Publications by authors named "Himanshu Khandelia"

68 Publications

An Intracellular Pathway Controlled by the N-terminus of the Pump Subunit Inhibits the Bacterial KdpFABC Ion Pump in High K Conditions.

J Mol Biol 2021 May 2;433(15):167008. Epub 2021 May 2.

PHYLIFE: Physical Life Science, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, Odense 5230 M, Denmark. Electronic address:

The heterotetrameric bacterial KdpFABC transmembrane protein complex is an ion channel-pump hybrid that consumes ATP to import K against its transmembrane chemical potential gradient in low external K environments. The KdpB ion-pump subunit of KdpFABC is a P-type ATPase, and catalyses ATP hydrolysis. Under high external K conditions, K can diffuse into the cells through passive ion channels. KdpFABC must therefore be inhibited in high K conditions to conserve cellular ATP. Inhibition is thought to occur via unusual phosphorylation of residue Ser162 of the TGES motif of the cytoplasmic A domain. It is proposed that phosphorylation most likely traps KdpB in an inactive E1-P like conformation, but the molecular mechanism of phosphorylation-mediated inhibition remains unknown. Here, we employ molecular dynamics (MD) simulations of the dephosphorylated and phosphorylated versions of KdpFABC to demonstrate that phosphorylated KdpB is trapped in a conformation where the ion-binding site is hydrated by an intracellular pathway between transmembrane helices M1 and M2 which opens in response to the rearrangement of cytoplasmic domains resulting from phosphorylation. Cytoplasmic access of water to the ion-binding site is accompanied by a remarkable loss of secondary structure of the KdpB N-terminus and disruption of a key salt bridge between Glu87 in the A domain and Arg212 in the P domain. Our results provide the molecular basis of a unique mechanism of regulation amongst P-type ATPases, and suggest that the N-terminus has a significant role to play in the conformational cycle and regulation of KdpFABC.
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http://dx.doi.org/10.1016/j.jmb.2021.167008DOI Listing
May 2021

EnCurv: Simple Technique of Maintaining Global Membrane Curvature in Molecular Dynamics Simulations.

J Chem Theory Comput 2021 Feb 29;17(2):1181-1193. Epub 2021 Jan 29.

PHYLIFE: Physical Life Science, Department of Physics Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, Odense 5230 M, Denmark.

The EnCurv method for maintaining membrane curvature in molecular dynamics simulations is introduced. The method allows maintaining any desired curvature in a sector of lipid membrane bent in a single plane without adding any unphysical interactions into the system and without restrictions on lateral and transversal lipid diffusion and distribution. The current implementation is limited to the membranes curved in a single plane but generalization to arbitrary curvature and membrane topology is possible. The method is simple, easy to implement, and scales linearly with the system size. EnCurv is agnostic to the force field, simulation parameters, and membrane composition. The proof of principle implementation (https://github.com/yesint/EnCurv) is compatible with the majority of modern simulation packages and shows consistent results on the model systems.
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http://dx.doi.org/10.1021/acs.jctc.0c00800DOI Listing
February 2021

Cholesterol binding to the sterol-sensing region of Niemann Pick C1 protein confines dynamics of its N-terminal domain.

PLoS Comput Biol 2020 10 6;16(10):e1007554. Epub 2020 Oct 6.

PhyLife Physical Life Sciences, Department of Physics Chemistry, and Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.

Lysosomal accumulation of cholesterol is a hallmark of Niemann Pick type C (NPC) disease caused by mutations primarily in the lysosomal membrane protein NPC1. NPC1 contains a transmembrane sterol-sensing domain (SSD), which is supposed to regulate protein activity upon cholesterol binding, but the mechanisms underlying this process are poorly understood. Using atomistic simulations, we show that in the absence of cholesterol in the SSD, the luminal domains of NPC1 are highly dynamic, resulting in the disengagement of the NTD from the rest of the protein. The disengaged NPC1 adopts a flexed conformation that approaches the lipid bilayer, and could represent a conformational state primed to receive a sterol molecule from the soluble lysosomal cholesterol carrier NPC2. The binding of cholesterol to the SSD of NPC1 allosterically suppresses the conformational dynamics of the luminal domains resulting in an upright NTD conformation. The presence of an additional 20% cholesterol in the membrane has negligible impact on this process. The additional presence of an NTD-bound cholesterol suppresses the flexing of the NTD. We propose that cholesterol acts as an allosteric effector, and the modulation of NTD dynamics by the SSD-bound cholesterol constitutes an allosteric feedback mechanism in NPC1 that controls cholesterol abundance in the lysosomal membrane.
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http://dx.doi.org/10.1371/journal.pcbi.1007554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7537887PMC
October 2020

Serine phosphorylation regulates the P-type potassium pump KdpFABC.

Elife 2020 09 21;9. Epub 2020 Sep 21.

Skirball Institute, Dept. of Cell Biology, New York University School of Medicine, New York, United States.

