Publications by authors named "Carmen Domene"

91 Publications

Structure based analysis of K channel with a DEND syndrome mutation in murine skeletal muscle.

Sci Rep 2021 Mar 23;11(1):6668. Epub 2021 Mar 23.

Department of Bioregulation and Pharmacological Medicine, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima, 960-1295, Japan.

Developmental delay, epilepsy, and neonatal diabetes (DEND) syndrome, the most severe end of neonatal diabetes mellitus, is caused by mutation in the ATP-sensitive potassium (K) channel. In addition to diabetes, DEND patients present muscle weakness as one of the symptoms, and although the muscle weakness is considered to originate in the brain, the pathological effects of mutated K channels in skeletal muscle remain elusive. Here, we describe the local effects of the K channel on muscle by expressing the mutation present in the K channels of the DEND syndrome in the murine skeletal muscle cell line C2C12 in combination with computer simulation. The present study revealed that the DEND mutation can lead to a hyperpolarized state of the muscle cell membrane, and molecular dynamics simulations based on a recently reported high-resolution structure provide an explanation as to why the mutation reduces ATP sensitivity and reveal the changes in the local interactions between ATP molecules and the channel.
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http://dx.doi.org/10.1038/s41598-021-86121-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7988048PMC
March 2021

The ligand-bound state of a G protein-coupled receptor stabilizes the interaction of functional cholesterol molecules.

J Lipid Res 2021 Feb 26;62:100059. Epub 2021 Feb 26.

Univ. Grenoble Alpes, CNRS, CEA, IBS, Grenoble, France. Electronic address:

Cholesterol is a major component of mammalian plasma membranes that not only affects the physical properties of the lipid bilayer but also is the function of many membrane proteins including G protein-coupled receptors. The oxytocin receptor (OXTR) is involved in parturition and lactation of mammals and in their emotional and social behaviors. Cholesterol acts on OXTR as an allosteric modulator inducing a high-affinity state for orthosteric ligands through a molecular mechanism that has yet to be determined. Using the ion channel-coupled receptor technology, we developed a functional assay of cholesterol modulation of G protein-coupled receptors that is independent of intracellular signaling pathways and operational in living cells. Using this assay, we discovered a stable binding of cholesterol molecules to the receptor when it adopts an orthosteric ligand-bound state. This stable interaction preserves the cholesterol-dependent activity of the receptor in cholesterol-depleted membranes. This mechanism was confirmed using time-resolved FRET experiments on WT OXTR expressed in CHO cells. Consequently, a positive cross-regulation sequentially occurs in OXTR between cholesterol and orthosteric ligands.
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http://dx.doi.org/10.1016/j.jlr.2021.100059DOI Listing
February 2021

Conduction and Gating Properties of the TRAAK Channel from Molecular Dynamics Simulations with Different Force Fields.

J Chem Inf Model 2020 12 9;60(12):6532-6543. Epub 2020 Dec 9.

Department of Pharmacy and Biotechnology, Alma Mater Studiorum-Università di Bologna, via Belmeloro 6, 40126 Bologna, Italy.

In recent years, the K2P family of potassium channels has been the subject of intense research activity. Owing to the complex function and regulation of this family of ion channels, it is common practice to complement experimental findings with the atomistic description provided by computational approaches such as molecular dynamics (MD) simulations, especially, in light of the unprecedented timescales accessible at present. However, despite recent substantial improvements, the accuracy of MD simulations is still undermined by the intrinsic limitations of force fields. Here, we systematically assessed the performance of the most popular force fields employed to study ion channels at timescales that are orders of magnitude greater than the ones accessible when these energy functions were first developed. Using 32 μs of trajectories, we investigated the dynamics of a member of the K2P ion channel family, the TRAAK channel, using two established force fields in simulations of biological systems: AMBER and CHARMM. We found that while results are comparable on the nanosecond timescales, significant inconsistencies arise at microsecond timescales.
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http://dx.doi.org/10.1021/acs.jcim.0c01179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8016162PMC
December 2020

Critical Assessment of Common Force Fields for Molecular Dynamics Simulations of Potassium Channels.

J Chem Theory Comput 2020 Nov 15;16(11):7148-7159. Epub 2020 Oct 15.

Department of Chemistry, University of Bath, Claverton Down, Bath BA2 7AY, U.K.

For the last two decades, the KcsA K channel has served as a case study to understand how potassium channels operate at the atomic scale, and molecular dynamics simulations have contributed significantly to the current knowledge of the atomic mechanisms of conduction, inactivation, and gating in this family of membrane proteins. Currently, microsecond trajectories are becoming the new standard in the field, and consequently, it is critical to assess and compare the performance of the classical force fields ordinarily used in simulations of biological systems as well as to quantitatively assess the agreement with experimental data for trajectories of this order of magnitude. To that extent, we performed classical molecular dynamics simulations with CHARMM36 and AMBER-ff14sb force fields using atomic models based on the experimental structure of the KcsA channel in the open/conductive state, at conditions of ionic concentrations and membrane potentials resembling the ones adopted in experiments. In simulations using the CHARMM force field, the experimental open/conductive structure of the channel exhibited high conformational plasticity and fast collapse toward an occluded state. In contrast, in an identical set of simulations using the AMBER force field, no major deviations from the experimental structure were recorded. Force field development is a complex process in which many approximations are typically required and adopted. The results presented here provide additional motivation to further improve the existing models and, crucially, alert practitioners about limitations.
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http://dx.doi.org/10.1021/acs.jctc.0c00331DOI Listing
November 2020

Differential Stability of Aurein 1.2 Pores in Model Membranes of Two Probiotic Strains.

