group of the lipid through a previously uncharacterized phosphate-binding site formed by the backbone Phe10-Abu11-Phe12-Val13 moieties (Abu = 1-α-aminobutyric acid). In addition, hydroxylation of Asp15 at Cβ plays a role in selective binding of PE due to its tight interaction with the charged amine of the lipid head group. The simulations reveal that the position and orientation of the peptide in the membrane depend on the type of lipid to which it binds, suggesting a reason for why cinnamycin selectively permeabilizes PE-containing membranes.

Hedgehog (HH) ligands, classical morphogens that pattern embryonic tissues in all animals, are covalently coupled to two lipids—a palmitoyl group at the N terminus and a cholesteroyl group at the C terminus. While the palmitoyl group binds and inactivates Patched 1 (PTCH1), the main receptor for HH ligands, the function of the cholesterol modification has remained mysterious. Using structural and biochemical studies, along with reassessment of previous cryo-electron microscopy structures, we find that the C-terminal cholesterol attached to Sonic hedgehog (Shh) binds the first extracellular domain of PTCH1 and promotes its inactivation, thus triggering HH signaling. Molecular dynamics simulations show that this interaction leads to the closure of a tunnel through PTCH1 that serves as the putative conduit for sterol transport. Thus, Shh inactivates PTCH1 by grasping its extracellular domain with two lipidic pincers, the N-terminal palmitate and the C-terminal cholesterol, which are both inserted into the PTCH1 protein core. Cholesterol can function as both a substrate and an inhibitor of the Hedgehog receptor Patched. Structural analysis and molecular dynamics simulations reveal that cholesterol inhibits Patched by inserting into its extracellular domain

Otopetrins (Otop1–Otop3) comprise one of two known eukaryotic proton-selective channel families. Otop1 is required for otoconia formation and a candidate mammalian sour taste receptor. Here we report cryo-EM structures of zebrafish Otop1 and chicken Otop3 in lipid nanodiscs. The structures reveal a dimeric architecture, with each subunit forming 12 transmembrane helices divided into structurally similar amino (N) and carboxy (C) domains. Cholesterol-like molecules occupy various sites in Otop1 and Otop3 and occlude a central tunnel. In molecular dynamics simulations, hydrophilic vestibules formed by the N and C domains and in the intrasubunit interface between N and C domains form conduits for water entry into the membrane core, suggesting three potential proton conduction pathways. By mutagenesis, we tested the roles of charged residues in each putative permeation pathway. Our results provide a structural basis for understanding selective proton permeation and gating of this conserved family of proton channels. Otopetrins are proton channels and candidate sour taste receptors. Cryo-EM structures of zebrafish Otop1 and chicken Otop3 reveal potential proton conduction pathways.

[Image: see text] Cell membranes contain a large variety of lipid types and are crowded with proteins, endowing them with the plasticity needed to fulfill their key roles in cell functioning. The compositional complexity of cellular membranes gives rise to a heterogeneous lateral organization, which is still poorly understood. Computational models, in particular molecular dynamics simulations and related techniques, have provided important insight into the organizational principles of cell membranes over the past decades. Now, we are witnessing a transition from simulations of simpler membrane models to multicomponent systems, culminating in realistic models of an increasing variety of cell types and organelles. Here, we review the state of the art in the field of realistic membrane simulations and discuss the current limitations and challenges ahead.

Extracellular glycan biosynthesis is a widespread microbial protection mechanism. In Gram-negative bacteria, the O antigen polysaccharide represents the variable region of outer membrane lipopolysaccharides. Fully assembled lipid-linked O antigens are translocated across the inner membrane by the WzmWzt ABC transporter for ligation to the lipopolysaccharide core, with the transporter forming a continuous transmembrane channel in a nucleotide-free state. Here, we report its structure in an ATP-bound conformation. Large structural changes within the nucleotide-binding and transmembrane regions push conserved hydrophobic residues at the substrate entry site towards the periplasm and provide a model for polysaccharide translocation. With ATP bound, the transporter forms a large transmembrane channel with openings toward the membrane and periplasm. The channel’s periplasmic exit is sealed by detergent molecules that block solvent permeation. Molecular dynamics simulation data suggest that, in a biological membrane, lipid molecules occupy this periplasmic exit and prevent water flux in the transporter’s resting state., Bacterial O antigen polysaccharides play a role in innate immune response evasion. Here, the authors uncover the ATP-bound structure of the Aquifex aeolicus WzmWzt O antigen ABC transporter, shedding light onto the mechanism of lipid-linked polysaccharide translocation.