KdpFABC is an ATP-dependent K pump that ensures bacterial survival in K-deficient environments. Whereas transcriptional activation of kdpFABC expression is well studied, a mechanism for down-regulation when K levels are restored has not been described. Here, we show that KdpFABC is inhibited when cells return to a K-rich environment. The mechanism of inhibition involves phosphorylation of Ser162 on KdpB, which can be reversed in vitro by treatment with serine phosphatase. Mutating Ser162 to Alanine produces constitutive activity, whereas the phosphomimetic Ser162Asp mutation inactivates the pump. Analyses of the transport cycle show that serine phosphorylation abolishes the K-dependence of ATP hydrolysis and blocks the catalytic cycle after formation of the aspartyl phosphate intermediate (E1~P). This regulatory mechanism is unique amongst P-type pumps and this study furthers our understanding of how bacteria control potassium homeostasis to maintain cell volume and osmotic potential.
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http://dx.doi.org/10.7554/eLife.55480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7535926PMC
September 2020

Annexin A4 trimers are recruited by high membrane curvatures in giant plasma membrane vesicles.

Soft Matter 2021 Jan;17(2):308-318

Niels Bohr Institute, University of Copenhagen, Denmark.

The plasma membrane (PM) of eukaryotic cells consists of a crowded environment comprised of a high diversity of proteins in a complex lipid matrix. The lateral organization of membrane proteins in the PM is closely correlated with biological functions such as endocytosis, membrane budding and other processes which involve protein mediated shaping of the membrane into highly curved structures. Annexin A4 (ANXA4) is a prominent player in a number of biological functions including PM repair. Its binding to membranes is activated by Ca2+ influx and it is therefore rapidly recruited to the cell surface near rupture sites where Ca2+ influx takes place. However, the free edges near rupture sites can easily bend into complex curvatures and hence may accelerate recruitment of curvature sensing proteins to facilitate rapid membrane repair. To analyze the curvature sensing behavior of curvature inducing proteins in crowded membranes, we quantifify the affinity of ANXA4 monomers and trimers for high membrane curvatures by extracting membrane nanotubes from giant PM vesicles (GPMVs). ANXA4 is found to be a sensor of negative membrane curvatures. Multiscale simulations, in which we extract molecular information from atomistic scale simulations as input to our macroscopic scale simulations, furthermore predicted that ANXA4 trimers generate membrane curvature upon binding and have an affinity for highly curved membrane regions only within a well defined membrane curvature window. Our results indicate that curvature sensing and mobility of ANXA4 depend on the trimer structure of ANXA4 which could provide new biophysical insight into the role of ANXA4 in membrane repair and other biological processes.
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http://dx.doi.org/10.1039/d0sm00241kDOI Listing
January 2021

Shuffled lipidation pattern and degree of lipidation determines the membrane interaction behavior of a linear cationic membrane-active peptide.

J Colloid Interface Sci 2020 Oct 3;578:584-597. Epub 2020 Jun 3.

Centerfor Biopharmaceuticals and Biobarriers in Drug Delivery, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark. Electronic address:

Hypothesis: Permeation of macromolecular drugs across biological plasma membranes is a major challenge in drug delivery. Cationic cell-penetrating peptides (CPPs) are attractive functional excipient candidates for the delivery of macromolecules across membrane barriers, due to their membrane translocating ability. The properties of CPPs can be tailored by lipidation, a promising approach to facilitate enhanced membrane insertion, potentially promoting increased translocation of the CPP and cargo.

Experiments: To explore the impact that site and degree of lipidation have on the membrane interaction of a cationic CPP, we designed and investigated CPP conjugates with one or two fatty acid chains.

Findings: Compared to the parent CPP and the single-lipidated conjugates, the double-lipidated conjugate exhibited the most pronounced membrane perturbation effects, as measured by several biophysical techniques. The experimental findings were supported by molecular dynamics (MD) simulations, demonstrating that all CPP conjugates interacted with the membrane by insertion of the lipid chain(s) into the core of the bilayer. Moreover, membrane-thinning effects and induced membrane curvature were displayed upon CPP interaction. Our results demonstrate that the impact exerted by the CPP on the membrane is notably affected by positioning and especially the degree of lipidation, which might influence the properties of CPPs as functional excipients.
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http://dx.doi.org/10.1016/j.jcis.2020.05.121DOI Listing
October 2020

Clearance of activity-evoked K transients and associated glia cell swelling occur independently of AQP4: A study with an isoform-selective AQP4 inhibitor.

Glia 2021 Jan 7;69(1):28-41. Epub 2020 Jun 7.

Department of Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

The mammalian brain consists of 80% water, which is continuously shifted between different compartments and cellular structures by mechanisms that are, to a large extent, unresolved. Aquaporin 4 (AQP4) is abundantly expressed in glia and ependymal cells of the mammalian brain and has been proposed to act as a gatekeeper for brain water dynamics, predominantly based on studies utilizing AQP4-deficient mice. However, these mice have a range of secondary effects due to the gene deletion. An efficient and selective AQP4 inhibitor has thus been sorely needed to validate the results obtained in the AQP4 mice to quantify the contribution of AQP4 to brain fluid dynamics. In AQP4-expressing Xenopus laevis oocytes monitored by a high-resolution volume recording system, we here demonstrate that the compound TGN-020 is such a selective AQP4 inhibitor. TGN-020 targets the tested species of AQP4 with an IC of ~3.5 μM, but displays no inhibitory effect on the other AQPs (AQP1-AQP9). With this tool, we employed rat hippocampal slices and ion-sensitive microelectrodes to determine the role of AQP4 in glia cell swelling following neuronal activity. TGN-020-mediated inhibition of AQP4 did not prevent stimulus-induced extracellular space shrinkage, nor did it slow clearance of the activity-evoked K transient. These data, obtained with a verified isoform-selective AQP4 inhibitor, indicate that AQP4 is not required for the astrocytic contribution to the K clearance or the associated extracellular space shrinkage.
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http://dx.doi.org/10.1002/glia.23851DOI Listing
January 2021

Interdisciplinary Synergy to Reveal Mechanisms of Annexin-Mediated Plasma Membrane Shaping and Repair.