J Chem Inf Model 2020 10 3;60(10):5142-5152. Epub 2020 Sep 3.

Departamento de Física Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 1, Buenos Aires 1428, Argentina.

Aurein 1.2 is an antimicrobial peptide from the skin secretion of an Australian frog. In the previous experimental work, we reported a differential action of aurein 1.2 on two probiotic strains subsp. (CIDCA 331) and subsp. (CIDCA 133). The differences found were attributed to the bilayer compositions. Cell cultures and CIDCA 331-derived liposomes showed higher susceptibility than the ones derived from the CIDCA 133 strain, leading to content leakage and structural disruption. Here, we used molecular dynamics simulations to explore these systems at the atomistic level. We hypothesize that if the antimicrobial peptides organized themselves to form a pore, it will be more stable in membranes that emulate the CIDCA 331 strain than in those of the CIDCA 133 strain. To test this hypothesis, we simulated preassembled aurein 1.2 pores embedded into bilayer models that emulate the two probiotic strains. It was found that the general behavior of the systems depends on the composition of the membrane rather than the preassemble system characteristics. Overall, it was observed that aurein 1.2 pores are more stable in the CIDCA 331 model membranes. This fact coincides with the high susceptibility of this strain against antimicrobial peptide. In contrast, in the case of the CIDCA 133 model membranes, peptides migrate to the water-lipid interphase, the pore shrinks, and the transport of water through the pore is reduced. The tendency of glycolipids to make hydrogen bonds with peptides destabilizes the pore structures. This feature is observed to a lesser extent in CIDCA 331 due to the presence of anionic lipids. Glycolipid transverse diffusion (flip-flop) between monolayers occurs in the pore surface region in all the cases considered. These findings expand our understanding of the antimicrobial peptide resistance properties of probiotic strains.
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http://dx.doi.org/10.1021/acs.jcim.0c00855DOI Listing
October 2020

Affinity for the Interface Underpins Potency of Antibodies Operating In Membrane Environments.

Cell Rep 2020 08;32(7):108037

Instituto Biofisika (CSIC, UPV/EHU) and Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), PO Box 644, 48080 Bilbao, Spain. Electronic address:

The contribution of membrane interfacial interactions to recognition of membrane-embedded antigens by antibodies is currently unclear. This report demonstrates the optimization of this type of antibodies via chemical modification of regions near the membrane but not directly involved in the recognition of the epitope. Using the HIV-1 antibody 10E8 as a model, linear and polycyclic synthetic aromatic compounds are introduced at selected sites. Molecular dynamics simulations predict the favorable interactions of these synthetic compounds with the viral lipid membrane, where the epitope of the HIV-1 glycoprotein Env is located. Chemical modification of 10E8 with aromatic acetamides facilitates the productive and specific recognition of the native antigen, partially buried in the crowded environment of the viral membrane, resulting in a dramatic increase of its capacity to block viral infection. These observations support the harnessing of interfacial affinity through site-selective chemical modification to optimize the function of antibodies that target membrane-proximal epitopes.
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http://dx.doi.org/10.1016/j.celrep.2020.108037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7861656PMC
August 2020

Effect of anionic lipids on ion permeation through the KcsA K-channel.

Biochim Biophys Acta Biomembr 2020 11 13;1862(11):183406. Epub 2020 Jul 13.

Department of Chemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK; Department of Chemistry, University of Oxford, Oxford OX1 3TA, UK. Electronic address:

K-channels are responsible for the efficient and selective conduction of K ions across the plasma membrane. The bacterial K-channel KcsA has historically been used to characterize various aspects of K conduction via computational means. The energetic barriers associated with ion translocation across the KcsA selectivity filter have been computed in various studies, leading to the proposal of two alternate mechanisms of conduction, involving or neglecting the presence of water molecules in between the permeating ions. Here, the potential of mean force of K permeation is evaluated for KcsA in lipid bilayers containing anionic lipids, which is known to increase the open probability of the channel. In addition, the effect of the protonation/deprotonation of residue E71, which directly interacts with the selectivity filter sequence, is assessed. Both conduction mechanisms are considered throughout. The results obtained provide novel insights into the molecular functioning of K-channels including the inactivation process.
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http://dx.doi.org/10.1016/j.bbamem.2020.183406DOI Listing
November 2020

Mechanism of Molecular Oxygen Diffusion in a Hypoxia-Sensing Prolyl Hydroxylase Using Multiscale Simulation.

J Am Chem Soc 2020 02 23;142(5):2253-2263. Epub 2020 Jan 23.

Chemistry Research Laboratory, Mansfield Road , University of Oxford , Oxford OX1 3TA , United Kingdom.