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.

Flycatcher1 (FLYC1), a MscS homolog, has recently been identified as a candidate mechanosensitive (MS) ion channel involved in Venus flytrap prey recognition. FLYC1 is larger and its sequence diverges from previously studied MscS homologs, suggesting it has unique structural features that contribute to its function. Here, we characterized FLYC1 by cryo-electron microscopy, molecular dynamics simulations, and electrophysiology. Akin to bacterial MscS and plant MSL1 channels, we find that FLYC1 central core includes side portals in the cytoplasmic cage that regulate ion conduction, by identifying critical residues that modulate channel conductance. Topologically unique cytoplasmic flanking regions can adopt ‘up’ or ‘down’ conformations, making the channel asymmetric. Disruption of an up conformation-specific interaction severely delays channel deactivation by 40-fold likely due to stabilization of the channel open state. Our results illustrate novel structural features and likely conformational transitions that regulate mechano-gating of FLYC1.

Summary 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., Graphical abstract, Highlights • High-resolution cryo-EM structure of HHAT in complex with a SHH-mimetic megabody • Heme is bound to HHAT Cys324 in a membrane cavity and is essential for function • Palm-CoA binds in a central HHAT cavity adjacent to the catalytic histidine • The competitive inhibitor IMP-1575 causes conformational changes in the active site, HHAT is a key enzyme in the Hedgehog signaling pathway and protein-substrate member of the membrane-bound O-acyltransferase (MBOAT) superfamily. Coupland et al. report the cryo-EM structures of HHAT bound to acyl-donor substrate, megabody, and inhibitor IMP-1575, providing insight into the structure-function relationship of HHAT, mechanism, and the basis for inhibition.

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.\ud \ud \ud

Graphical abstract, 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 Ca2+ and Na+, respectively, were examined using neutron reflectivity and quartz crystal microbalance measurements, alongside molecular dynamics simulations. In neutron reflectivity the variation of Ca2+ and Na+ concentration enabled precision manipulation of the membrane-to-surface distance. Simulations suggest that Ca2+ 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.

Integral membrane proteins are localised and/or regulated by lipids present in the surrounding bilayer. Whilst bacteria such as E. coli have relatively simple membranes when compared to eukaryotic cells, 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 E. coli inner membrane proteins. Our data reveals a strong asymmetry between the membrane leaflets, with a marked increase of anionic lipid binding to the inner leaflet regions of membrane proteins, particularly for cardiolipin. From our simulations we identify over 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 more complex protein-lipid interactions.

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.

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.

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 via 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 This work was supported by the Spanish Agencia Estatal de Investigación (AEI) and the ERDF (CTQ2016-78423-R, PID2019-111126RB-100, and RTI2018-098795-A-I00) and by the Xunta de Galicia and the ERDF (ED431F 2020/05, ED431C 2017/25, and Centro singular de investigación de Galicia accreditation 2016-2019, ED431G/09). M.C. thanks Xunta de Galicia for a predoctoral fellowship (ED481A-2017/068). R.G.-F. thanks Ministerio de Ciencia, Innovación y Universidades for a Ramón y Cajal contract (RYC-2016- 20335). Research in MSPS’s group is supported by EPSRC (EP/R004722/1; EP/V010948/1) BBSRC (BB/R00126X/1) and Wellcome Trust (208361/Z/17/Z) SI

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.

Local structural frustration, the existence of mutually exclusive competing interactions, may explain why some proteins are dynamic while others are rigid. Frustration is thought to underpin biomolecular recognition and the flexibility of protein-binding sites. Here, we show how a small chemical modification, the oxidation of two cysteine thiols to a disulfide bond, during the catalytic cycle of the N-terminal domain of the key bacterial oxidoreductase DsbD (nDsbD), introduces frustration ultimately influencing protein function. In oxidized nDsbD, local frustration disrupts the packing of the protective cap-loop region against the active site allowing loop opening. By contrast, in reduced nDsbD the cap loop is rigid, always protecting the active-site thiols from the oxidizing environment of the periplasm. Our results point toward an intricate coupling between the dynamics of the active-site cysteines and of the cap loop which modulates the association reactions of nDsbD with its partners resulting in optimized protein function.