Cells 2020 04 21;9(4). Epub 2020 Apr 21.

Membrane Integrity, Cell Death and Metabolism Unit, Center for Autophagy, Recycling and Disease, Danish Cancer Society Research Center, DK-2100 Copenhagen, Denmark.

The plasma membrane surrounds every single cell and essentially shapes cell life by separating the interior from the external environment. Thus, maintenance of cell membrane integrity is essential to prevent death caused by disruption of the plasma membrane. To counteract plasma membrane injuries, eukaryotic cells have developed efficient repair tools that depend on Ca- and phospholipid-binding annexin proteins. Upon membrane damage, annexin family members are activated by a Ca influx, enabling them to quickly bind at the damaged membrane and facilitate wound healing. Our recent studies, based on interdisciplinary research synergy across molecular cell biology, experimental membrane physics, and computational simulations show that annexins have additional biophysical functions in the repair response besides enabling membrane fusion. Annexins possess different membrane-shaping properties, allowing for a tailored response that involves rapid bending, constriction, and fusion of membrane edges for resealing. Moreover, some annexins have high affinity for highly curved membranes that appear at free edges near rupture sites, a property that might accelerate their recruitment for rapid repair. Here, we discuss the mechanisms of annexin-mediated membrane shaping and curvature sensing in the light of our interdisciplinary approach to study plasma membrane repair.
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http://dx.doi.org/10.3390/cells9041029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226303PMC
April 2020

Thermodynamic Investigation of the Mechanism of Heat Production During Membrane Depolarization.

J Phys Chem B 2020 04 30;124(14):2815-2822. Epub 2020 Mar 30.

MEMPHYS-Center for Biomembrane Physics, http://phylife.sdu.dk.

When an action potential passes through a neuron, heat is first produced and then reabsorbed by the neuronal membrane, resulting in a small measurable temperature spike. Here, we describe the thermodynamics and molecular features of the heat production using a coarse-grained molecular dynamics approach. We study a simple unicomponent lipid bilayer membrane surrounded by physiological salt solution with and without an external electric field, which represents an imbalanced charge across the membrane. We show that the temperature increases significantly upon removal of the electric field under constant pressure conditions. The potential energy converted to heat is initially stored mainly in the imbalanced ion distribution across the membrane and the elastic energy of the membrane has only a minor role to play. We demonstrate that the mechanism of heat production involves interaction between ions as well as lipid headgroup dipoles while the interactions between polar water molecules and lipid headgroup dipoles absorbs a considerable portion of such produced heat upon removal of the electric field. Our data provide novel thermodynamic insights into the molecular processes governing membrane reorganization upon discharging of lipid membranes and insight into energy metabolism in nerves.
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http://dx.doi.org/10.1021/acs.jpcb.9b11456DOI Listing
April 2020

Interaction of the mononucleotide UMP with a fluid phospholipid bilayer.

Soft Matter 2019 Oct 7;15(40):8129-8136. Epub 2019 Oct 7.

Raman Research Institute, Bangalore-560080, India.

Interaction between mononucleotides and lipid membranes is believed to have played an important role in the origin of life on Earth. Studies on mononucleotide-lipid systems hitherto have focused on the influence of the lipid environment on the organization of the mononucleotide molecules, and the effect of the latter on the confining medium has not been investigated in detail. We have probed the interaction of the mononucleotide, uridine 5'-monophosphate (UMP), and its disodium salt (UMPDSS) with fluid dimyristoylphosphatidylcholine (DMPC) membranes, using small-angle X-ray scattering (SAXS), cryogenic scanning electron microscopy (cryo-SEM) and computer simulations. UMP adsorbs and charges the lipid membrane, resulting in the formation of unilamellar vesicles in dilute solutions. Adsorption of UMP reduces the bilayer thickness of DMPC. UMPDSS has a much weaker effect on interbilayer interactions. These observations are in very good agreement with the results of an all-atom molecular dynamics simulation of these systems. In the presence of counterions, such as Na, UMP forms small aggregates in water, which bind to the bilayer without significantly perturbing it. The phosphate moiety in the lipid headgroup is found to bind to the hydrogens from the sugar ring of UMP, while the choline group tends to bind to the two oxygens from the nucleotide base. These studies provide important insights into lipid-nucleotide interactions and the effect of the nucleotide on lipid membranes.
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http://dx.doi.org/10.1039/c9sm01257eDOI Listing
October 2019

A single K-binding site in the crystal structure of the gastric proton pump.

Elife 2019 08 22;8. Epub 2019 Aug 22.

Cellular and Structural Physiology Institute, Nagoya University, Nagoya, Japan.