The chronic response of animals to hypoxia is mediated by the αβ-heterodimeric hypoxia-inducible transcription factors (α,β-HIFs) which upregulate the expression of sets of genes that work to ameliorate the effects of limiting dioxygen. The HIF prolyl hydroxylase domain enzymes (PHDs) are Fe(II)- and 2-oxoglutarate-dependent oxygenases that act as hypoxia-sensing components of the HIF system: prolyl-hydroxylation signals for dioxgen availability-dependent HIF-α degradation via the ubiquitin proteasome system. The unusual kinetic properties of the PHDs, in particular a high for dioxygen and slow reaction with dioxygen, are proposed to enable their hypoxia-sensing role. An understanding of how dioxygen is delivered to, and binds at, the active site of the PHDs is important for the development of a chemical understanding of the hypoxic response. We employed a combined multiscale approach involving classical atomistic equilibrium and nonequilibrium MD simulations combined with QM/MM trajectories to investigate dioxygen diffusion to, and binding at, the active site in the PHD2.Fe(II).2OG.HIF substrate complex; PHD2 is the most important of the three human PHDs. The transport of dioxygen to the active site is described; dioxygen transport follows a single well-defined hydrophobic tunnel, formed from both enzyme and substrate elements to reach the PHD2 active site. The results provide estimates for rate constants that define a diffusion-reaction model for dioxygen:PHD2 interactions; in combination with reported biophysical analyses they provide chemical insight into the basis of the slow reaction of PHD2 with dioxygen. They imply that the reversible binding of dioxygen is central to the hypoxia-sensing capacity of the PHDs and that different PHD HIF-α substrate combinations might have different dioxygen sensitivity profiles. The extent of HIF-α substrate prolyl hydroxylation, which signals for subsequent HIF-α degradation, may thus be a manifestation of the equilibrium between dioxygen in bulk solution and dioxygen bound to the PHD2.Fe.2OG.HIF-α substrate complex.
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http://dx.doi.org/10.1021/jacs.9b09236DOI Listing
February 2020

Insights into the Mechanisms of K Permeation in K Channels from Computer Simulations.

J Chem Theory Comput 2020 Jan 20;16(1):794-799. Epub 2019 Dec 20.

Department of Chemistry , University of Bath , Claverton Down, Bath BA2 7AY , U.K.

Ion permeation, selectivity, and the behavior of the K channel selectivity filter have been studied intensively in the previous two decades. The agreement among multiple approaches used to study ion flux in K channels suggests a consensus mechanism of ion permeation across the selectivity that has been put to the test in recent years with the proposal of an alternative way by which ions can cross the selectivity filter of K channels via direct Coulomb repulsion between contacting cations. Past experimental work by Zhou and MacKinnon (, , 839) showed that mutation of the site S4 reduces the total occupancy of the selectivity filter to less than two ions on average by lowering the occupancy of the S2-S4 configuration without changing the S1-S3 configuration much, and this reduction of occupancy means that ion configurations different from the ones involved in the canonical mechanism are likely to be involved. At that time, calculations using complicated kinetic networks to relate occupancy to conduction did not provide deeper insight into the conduction mechanism. Here, to help solve this enigma, umbrella sampling simulations have been performed to evaluate the potential of mean force of two KcsA mutant channels where the S4 site is substituted. Our new results provide insights into the significance of threonine in this position, revealing the effect of substitution on the alternate mechanisms of conduction proposed, involving either water or vacant sites.
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http://dx.doi.org/10.1021/acs.jctc.9b00971DOI Listing
January 2020

The Bilayer Collective Properties Govern the Interaction of an HIV-1 Antibody with the Viral Membrane.

Biophys J 2020 01 14;118(1):44-56. Epub 2019 Nov 14.

Instituto Biofisika (CSIC, UPV/EHU), Barrio Sarriena s/n, Leioa, Spain; Centro Nacional de Biotecnología, CSIC, Madrid, Spain; Unidad de Nanobiotecnología, CNB-CSIC-IMDEA Nanociencia Associated Unit, Madrid, Spain. Electronic address:

Efficient engagement with the envelope glycoprotein membrane-proximal external region (MPER) results in robust blocking of viral infection by a class of broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV). Developing an accommodation surface that engages with the viral lipid envelope appears to correlate with the neutralizing potency displayed by these bnAbs. The nature of the interactions established between the antibody and the lipid is nonetheless a matter of debate, with some authors arguing that anti-MPER specificity arises only under pathological conditions in autoantibodies endowed with stereospecific binding sites for phospholipids. However, bnAb-lipid interactions are often studied in systems that do not fully preserve the biophysical properties of lipid bilayers, and therefore, questions on binding specificity and the effect of collective membrane properties on the interaction are still open. Here, to evaluate the specificity of lipid interactions of an anti-MPER bnAb (4E10) in an intact membrane context, we determine quantitatively its association with lipid bilayers by means of scanning fluorescence correlation spectroscopy and all-atom molecular dynamic simulations. Our data support that 4E10 establishes electrostatic and hydrophobic interactions with the viral membrane surface and that the collective physical properties of the lipid bilayer influence 4E10 dynamics therein. We conclude that establishment of peripheral, nonspecific electrostatic interactions with the viral membrane through accommodation surfaces may assist high-affinity binding of HIV-1 MPER epitope at membrane interfaces. These findings highlight the importance of considering antibody-lipid interactions in the design of antibody-based anti-HIV strategies.
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http://dx.doi.org/10.1016/j.bpj.2019.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6950647PMC
January 2020

Modulation of the potassium channel KcsA by anionic phospholipids: Role of arginines at the non-annular lipid binding sites.

Biochim Biophys Acta Biomembr 2019 10 24;1861(10):183029. Epub 2019 Jul 24.