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-HT3receptor in its closed state, with a field of at least ∼100 mV nm−1was 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

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.

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 PIP2interaction profile near ICL2/TM3 at the G-protein coupling interface, suggesting a mechanism of engaging G-proteins which may have a distinct dependence on PIP2compared to Class A GPCRs. Given the structural conservation of Class B1 GPCRs, the modulatory effects of GM3 and PIP2on 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 PIP2interaction 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.

Significance Ion channels form pores that allow for the selective transport of ions across cell membranes, generating electrical signals in response to a variety of signals. Inward rectifier potassium (Kir) channels in particular are regulated by direct interactions with the complex mixture of lipids that are present in eukaryotic cell membranes. However, the molecular details of these concurrent lipid interactions with Kir channels are not clear and difficult to access via experimental methods. Here, we simulate the Kir2.2 channel in a complex lipid mixture to explore how anionic phospholipids and cholesterol dynamically organize around the membrane protein. In particular we demonstrate a synergy between binding interactions of different anionic phospholipid species which are known to activate Kir channels., 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 (PIP2) activates the channel; a secondary anionic lipid site has been identified, which augments the activation by PIP2; 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 PIP2 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 PIP2 concentration-dependent manner. There is interplay between these anionic lipids: PS interactions are diminished when PIP2 is not present in the membrane, underlining the need to consider multiple lipid species when investigating protein–lipid interactions.

Ion channel proteins form water-filled nanoscale pores within lipid bilayers and their properties are dependent on the complex behavior of water in a nano-confined environment. Using the pore of the 5HT3 receptor (5HT3R) 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− whilst 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.ToC Graphic

Molecular simulations reveal that multiple lipid interactions are needed for high-affinity binding of protein domains to membranes., 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 PIP3-containing membranes. The free energy of interaction of the PH domain with more than two PIP3 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.

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.

The 5-HT3A serotonin receptor1, a cationic pentameric ligand-gated ion channel (pLGIC), is the clinical target for management of nausea and vomiting associated with radiation and chemotherapies2. Upon binding, serotonin induces a global conformational change that encompasses the ligand-binding extracellular domain (ECD), the transmembrane domain (TMD) and the intracellular domain (ICD), the molecular details of which are unclear. Here we present two serotonin-bound structures of the full-length 5-HT3A receptor in distinct conformations at 3.32 A and 3.89 A resolution that reveal the mechanism underlying channel activation. In comparison to the apo 5-HT3A receptor, serotonin-bound states underwent a large twisting motion in the ECD and TMD, leading to the opening of a 165 A permeation pathway. Notably, this motion results in the creation of lateral portals for ion permeation at the interface of the TMD and ICD. Combined with molecular dynamics simulations, these structures provide novel insights into conformational coupling across domains and functional modulation. Cryo-electron microscopy structures of the serotonin-bound 5-HT3A serotonin receptor show the receptor populating two distinct states, characterized by twisting in the extracellular and transmembrane domains relative to the apo state, which creates pathways for ion permeation.

The accurate sequencing of DNA using nanopores requires control over the speed of DNA translocation through the pores and also of the DNA conformation. Our studies show that ssDNA translocates through hourglass-shaped pores with hydrophobic constriction regions when an electric field is applied. The constriction provides a barrier to translocation and thereby slows down DNA movement through the pore compared with pores without the constriction. We show that ssDNA moves through these hydrophobic pores in an extended conformation and therefore does not form undesirable secondary structures that may affect the accuracy of partial current blockages for DNA sequencing.

In the present contribution, we report a combined spectroscopic and computational approach aiming to unravel at atomic resolution the effect of the anionic SDS detergent on the structure of two model peptides, the α-helix TrpCage and the β-stranded TrpZip. A detailed characterization of the specific amino acids involved is performed. Monomeric (single molecules) and micellar SDS species differently interact with the α-helix and β-stranded peptides, emphasizing the different mechanisms occurring below and above the critical aggregation concentration (CAC). Below the CAC, the α-helix peptide is fully unfolded, losing its hydrophobic core and its Asp-Arg salt bridge, while the β-stranded peptide keeps its native structure with its four Trp well oriented. Above the CAC, the SDS micelles have the same effect on both peptides, that is, destabilizing the tertiary structure while keeping their secondary structure. Our studies will be helpful to deepen our understanding of the action of the denaturant SDS on peptides and proteins.

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 behaviour within a biological nanopore.