The gastric proton pump (H,K-ATPase), a P-type ATPase responsible for gastric acidification, mediates electro-neutral exchange of H and K coupled with ATP hydrolysis, but with an as yet undetermined transport stoichiometry. Here we show crystal structures at a resolution of 2.5 Å of the pump in the E2-P transition state, in which the counter-transporting cation is occluded. We found a single K bound to the cation-binding site of the H,K-ATPase, indicating an exchange of 1H/1K per hydrolysis of one ATP molecule. This fulfills the energy requirement for the generation of a six pH unit gradient across the membrane. The structural basis of K recognition is resolved and supported by molecular dynamics simulations, establishing how the H,K-ATPase overcomes the energetic challenge to generate an H gradient of more than a million-fold-one of the highest cation gradients known in mammalian tissue-across the membrane.
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http://dx.doi.org/10.7554/eLife.47701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6706254PMC
August 2019

Evidence for ATP Interaction with Phosphatidylcholine Bilayers.

Langmuir 2019 07 19;35(30):9944-9953. Epub 2019 Jul 19.

School of Chemistry , University of Sydney , Sydney , NSW 2006 , Australia.

ATP is a fundamental intracellular molecule and is thought to diffuse freely throughout the cytosol. Evidence obtained from nucleotide-sensing sarcolemmal ion channels and red blood cells, however, suggest that ATP is compartmentalized or buffered, especially beneath the sarcolemma, but no definitive mechanism for restricted diffusion or potential buffering system has been postulated. In this study, we provide evidence from alterations to membrane dipole potential, membrane conductance, changes in enthalpy of phospholipid phase transition, and from free energy calculations that ATP associates with phospholipid bilayers. Furthermore, all-atom molecular dynamics simulations show that ATP can form aggregates in the aqueous phase at high concentrations. ATP interaction with membranes provides a new model to understand the diffusion of ATP through the cell. Coupled with previous reports of diffusion restriction in the subsarcolemmal space, these findings support the existence of compartmentalized or buffered pools of ATP.
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http://dx.doi.org/10.1021/acs.langmuir.9b01240DOI Listing
July 2019

On identifying collective displacements in apo-proteins that reveal eventual binding pathways.

PLoS Comput Biol 2019 01 15;15(1):e1006665. Epub 2019 Jan 15.

Tata Institute of Fundamental Research, Center for Interdisciplinary Sciences, Hyderabad, India.

Binding of small molecules to proteins often involves large conformational changes in the latter, which open up pathways to the binding site. Observing and pinpointing these rare events in large scale, all-atom, computations of specific protein-ligand complexes, is expensive and to a great extent serendipitous. Further, relevant collective variables which characterise specific binding or un-binding scenarios are still difficult to identify despite the large body of work on the subject. Here, we show that possible primary and secondary binding pathways can be discovered from short simulations of the apo-protein without waiting for an actual binding event to occur. We use a projection formalism, introduced earlier to study deformation in solids, to analyse local atomic displacements into two mutually orthogonal subspaces-those which are "affine" i.e. expressible as a homogeneous deformation of the native structure, and those which are not. The susceptibility to non-affine displacements among the various residues in the apo- protein is then shown to correlate with typical binding pathways and sites crucial for allosteric modifications. We validate our observation with all-atom computations of three proteins, T4-Lysozyme, Src kinase and Cytochrome P450.
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http://dx.doi.org/10.1371/journal.pcbi.1006665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333327PMC
January 2019

K binding and proton redistribution in the EP state of the H, K-ATPase.

Sci Rep 2018 08 24;8(1):12732. Epub 2018 Aug 24.

Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, 5230 M, Denmark.

The H, K-ATPase (HKA) uses ATP to pump protons into the gastric lumen against a million-fold proton concentration gradient while counter-transporting K from the lumen. The mechanism of release of a proton into a highly acidic stomach environment, and the subsequent binding of a K ion necessitates a network of protonable residues and dynamically changing protonation states in the cation binding pocket dominated by five acidic amino acid residues E343, E795, E820, D824, and D942. We perform molecular dynamics simulations of spontaneous K binding to all possible protonation combinations of the acidic amino acids and carry out free energy calculations to determine the optimal protonation state of the luminal-open EP state of the pump which is ready to bind luminal K. A dynamic pK correlation analysis reveals the likelihood of proton transfer events within the cation binding pocket. In agreement with in-vitro measurements, we find that E795 is likely to be protonated, and that E820 is at the center of the proton transfer network in the luminal-open EP state. The acidic residues D942 and D824 are likely to remain protonated, and the proton redistribution occurs predominantly amongst the glutamate residues exposed to the lumen. The analysis also shows that a lower number of K ions bind at lower pH, modeled by a higher number of protons in the cation binding pocket, in agreement with the 'transport stoichiometry variation' hypothesis.
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http://dx.doi.org/10.1038/s41598-018-30885-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6109069PMC
August 2018

Different footprints of the Zika and dengue surface proteins on viral membranes.

Soft Matter 2018 Jul;14(27):5615-5621

MEMPHYS: Center for Biomembrane Physics, Department of Physics Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark.