Department of Chemistry, University of Bath, 1 South Bldg., Claverton Down, Bath BA2 7AY, United Kingdom; Department of Chemistry, University of Oxford, Oxford OX1 3TA, Oxford, United Kingdom. Electronic address:

The role of arginines R64 and R89 at non-annular lipid binding sites of KcsA, on the modulation of channel activity by anionic lipids has been investigated. In wild-type (WT) KcsA reconstituted into asolectin lipid membranes, addition of phosphatidic acid (PA) drastically reduces inactivation in macroscopic current recordings. Consistent to this, PA increases current amplitude, mean open time and open probability at the single channel level. Moreover, kinetic analysis reveals that addition of PA causes longer open channel lifetimes and decreased closing rate constants. Effects akin to those of PA on WT-KcsA are observed when R64 and/or R89 are mutated to alanine, regardless of the added anionic lipids. We interpret these results as a consequence of interactions between the arginines and the anionic PA bound to the non-annular sites. NMR data shows indeed that at least R64 is involved in binding PA. Moreover, molecular dynamics (MD) simulations predict that R64, R89 and surrounding residues such as T61, mediate persistent binding of PA to the non-annular sites. Channel inactivation depends on interactions within the inactivation triad (E71-D80-W67) behind the selectivity filter. Therefore, it is expected that such interactions are affected when PA binds the arginines at the non-annular sites. In support of this, MD simulations reveal that PA binding prevents interaction between R89 and D80, which seems critical to the effectiveness of the inactivation triad. This mechanism depends on the stability of the bound lipid, favoring anionic headgroups such as that of PA, which thrive on the positive charge of the arginines.
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http://dx.doi.org/10.1016/j.bbamem.2019.183029DOI Listing
October 2019

Combining Structural Data with Computational Methodologies to Investigate Structure-Function Relationships in TRP Channels.

Methods Mol Biol 2019 ;1987:65-82

Department of Chemistry, University of Bath, Bath, UK.

Since the emergence of high-resolution three-dimensional structures of membrane proteins, and the increasing availability of state-of-the-art algorithms and high-performance-computing facilities, classical molecular dynamics (MD) simulations have become a routine device to explore the molecular behavior of these proteins. The rise of cryo-electron microscopy (cryo-EM) as a credible experimental tool to resolve structures at an atomic level has revolutionized structural biology in recent years, culminating in the disclosure of the first high-resolution three-dimensional structure of a transient receptor potential (TRP) channel, the vanilloid receptor 1 (TRPV1). As a result, the number of research articles investigating the molecular behavior of TRP channels using macromolecular simulation techniques has proliferated. This review provides an overview of the current state of this field, including our understanding of TRP channel structure, the framework of classical MD simulations, and how to perform such simulations to investigate structure-function relationships in TRP channels.
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http://dx.doi.org/10.1007/978-1-4939-9446-5_5DOI Listing
January 2020

Influence of Cholesterol and Its Stereoisomers on Members of the Serotonin Receptor Family.

J Mol Biol 2019 04 8;431(8):1633-1649. Epub 2019 Mar 8.

Department of Chemistry, University of Bath, Claverton Down, Bath, BA2 7AY, UK; Department of Chemistry, University of Oxford, Oxford, OX1 3TA, Oxford, UK. Electronic address:

Despite the ubiquity of cholesterol within the cell membrane, the mechanism by which it influences embedded proteins remains elusive. Numerous G-protein coupled receptors exhibit dramatic responses to membrane cholesterol with regard to the ligand-binding affinity and functional properties, including the 5-HT receptor family. Here, we use over 25 μs of unbiased atomistic molecular dynamics simulations to identify cholesterol interaction sites in the 5-HT and 5-HT receptors and evaluate their impact on receptor structure. Susceptibility to membrane cholesterol is shown to be subtype dependent and determined by the quality of interactions between the extracellular loops. Charged residues are essential for maintaining the arrangement of the extracellular surface in 5-HT; in the absence of such interactions, the extracellular surface of the 5-HT is malleable, populating a number of distinct conformations. Elevated cholesterol density near transmembrane helix 4 is considered to be conducive to the conformation of extracellular loop 2. Occupation of this site is also shown to be stereospecific, illustrated by differential behavior of nat-cholesterol isomers, ent- and epi-cholesterol. In simulations containing the endogenous agonist, serotonin, cholesterol binding at transmembrane helix 4 biases bound serotonin molecules toward an unexpected binding mode in the extended binding pocket. The results highlight the capability of membrane cholesterol to influence the mobility of the extracellular surface in the 5-HT receptor family and manipulate the architecture of the extracellular ligand-binding pocket.
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http://dx.doi.org/10.1016/j.jmb.2019.02.030DOI Listing
April 2019

Antibiotic resistance and host immune evasion in mediated by a metabolic adaptation.

Proc Natl Acad Sci U S A 2019 02 11;116(9):3722-3727. Epub 2019 Feb 11.

Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, VIC 3800, Australia;

is a notorious human bacterial pathogen with considerable capacity to develop antibiotic resistance. We have observed that human infections caused by highly drug-resistant are more prolonged, complicated, and difficult to eradicate. Here we describe a metabolic adaptation strategy used by clinical strains that leads to resistance to the last-line antibiotic, daptomycin, and simultaneously affects host innate immunity. This response was characterized by a change in anionic membrane phospholipid composition induced by point mutations in the phospholipid biosynthesis gene, , encoding cardiolipin synthase. Single point mutations were sufficient for daptomycin resistance, antibiotic treatment failure, and persistent infection. These phenotypes were mediated by enhanced cardiolipin biosynthesis, leading to increased bacterial membrane cardiolipin and reduced phosphatidylglycerol. The changes in membrane phospholipid profile led to modifications in membrane structure that impaired daptomycin penetration and membrane disruption. The point mutations also allowed to evade neutrophil chemotaxis, mediated by the reduction in bacterial membrane phosphatidylglycerol, a previously undescribed bacterial-driven chemoattractant. Together, these data illustrate a metabolic strategy used by to circumvent antibiotic and immune attack and provide crucial insights into membrane-based therapeutic targeting of this troublesome pathogen.
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http://dx.doi.org/10.1073/pnas.1812066116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397524PMC
February 2019

Capturing the Molecular Mechanism of Anesthetic Action by Simulation Methods.

Chem Rev 2019 05 25;119(9):5998-6014. Epub 2018 Oct 25.

Department of Chemistry , University of Bath , Claverton Down, Bath BA2 7AY , United Kingdom.

Significant computational efforts have been focused toward exposing the molecular mechanisms of anesthesia in recent years. In the past decade, this has been aided considerably by a momentous increase in the number of high-resolution structures of ion channels, which are putative targets for the anesthetic agents, as well as advancements in high-performance computing technologies. In this review, typical simulation methods to investigate the behavior of model membranes and membrane-protein systems are briefly reviewed, and related computational studies are surveyed. Both lipid- and protein-mediated mechanisms of anesthetic action are scrutinized, focusing on the behavior of ion channels in the latter case.
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http://dx.doi.org/10.1021/acs.chemrev.8b00366DOI Listing
May 2019

Location and Character of Volatile General Anesthetics Binding Sites in the Transmembrane Domain of TRPV1.

Mol Pharm 2018 09 16;15(9):3920-3930. Epub 2018 Aug 16.

Department of Chemistry , King's College London , Britannia House, 7 Trinity Street , London SE1 1DB , U.K.

It has been proposed that general anesthesia results from direct multisite interactions with multiple and diverse ion channels in the brain. An understanding of the mechanisms by which general anesthetics modulate ion channels is essential to clarify their underlying behavior and their role in reversible immobilization and amnesia. Despite the fact that volatile general anesthetics are drugs that primarily induce insensitivity to pain, they have been reported to sensitize and active the vanilloid-1 receptor, TRPV1, which is known to mediate the response of the nervous system to certain harmful stimuli and which plays a crucial role in the pain pathway. Currently, the mechanism of action of anesthetics is unknown and the precise molecular sites of interaction have not been identified. Here, using ∼2.5 μs of classical molecular dynamics simulations and metadynamics, we explore these enigmas. Binding sites are identified and the strength of the association is further characterized using alchemical free-energy calculations. Anesthetic binding/unbinding proceeds primarily through a membrane-embedded pathway, and subsequently, a complex scenario is established involving multiple binding sites featuring single or multiple occupancy states of two small volatile drugs. One of the five anesthetic binding sites reported was previously identified experimentally, and another one, importantly, is identical to that of capsaicin, one of the chemical stimuli that activate TRPV1. However, in contrast to capsaicin, isoflurane and chloroform binding free-energies render modest to no association compared to capsaicin, suggesting a different activation mechanism. Uncovering chloroform and isoflurane modulatory sites will further our understanding of the TRPV1 molecular machinery and open the possibility of developing site-specific drugs.
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http://dx.doi.org/10.1021/acs.molpharmaceut.8b00381DOI Listing
September 2018

Ion-triggered selectivity in bacterial sodium channels.

Proc Natl Acad Sci U S A 2018 05 7;115(21):5450-5455. Epub 2018 May 7.

Department of Chemistry, University of Bath, Claverton Down, BA2 7AY Bath, United Kingdom.

Since the availability of the first crystal structure of a bacterial Na channel in 2011, understanding selectivity across this family of membrane proteins has been the subject of intense research efforts. Initially, free energy calculations based on molecular dynamics simulations revealed that although sodium ions can easily permeate the channel with their first hydration shell almost intact, the selectivity filter is too narrow for efficient conduction of hydrated potassium ions. This steric view of selectivity was subsequently questioned by microsecond atomic trajectories, which proved that the selectivity filter appears to the permeating ions as a highly degenerate, liquid-like environment. Although this liquid-like environment looks optimal for rapid conduction of Na, it seems incompatible with efficient discrimination between similar ion species, such as Na and K, through steric effects. Here extensive molecular dynamics simulations, combined with Markov state model analyses, reveal that at positive membrane potentials, potassium ions trigger a conformational change of the selectivity toward a nonconductive metastable state. It is this transition of the selectivity filter, and not steric effects, that prevents the outward flux of K at positive membrane potentials. This description of selectivity, triggered by the nature of the permeating ions, might have implications on the current understanding of how ion channels, and in particular bacterial Na channels, operate at the atomic scale.
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http://dx.doi.org/10.1073/pnas.1722516115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003493PMC
May 2018

2-Oxoglutarate regulates binding of hydroxylated hypoxia-inducible factor to prolyl hydroxylase domain 2.

Chem Commun (Camb) 2018 Mar 9;54(25):3130-3133. Epub 2018 Mar 9.

Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, UK.

Prolyl hydroxylation of hypoxia inducible factor (HIF)-α, as catalysed by the Fe(ii)/2-oxoglutarate (2OG)-dependent prolyl hydroxylase domain (PHD) enzymes, has a hypoxia sensing role in animals. We report that binding of prolyl-hydroxylated HIF-α to PHD2 is ∼50 fold hindered by prior 2OG binding; thus, when 2OG is limiting, HIF-α degradation might be inhibited by PHD binding.
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http://dx.doi.org/10.1039/c8cc00387dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5885369PMC
March 2018

Role of Zinc and Magnesium Ions in the Modulation of Phosphoryl Transfer in Protein Tyrosine Phosphatase 1B.