The flavivirus Zika virus (ZV) became an international emergency within two years of its outbreak in the Americas. Dengue virus (DENV), which is also a flavivirus, causes significant clinical harm in equatorial regions. A common feature amongst flaviviruses like ZV and DENV is an icosahedral shell of exactly 180 copies of the envelope (E) and membrane (M) proteins anchored in a lipid membrane, which engulfs the viral RNA and capsid proteins. Host recognition by both ZV and DENV is linked to the presence of phosphatidylserine (PS) and phosphatidylethanolamine (PE) lipids in the viral lipidome. Glycosylation of Asn residues on the Zika E protein may be linked to ZV induced neuropathies. We carry out coarse grained molecular dynamics simulations of the E3M3 hexamer embedded in the ZV and DENV lipidomes, and we show that the proteins have a significantly different lipid footprint in the viral lipidome. PE lipids in DENV and PS lipids in ZV enrich near the protein hexamer. We attribute the difference to a higher number of cationic amino acids in the ZV M protein. We also show that the three glycosylation sites on ZV, but not on DENV, are conformationally variant. Our data shed new light on the lipid interactions, and thus the host recognition mechanisms of the two viruses, which may be molecular determinants of the neuropathies caused by the ZV.
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http://dx.doi.org/10.1039/c8sm00223aDOI Listing
July 2018

Faster Simulations with a 5 fs Time Step for Lipids in the CHARMM Force Field.

J Chem Theory Comput 2018 Jun 25;14(6):3342-3350. Epub 2018 May 25.

MEMPHYS: Center for Biomembrane Physics, Department of Physics, Chemistry and Pharmacy , University of Southern Denmark , M 5230 Odense , Denmark.

The performance of all-atom molecular dynamics simulations is limited by an integration time step of 2 fs, which is needed to resolve the fastest degrees of freedom in the system, namely, the vibration of bonds and angles involving hydrogen atoms. The virtual interaction sites (VIS) method replaces hydrogen atoms by massless virtual interaction sites to eliminate these degrees of freedom while keeping intact nonbonded interactions and the explicit treatment of hydrogen atoms. We have modified the existing VIS algorithm for most lipids in the popular CHARMM36 force field by increasing the hydrogen atom masses at regular intervals in the lipid acyl chains and obtained lipid properties and pore formation free energies in very good agreement with those calculated in simulations without VIS. Our modified VIS scheme enables a 5 fs time step resulting in a significant performance gain for all-atom simulations of membranes. The method has the potential to make longer time and length scales accessible in all-atom simulations of membrane-protein complexes.
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http://dx.doi.org/10.1021/acs.jctc.8b00267DOI Listing
June 2018

Lipid Configurations from Molecular Dynamics Simulations.

Biophys J 2018 04;114(8):1895-1907

MEMPHYS-Centre for Biomembrane Physics, University of Southern Denmark, Odense M, Denmark; Max-Planck-Institut für biophysikalische Chemie, Göttingen, Germany. Electronic address:

The extent to which current force fields faithfully reproduce conformational properties of lipids in bilayer membranes, and whether these reflect the structural principles established for phospholipids in bilayer crystals, are central to biomembrane simulations. We determine the distribution of dihedral angles in palmitoyl-oleoyl phosphatidylcholine from molecular dynamics simulations of hydrated fluid bilayer membranes. We compare results from the widely used lipid force field of Berger et al. with those from the most recent C36 release of the CHARMM force field for lipids. Only the CHARMM force field produces the chain inequivalence with sn-1 as leading chain that is characteristic of glycerolipid packing in fluid bilayers. The exposure and high partial charge of the backbone carbonyls in Berger lipids leads to artifactual binding of Na ions reported in the literature. Both force fields predict coupled, near-symmetrical distributions of headgroup dihedral angles, which is compatible with models of interconverting mirror-image conformations used originally to interpret NMR order parameters. The Berger force field produces rotamer populations that correspond to the headgroup conformation found in a phosphatidylcholine lipid bilayer crystal, whereas CHARMM36 rotamer populations are closer to the more relaxed crystal conformations of phosphatidylethanolamine and glycerophosphocholine. CHARMM36 alone predicts the correct relative signs of the time-average headgroup order parameters, and reasonably reproduces the full range of NMR data from the phosphate diester to the choline methyls. There is strong motivation to seek further experimental criteria for verifying predicted conformational distributions in the choline headgroup, including the P chemical shift anisotropy and N and CD NMR quadrupole splittings.
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http://dx.doi.org/10.1016/j.bpj.2018.02.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5937052PMC
April 2018

Interaction of N-terminal peptide analogues of the Na,K-ATPase with membranes.

Biochim Biophys Acta Biomembr 2018 Jun 6;1860(6):1282-1291. Epub 2018 Mar 6.