J Am Chem Soc 2018 03 19;140(12):4446-4454. Epub 2018 Mar 19.

Department of Chemistry , King's College London , Britannia House, 7 Trinity Street , London SE1 1DB , United Kingdom.

While the majority of phosphatases are metalloenzymes, the prevailing model for the reactions catalyzed by protein tyrosine phosphatases does not involve any metal ion, yet both metal cations and oxoanions affect their enzymatic activity. Mg and Zn activate and inhibit, respectively, protein tyrosine phosphatase 1B (PTP1B). Molecular dynamics simulations, metadynamics, and quantum chemical calculations in combination with experimental investigations demonstrate that Mg and Zn compete for the same binding site in the active site only in the closed conformation of the enzyme in its phosphorylated state. The two cations have different effects on the arrangements and activities of water molecules that are necessary for the hydrolysis of the phosphocysteine intermediate in the second catalytic step of the reaction. Remarkable differences between the established structural enzymology of PTP1B investigated ex vivo and the function of PTP1B in vivo become evident. Different reaction pathways are viable when the presence of metal ions and their cellular concentrations are considered. The findings suggest that the substrate delivers the inhibitory Zn ion to the active site. The inhibition and activation can be ascribed to the different coordination chemistries of Zn and Mg ions and the orientation of the metal-coordinated water molecules. Metallochemistry adds an additional dimension to the regulation of PTP1B and presumably other members of this enzyme family.
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http://dx.doi.org/10.1021/jacs.8b01534DOI Listing
March 2018

Exposure of the HIV-1 broadly neutralizing antibody 10E8 MPER epitope on the membrane surface by gp41 transmembrane domain scaffolds.

Biochim Biophys Acta Biomembr 2018 Jun 23;1860(6):1259-1271. Epub 2018 Feb 23.

Biofisika Institute (CSIC, UPV/EHU), Biochemistry and Molecular Biology Department, University of the Basque Country (UPV/EHU), PO Box 644, 48080 Bilbao, Spain. Electronic address:

The 10E8 antibody achieves near-pan neutralization of HIV-1 by targeting the remarkably conserved gp41 membrane-proximal external region (MPER) and the connected transmembrane domain (TMD) of the HIV-1 envelope glycoprotein (Env). Thus, recreating the structure that generates 10E8-like antibodies is a major goal of the rational design of anti-HIV vaccines. Unfortunately, high-resolution information of this segment in the native Env is lacking, limiting our understanding of the behavior of the crucial 10E8 epitope residues. In this report, two sequences, namely, MPER-TMD1 (gp41 residues 671-700) and MPER-TMD2 (gp41 residues 671-709) were compared both experimentally and computationally, to assess the TMD as a potential membrane integral scaffold for the 10E8 epitope. These sequences were selected to represent a minimal (MPER-TMD1) or full-length (MPER-TMD2) TMD membrane anchor according to mutagenesis results reported by Yue et al. (2009) J. Virol. 83, 11,588. Immunochemical assays revealed that MPER-TMD1, but not MPER-TMD2, effectively exposed the MPER C-terminal stretch, harboring the 10E8 epitope on the surface of phospholipid bilayers containing a cholesterol concentration equivalent to that of the viral envelope. Molecular dynamics simulations, using the recently resolved TMD trimer structure combined with the MPER in a cholesterol-enriched model membrane confirmed these results and provided an atomistic mechanism of epitope exposure which revealed that TMD truncation at position A700 combined with N-terminal addition of lysine residues positively impacts epitope exposure. Overall, these results provide crucial insights into the design of effective MPER-TMD derived immunogens.
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http://dx.doi.org/10.1016/j.bbamem.2018.02.019DOI Listing
June 2018

Stereospecific Interactions of Cholesterol in a Model Cell Membrane: Implications for the Membrane Dipole Potential.

J Membr Biol 2018 06 30;251(3):507-519. Epub 2018 Jan 30.

Department of Chemistry, University of Bath, 1 South Building, Claverton Down Road, Bath, BA2 7AY, UK.

Cholesterol is a major constituent of the plasma membrane in higher order eukaryotes. The effect of cholesterol on the structure and organisation of cell membranes has been studied extensively by both experimental and computational means. In recent years, a wealth of data has been accumulated illustrating how subtle differences in the structure of cholesterol equate to considerable changes in the physical properties of the membrane. The effect of cholesterol stereoisomers, in particular, has been established, identifying a direct link with the activity of specific membrane proteins. In this study, we perform extensive molecular dynamics simulations of phospholipid bilayers containing three isomers of cholesterol, the native form (nat-cholesterol), the enantiomer of the native form (ent-cholesterol), and an epimer of cholesterol that differs by the orientation of the polar hydroxyl group (epi-cholesterol). Based on these simulations, an atomic-level description of the stereospecific cholesterol-phospholipid interactions is provided, establishing a potential mechanism for the perturbation of membrane properties, specifically the membrane dipole potential.
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http://dx.doi.org/10.1007/s00232-018-0016-0DOI Listing
June 2018

Oxytocin Modulates Nociception as an Agonist of Pain-Sensing TRPV1.