School of Chemistry, University of Sydney, Sydney, NSW 2006, Australia; The University of Sydney Nano Institute, Sydney, NSW 2006, Australia. Electronic address:

The Na,K-ATPase, which is present in the plasma membrane of all animal cells, plays a crucial role in maintaining the Na and K electrochemical potential gradients across the membrane. Recent studies have suggested that the N-terminus of the protein's catalytic α-subunit is involved in an electrostatic interaction with the surrounding membrane, which controls the protein's conformational equilibrium. However, because the N-terminus could not yet be resolved in any X-ray crystal structures, little information about this interaction is so far available. In measurements utilising poly-l-lysine as a model of the protein's lysine-rich N-terminus and using lipid vesicles of defined composition, here we have identified the most likely origin of the interaction as one between positively charged lysine residues of the N-terminus and negatively charged headgroups of phospholipids (notably phosphatidylserine) in the surrounding membrane. Furthermore, to isolate which segments of the N-terminus could be involved in membrane binding, we chemically synthesized N-terminal fragments of various lengths. Based on a combination of results from RH421 UV/visible absorbance measurements and solid-state P and H NMR using these N-terminal fragments as well as MD simulations it appears that the membrane interaction arises from lysine residues prior to the conserved LKKE motif of the N-terminus. The MD simulations indicate that the strength of the interaction varies significantly between different enzyme conformations.
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http://dx.doi.org/10.1016/j.bbamem.2018.03.002DOI Listing
June 2018

The CAPOS mutation in ATP1A3 alters Na/K-ATPase function and results in auditory neuropathy which has implications for management.

Hum Genet 2018 Feb 5;137(2):111-127. Epub 2018 Jan 5.

Institute for Auditory Neuroscience and InnerEarLab, University Medical Center, Göttingen, Germany.

Cerebellar ataxia, areflexia, pes cavus, optic atrophy and sensorineural hearing impairment (CAPOS) is a rare clinically distinct syndrome caused by a single dominant missense mutation, c.2452G>A, p.Glu818Lys, in ATP1A3, encoding the neuron-specific alpha subunit of the Na+/K+-ATPase α3. Allelic mutations cause the neurological diseases rapid dystonia Parkinsonism and alternating hemiplegia of childhood, disorders which do not encompass hearing or visual impairment. We present detailed clinical phenotypic information in 18 genetically confirmed patients from 11 families (10 previously unreported) from Denmark, Sweden, UK and Germany indicating a specific type of hearing impairment-auditory neuropathy (AN). All patients were clinically suspected of CAPOS and had hearing problems. In this retrospective analysis of audiological data, we show for the first time that cochlear outer hair cell activity was preserved as shown by the presence of otoacoustic emissions and cochlear microphonic potentials, but the auditory brainstem responses were grossly abnormal, likely reflecting neural dyssynchrony. Poor speech perception was observed, especially in noise, which was beyond the hearing level obtained in the pure tone audiograms in several of the patients presented here. Molecular modelling and in vitro electrophysiological studies of the specific CAPOS mutation were performed. Heterologous expression studies of α3 with the p.Glu818Lys mutation affects sodium binding to, and release from, the sodium-specific site in the pump, the third ion-binding site. Molecular dynamics simulations confirm that the structure of the C-terminal region is affected. In conclusion, we demonstrate for the first time evidence for auditory neuropathy in CAPOS syndrome, which may reflect impaired propagation of electrical impulses along the spiral ganglion neurons. This has implications for diagnosis and patient management. Auditory neuropathy is difficult to treat with conventional hearing aids, but preliminary improvement in speech perception in some patients suggests that cochlear implantation may be effective in CAPOS patients.
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http://dx.doi.org/10.1007/s00439-017-1862-zDOI Listing
February 2018

Structural design of intrinsically fluorescent oxysterols.

Chem Phys Lipids 2018 05 26;212:26-34. Epub 2017 Dec 26.

Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense, Denmark. Electronic address:

Oxysterols are oxidized derivatives of cholesterol with many important biological functions. Trafficking of oxysterols in and between cells is not well studied, largely due to the lack of appropriate oxysterol analogs. Intrinsically fluorescent oxysterols present a new route towards direct observation of intracellular oxysterol trafficking by fluorescence microscopy. We characterize the fluorescence properties of the existing fluorescent 25-hydroxycholesterol analog 25-hydroxycholestatrienol, and propose a new probe with an extended conjugated system. The location of both probes inside a membrane is analyzed and compared with that of 25-hydroxycholesterol using molecular dynamics simulations. The analogs' one- and two-photon absorption properties inside the membrane are evaluated using electronic structure calculations with polarizable embedding. Due to predicted keto-enol tautomerisation of the new oxysterol analog, we also evaluate the keto form. Both analogs are found to be good probe candidates for 25-hydroxycholesterol, provided that the new analog remains in the enol-form. Only the new analog with extended conjugated system shows significant two-photon absorption, which is strongly enhanced by the presence of the membrane.
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http://dx.doi.org/10.1016/j.chemphyslip.2017.12.005DOI Listing
May 2018

The role of caveolin-1 in lipid droplets and their biogenesis.

Chem Phys Lipids 2018 03 21;211:93-99. Epub 2017 Nov 21.

MEMPHYS: Center for Biomembrane Physics, University of Southern Denmark, Campusvej 55, Odense 5230 M, Denmark. Electronic address:

We address unresolved questions of the energetics and mechanism of lipid droplet (LD) biogenesis, and of the role of caveolins in the endoplasmic reticulum (ER) and in mature LDs. LDs are eukaryotic repositories of neutral lipids, which are believed to be synthesised in the ER. We investigate the effects of a curvature-inducing protein, caveolin-1, on the formation and structure of a spontaneously aggregated triolein (TO) lipid lens in a flat lipid bilayer using molecular dynamics (MD) simulations. A truncated form of caveolin-1 (Cav1) localises on the interface between the spontaneously formed TO aggregate and the bulk bilayer, and thins the bilayer at the edge of the aggregate, which may contribute to lowering the energy barrier for pinching off the aggregate from the host bilayer. Simulations of fully mature LDs do not conclusively establish the optimal localisation of Cav1 in LDs, but when Cav1 is in the LD core, the distribution of both neutral lipids in the LD core, and of phospholipids on the engulfing monolayer are altered significantly. Our simulations provide an unprecedented molecular description of the distribution and dynamics of various lipid species in both mature LDs and in the nascent LD inside the bilayer.
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http://dx.doi.org/10.1016/j.chemphyslip.2017.11.010DOI Listing
March 2018

Membrane Tubulation in Lipid Vesicles Triggered by the Local Application of Calcium Ions.

Langmuir 2017 10 2;33(41):11010-11017. Epub 2017 Oct 2.

Department of Chemistry and Chemical Engineering, Chalmers University of Technology , SE-412 96 Gothenburg, Sweden.

Experimental and theoretical studies on ion-lipid interactions predict that binding of calcium ions to cell membranes leads to macroscopic mechanical effects and membrane remodeling. Herein, we provide experimental evidence that a point source of Ca acting upon a negatively charged membrane generates spontaneous curvature and triggers the formation of tubular protrusions that point away from the ion source. This behavior is rationalized by strong binding of the divalent cations to the surface of the charged bilayer, which effectively neutralizes the surface charge density of outer leaflet of the bilayer. The mismatch in the surface charge density of the two leaflets leads to nonzero spontaneous curvature. We probe this mismatch through the use of molecular dynamics simulations and validate that calcium ion binding to a lipid membrane is sufficient to generate inward spontaneous curvature, bending the membrane. Additionally, we demonstrate that the formed tubular protrusions can be translated along the vesicle surface in a controlled manner by repositioning the site of localized Ca exposure. The findings demonstrate lipid membrane remodeling in response to local chemical gradients and offer potential insights into the cell membrane behavior under conditions of varying calcium ion concentrations.
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http://dx.doi.org/10.1021/acs.langmuir.7b01461DOI Listing
October 2017

A novel role for methyl cysteinate, a cysteine derivative, in cesium accumulation in Arabidopsis thaliana.

Sci Rep 2017 02 23;7:43170. Epub 2017 Feb 23.

RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.

Phytoaccumulation is a technique to extract metals from soil utilising ability of plants. Cesium is a valuable metal while radioactive isotopes of cesium can be hazardous. In order to establish a more efficient phytoaccumulation system, small molecules which promote plants to accumulate cesium were investigated. Through chemical library screening, 14 chemicals were isolated as 'cesium accumulators' in Arabidopsis thaliana. Of those, methyl cysteinate, a derivative of cysteine, was found to function within the plant to accumulate externally supplemented cesium. Moreover, metabolite profiling demonstrated that cesium treatment increased cysteine levels in Arabidopsis. The cesium accumulation effect was not observed for other cysteine derivatives or amino acids on the cysteine metabolic pathway tested. Our results suggest that methyl cysteinate, potentially metabolised from cysteine, binds with cesium on the surface of the roots or inside plant cells and improve phytoaccumulation.
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http://dx.doi.org/10.1038/srep43170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5322390PMC
February 2017

Electrostatic Stabilization Plays a Central Role in Autoinhibitory Regulation of the Na,K-ATPase.

Biophys J 2017 Jan;112(2):288-299

School of Chemistry, The University of Sydney, Sydney, New South Wales, Australia. Electronic address:

The Na,K-ATPase is present in the plasma membrane of all animal cells. It plays a crucial role in maintaining the Na and K electrochemical potential gradients across the membrane, which are essential in numerous physiological processes, e.g., nerve, muscle, and kidney function. Its cellular activity must, therefore, be under tight metabolic control. Consideration of eosin fluorescence and stopped-flow kinetic data indicates that the enzyme's E2 conformation is stabilized by electrostatic interactions, most likely between the N-terminus of the protein's catalytic α-subunit and the adjacent membrane. The electrostatic interactions can be screened by increasing ionic strength, leading to a more evenly balanced equilibrium between the E1 and E2 conformations. This represents an ideal situation for effective regulation of the Na,K-ATPase's enzymatic activity, because protein modifications, which perturb this equilibrium in either direction, can then easily lead to activation or inhibition. The effect of ionic strength on the E1:E2 distribution and the enzyme's kinetics can be mathematically described by the Gouy-Chapman theory of the electrical double layer. Weakening of the electrostatic interactions and a shift toward E1 causes a significant increase in the rate of phosphorylation of the enzyme by ATP. Electrostatic stabilization of the Na,K-ATPase's E2 conformation, thus, could play an important role in regulating the enzyme's physiological catalytic turnover.
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http://dx.doi.org/10.1016/j.bpj.2016.12.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5266142PMC
January 2017

Glutamate Water Gates in the Ion Binding Pocket of Na Bound Na, K-ATPase.

Sci Rep 2017 01 13;7:39829. Epub 2017 Jan 13.

MEMPHYS-Center for Biomembrane Physics, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.