Cell Rep 2017 Nov;21(6):1681-1691

Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine, 1 Illini Drive, Peoria, IL 61605, USA. Electronic address:

Oxytocin is a hormone with various actions. Oxytocin-containing parvocellular neurons project to the brainstem and spinal cord. Oxytocin release from these neurons suppresses nociception of inflammatory pain, the molecular mechanism of which remains unclear. Here, we report that the noxious stimulus receptor TRPV1 is an ionotropic oxytocin receptor. Oxytocin elicits TRPV1 activity in native and heterologous expression systems, regardless of the presence of the classical oxytocin receptor. In TRPV1 knockout mice, DRG neurons exhibit reduced oxytocin sensitivity relative to controls, and oxytocin injections significantly attenuate capsaicin-induced nociception in in vivo experiments. Furthermore, oxytocin potentiates TRPV1 in planar lipid bilayers, supporting a direct agonistic action. Molecular modeling and simulation experiments provide insight into oxytocin-TRPV1 interactions, which resemble DkTx. Together, our findings suggest the existence of endogenous regulatory pathways that modulate nociception via direct action of oxytocin on TRPV1, implying its analgesic effect via channel desensitization.
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http://dx.doi.org/10.1016/j.celrep.2017.10.063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5701661PMC
November 2017

Functional mapping of the N-terminal arginine cluster and C-terminal acidic residues of Kir6.2 channel fused to a G protein-coupled receptor.

Biochim Biophys Acta Biomembr 2017 Oct 28;1859(10):2144-2153. Epub 2017 Jul 28.

Institut de Biologie Structurale (IBS), Univ. Grenoble Alpes, CEA, CNRS, LabEx ICST, 71, avenue des Martyrs, CS10090, F-38044 Grenoble, France. Electronic address:

Ion channel-coupled receptors (ICCRs) are original man-made ligand-gated ion channels created by fusion of G protein-coupled receptors (GPCRs) to the inward-rectifier potassium channel Kir6.2. GPCR conformational changes induced by ligand binding are transduced into electrical current by the ion channel. This functional coupling is closely related to the length of the linker region formed by the GPCR C-terminus (C-ter) and Kir6.2N-terminus (N-ter). Manipulating the GPCR C-ter length allows to finely tune the channel regulation, both in amplitude and sign (opening or closing Kir6.2). In this work, we demonstrate that the primary sequence of the channel N-terminal domain is an additional parameter for the functional coupling with GPCRs. As for all Kir channels, a cluster of basic residues is present in the N-terminal domain of Kir6.2 and is composed of 5 arginines which are proximal to the GPCR C-ter in the fusion proteins. Using a functional mapping approach, we demonstrate the role of specific arginines (R27 and R32) for the function of ICCRs, indicating that the position and not the cluster of positively-charged arginines is critical for the channel regulation by the GPCR. Following observations provided by molecular dynamics simulation, we explore the hypothesis of interaction of these arginines with acidic residues, and using site-directed mutagenesis, we identified aspartate D307 and glutamate E308 residues as critical for the function of ICCRs. These results demonstrate the critical role of the N-terminal and C-terminal charged residues of Kir6.2 for its allosteric regulation by the fused GPCR.
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http://dx.doi.org/10.1016/j.bbamem.2017.07.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5599172PMC
October 2017

C-Carbamylation as a mechanistic probe for the inhibition of class D β-lactamases by avibactam and halide ions.

Org Biomol Chem 2017 Jul;15(28):6024-6032

Department of Chemistry, University of Oxford, Oxford, OX1 3TA, UK.

The class D (OXA) serine β-lactamases are a major cause of resistance to β-lactam antibiotics. The class D enzymes are unique amongst β-lactamases because they have a carbamylated lysine that acts as a general acid/base in catalysis. Previous crystallographic studies led to the proposal that β-lactamase inhibitor avibactam targets OXA enzymes in part by promoting decarbamylation. Similarly, halide ions are proposed to inhibit OXA enzymes via decarbamylation. NMR analyses, in which the carbamylated lysines of OXA-10, -23 and -48 were C-labelled, indicate that reaction with avibactam does not ablate lysine carbamylation in solution. While halide ions did not decarbamylate the C-labelled OXA enzymes in the absence of substrate or inhibitor, avibactam-treated OXA enzymes were susceptible to decarbamylation mediated by halide ions, suggesting halide ions may inhibit OXA enzymes by promoting decarbamylation of acyl-enzyme complex. Crystal structures of the OXA-10 avibactam complex were obtained with bromide, iodide, and sodium ions bound between Trp-154 and Lys-70. Structures were also obtained wherein bromide and iodide ions occupy the position expected for the 'hydrolytic water' molecule. In contrast with some solution studies, Lys-70 was decarbamylated in these structures. These results reveal clear differences between crystallographic and solution studies on the interaction of class D β-lactamases with avibactam and halides, and demonstrate the utility of C-NMR for studying lysine carbamylation in solution.
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http://dx.doi.org/10.1039/c7ob01514cDOI Listing
July 2017

Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication.

Mol Cell 2017 Apr 13;66(2):206-220.e9. Epub 2017 Apr 13.

Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford OX3 7DQ, UK. Electronic address:

Cells exposed to hypoxia experience replication stress but do not accumulate DNA damage, suggesting sustained DNA replication. Ribonucleotide reductase (RNR) is the only enzyme capable of de novo synthesis of deoxyribonucleotide triphosphates (dNTPs). However, oxygen is an essential cofactor for mammalian RNR (RRM1/RRM2 and RRM1/RRM2B), leading us to question the source of dNTPs in hypoxia. Here, we show that the RRM1/RRM2B enzyme is capable of retaining activity in hypoxia and therefore is favored over RRM1/RRM2 in order to preserve ongoing replication and avoid the accumulation of DNA damage. We found two distinct mechanisms by which RRM2B maintains hypoxic activity and identified responsible residues in RRM2B. The importance of RRM2B in the response to tumor hypoxia is further illustrated by correlation of its expression with a hypoxic signature in patient samples and its roles in tumor growth and radioresistance. Our data provide mechanistic insight into RNR biology, highlighting RRM2B as a hypoxic-specific, anti-cancer therapeutic target.
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http://dx.doi.org/10.1016/j.molcel.2017.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5405111PMC
April 2017

Membrane Phase-Dependent Occlusion of Intramolecular GLUT1 Cavities Demonstrated by Simulations.

Biophys J 2017 Mar;112(6):1176-1184

Department of Chemistry, School of Medicine, King's College London, London, United Kingdom; Chemistry Research Laboratory, University of Oxford, Oxford, United Kingdom. Electronic address:

Experimental evidence has shown a close correlation between the composition and physical state of the membrane bilayer and glucose transport activity via the glucose transporter GLUT1. Cooling alters the membrane lipids from the fluid to gel phase, and also causes a large decrease in the net glucose transport rate. The goal of this study is to investigate how the physical phase of the membrane alters glucose transporter structural dynamics using molecular-dynamics simulations. Simulations from an initial fluid to gel phase reduce the size of the cavities and tunnels traversing the protein and connecting the external regions of the transporter and the central binding site. These effects can be ascribed solely to membrane structural changes since in silico cooling of the membrane alone, while maintaining the higher protein temperature, shows protein structural and dynamic changes very similar to those observed with uniform cooling. These results demonstrate that the protein structure is sensitive to the membrane phase, and have implications for how transmembrane protein structures respond to their physical environment.
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http://dx.doi.org/10.1016/j.bpj.2017.01.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5376106PMC
March 2017

A perspective on structural and computational work on collagen.

Phys Chem Chem Phys 2016 Sep;18(36):24802-24811

Department of Chemistry, King's College London, Britannia House, 7 Trinity Street, London SE1 1DB, UK.

Collagen is the single most abundant protein in the extracellular matrix in the animal kingdom, with remarkable structural and functional diversity and regarded one of the most useful biomaterials. Etymologically, the term collagen comes from Greek kola 'glue' and gen 'giving birth to'. Thus, it is not surprising that the various collagens and the structures they form all serve the same purpose, to help tissues withstand stretching. Among the functions the various collagens are involved in are cell adhesion and migration, tissue repair, scaffolding and morphogenesis. Thus knowledge about the structure and properties of collagen, how they change depending on the nature of the local environment as well as the nature and specificity of collagen interactions with its partners is central to discerning the role of collagen in medical applications such as imaging, drug delivery and tissue engineering, and in the design and construction of synthetic collagen-like materials for tools in biomaterial science and nanotechnology. The main focus of this perspective is to review the molecular and packing structures of collagen and the computer simulations work performed up to now to further highlight the significance of collagen.
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http://dx.doi.org/10.1039/c6cp03403aDOI Listing
September 2016

Energetics of Ion Permeation in an Open-Activated TRPV1 Channel.

Biophys J 2016 Sep;111(6):1214-1222

Department of Chemistry, King's College London, London, United Kingdom; Chemistry Research Laboratory, University of Oxford, Oxford, United Kingdom. Electronic address:

Ion channels enable diffusion of ions down physiological electrochemical gradients. Modulation of ion permeation is crucial for the physiological functioning of cells, and misregulation of ion channels is linked to a myriad of channelopathies. The ion permeation mechanism in the transient receptor potential (TRP) ion channel family is currently not understood at an atomistic level. In this work, we employed a simulation strategy for ion permeation (molecular-dynamics simulations with bias-exchange metadynamics) to study and compare monovalent (Na(+), K(+)) ion permeation in the open-activated TRP vanniloid-1 (TRPV1) ion channel. Using ∼3.6 μs of simulation trajectories, we obtained atomistic evidence for the nonselective nature of TRPV1. Our analysis shows that solvated monovalent ions permeate through the selectivity filter with comparable energetic barriers via a two-site mechanism. Finally, we confirmed that an intracellular binding site is located between the intracellular gate residues I679 and E684.
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http://dx.doi.org/10.1016/j.bpj.2016.08.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5034363PMC
September 2016

Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases.

Nat Commun 2016 08 26;7:12673. Epub 2016 Aug 26.

Chemistry Research Laboratory, Department of Chemistry, Oxford Centre for Integrative Systems Biology, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK.

The response to hypoxia in animals involves the expression of multiple genes regulated by the αβ-hypoxia-inducible transcription factors (HIFs). The hypoxia-sensing mechanism involves oxygen limited hydroxylation of prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of HIFα isoforms, as catalysed by prolyl hydroxylases (PHD 1-3). Prolyl hydroxylation promotes binding of HIFα to the von Hippel-Lindau protein (VHL)-elongin B/C complex, thus signalling for proteosomal degradation of HIFα. We reveal that certain PHD2 variants linked to familial erythrocytosis and cancer are highly selective for CODD or NODD. Crystalline and solution state studies coupled to kinetic and cellular analyses reveal how wild-type and variant PHDs achieve ODD selectivity via different dynamic interactions involving loop and C-terminal regions. The results inform on how HIF target gene selectivity is achieved and will be of use in developing selective PHD inhibitors.
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http://dx.doi.org/10.1038/ncomms12673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007464PMC
August 2016