The dynamically changing protonation states of the six acidic amino acid residues in the ion binding pocket of the Na, K -ATPase (NKA) during the ion transport cycle are proposed to drive ion binding, release and possibly determine Na or K selectivity. We use molecular dynamics (MD) and density functional theory (DFT) simulations to determine the protonation scheme of the Na bound conformation of NKA. MD simulations of all possible protonation schemes show that the bound Na ions are most stably bound when three or four protons reside in the binding sites, and that Glu954 in site III is always protonated. Glutamic acid residues in the three binding sites act as water gates, and their deprotonation triggers water entry to the binding sites. From DFT calculations of Na binding energies, we conclude that three protons in the binding site are needed to effectively bind Na from water and four are needed to release them in the next step. Protonation of Asp926 in site III will induce Na release, and Glu327, Glu954 and Glu779 are all likely to be protonated in the Na bound occluded conformation. Our data provides key insights into the role of protons in the Na binding and release mechanism of NKA.
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http://dx.doi.org/10.1038/srep39829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5233988PMC
January 2017

Quantifying the Relationship between Curvature and Electric Potential in Lipid Bilayers.

J Phys Chem B 2016 06 23;120(21):4812-7. Epub 2016 May 23.

MEMPHYS - Center for Biomembrane Physics, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark , Campusvej 55, 5230 Odense M, Denmark.

Cellular membranes mediate vital cellular processes by being subject to curvature and transmembrane electrical potentials. Here we build upon the existing theory for flexoelectricity in liquid crystals to quantify the coupling between lipid bilayer curvature and membrane potentials. Using molecular dynamics simulations, we show that headgroup dipole moments, the lateral pressure profile across the bilayer, and spontaneous curvature all systematically change with increasing membrane potentials. In particular, there is a linear dependence between the bending moment (the product of bending rigidity and spontaneous curvature) and the applied membrane potentials. We show that biologically relevant membrane potentials can induce biologically relevant curvatures corresponding to radii of around 500 nm. The implications of flexoelectricity in lipid bilayers are thus likely to be of considerable consequence both in biology and in model lipid bilayer systems.
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http://dx.doi.org/10.1021/acs.jpcb.6b03439DOI Listing
June 2016

Perillyl alcohol: Dynamic interactions with the lipid bilayer and implications for long-term inhalational chemotherapy for gliomas.

Surg Neurol Int 2016 5;7. Epub 2016 Jan 5.

Department of Cellular and Molecular Biology, Institute of Biology, Fluminense Federal University, Rio de Janeiro, Brazil.

Background: Gliomas display a high degree of intratumor heterogeneity, including changes in physiological parameters and lipid composition of the plasma membrane, which may contribute to the development of drug resistance. Biophysical interactions between therapeutic agents and the lipid components at the outer plasma membrane interface are critical for effective drug uptake. Amphipathic molecules such as perillyl alcohol (POH) have a high partition coefficient and generally lead to altered lipid acyl tail dynamics near the lipid-water interface, impacting the lipid bilayer structure and transport dynamics. We therefore hypothesized that glioma cells may display enhanced sensitivity to POH-induced apoptosis due to plasma membrane alterations, while in non-transformed cells, POH may be expelled through thermal agitation.

Methods: Interactions between POH and the plasma membrane was studied using molecular dynamics simulations. In this phase I/II trial, we set up to evaluate the clinical effectiveness of long-term (up to 5 years) daily intranasal administration of POH in a cohort of 19 patients with low-grade glioma (LGG). Importantly, in a series of clinical studies previously published by our group, we have successfully established that intranasal delivery of POH to patients with malignant gliomas is a viable and effective therapeutic strategy.

Results: POH altered the plasma membrane potential of the lipid bilayer of gliomas and prolonged intranasal administration of POH in a cohort of patients with LGG halted disease progression with virtually no toxicity.

Conclusion: Altogether, the results suggest that POH-induced alterations of the plasma membrane might be contributing to its therapeutic efficacy in preventing LGG progression.
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http://dx.doi.org/10.4103/2152-7806.173301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4722523PMC
February 2016

Tuning of the Na,K-ATPase by the beta subunit.

Sci Rep 2016 Feb 5;6:20442. Epub 2016 Feb 5.

Danish Research Institute of Translational Neuroscience - DANDRITE, Nordic EMBL Partnership for Molecular Medicine, Aarhus University, DK-8000 Aarhus, Denmark.

The vital gradients of Na(+) and K(+) across the plasma membrane of animal cells are maintained by the Na,K-ATPase, an αβ enzyme complex, whose α subunit carries out the ion transport and ATP hydrolysis. The specific roles of the β subunit isoforms are less clear, though β2 is essential for motor physiology in mammals. Here, we show that compared to β1 and β3, β2 stabilizes the Na(+)-occluded E1P state relative to the outward-open E2P state, and that the effect is mediated by its transmembrane domain. Molecular dynamics simulations further demonstrate that the tilt angle of the β transmembrane helix correlates with its functional effect, suggesting that the relative orientation of β modulates ion binding at the α subunit. β2 is primarily expressed in granule neurons and glomeruli in the cerebellum, and we propose that its unique functional characteristics are important to respond appropriately to the cerebellar Na(+) and K(+) gradients.
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http://dx.doi.org/10.1038/srep20442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4742777PMC
February 2016