Enhancement of proton conductance by mutations of the selectivity filter of aquaporin-1

Prevention of cation permeation in wild-type aquaporin-1 (AQP1) is believed to be associated with the Asn-Pro-Ala (NPA) region and the aromatic/arginine selectivity filter (SF) domain. Previous work has suggested that the NPA region helps to impede proton permeation due to the protein backbone collective macrodipoles that create an environment favoring a directionally discontinuous channel hydrogen-bonded water chain and a large electrostatic barrier. The SF domain contributes to the proton permeation barrier by a spatial restriction mechanism and direct electrostatic interactions. To further explore these various effects, the free-energy barriers and the maximum cation conductance for the permeation of various cations through the AQP1-R195V and AQP1-R195S mutants are predicted computationally. The cations studied included the hydrated excess proton that utilizes the Grotthuss shuttling mechanism, a model “classical” charge localized hydronium cation that exhibits no Grotthuss shuttling, and a sodium cation. The hydrated excess proton was simulated using a specialized multi-state molecular dynamics method including a proper physical treatment of the proton shuttling and charge defect delocalization. Both AQP1 mutants exhibit a surprising cooperative effect leading to a reduction in the free-energy barrier for proton permeation around the NPA region due to altered water configurations in the SF region, with AQP1-R195S having a higher conductance than AQP1-R195V. The theoretical predictions are experimentally confirmed in wild-type AQP1 and the mutants expressed in Xenopus oocytes. The combined results suggest that the SF domain is a specialized structure that has evolved to impede proton permeation in aquaporins.     

Proton transfer along the external proton channel of bacteriorhodopsin

The process of proton transfer along a proton channel is considered using bacteriorhodopsin as a model system, for which a large body of experimental data is available. The possible amino acid composition of the external proton half-channel of bacteriorhodopsin and the stepwise scheme of proton transfer consistent with experimental data are proposed. The rate of proton transfer between fixed centers is assessed for certain regions of this channel for which spectroscopic data are available.     

The mechanism of proton transfer between adjacent sites on the molecular surface

The surface of a protein, or a membrane, is spotted with a multitude of proton binding sites, some of which are only few Å apart. When a proton is released from one site, it propagates through the water by a random walk under the bias of the local electrostatic potential determined by the distribution of the charges on the protein. Eventually, the released protons are dispersed in the bulk, but during the first few nanoseconds after the dissociation, the protons can be trapped by encounter with nearby acceptor sites. While the study of this reaction on the surface of a protein suffers from experimental and theoretical difficulties, it can be investigated with simple model compounds like derivatives of fluorescein. In the present study, we evaluate the mechanism of proton transfer reactions that proceed, preferentially, inside the Coulomb cage of the dye molecules. Kinetic analysis of the measured dynamics reveals the role of the dimension of the Coulomb cage on the efficiency of the reaction and how the ordering of the water molecules by the dye affects the kinetic isotope effect.     

Hypertonic induction of aquaporin-1 water channel independent of transcellular osmotic gradient

Aquaporin-1 (AQP1) water channel plays a critical role for water reabsorption in the urinary concentrating mechanism. AQP1 expression in renal cells is upregulated by hypertonicity, but not urea, suggesting the requirement of an osmotic gradient. To investigate whether AQP1 expression is regulated by apical and/or basolateral hypertonicity, murine renal medullary mIMCD-K2 cells grown on permeable support were exposed to hypertonic medium. When the medium on the apical or basolateral membrane side was switched to hypertonic, the transcellular osmotic gradient was dissipated within 8h. Basolateral hypertonicity increased AQP1 expression more than apical hypertonicity. Comparable apical and basolateral hypertonicity without a transcellular hypertonic gradient, however, increased AQP1 expression. Cell surface biotinylation experiments revealed that hypertonicity promoted AQP1 trafficking to both plasma cell membranes. These results indicate that AQP1 expression is predominantly mediated by basolateral hypertonicity but a transcellular osmotic gradient is not necessary for its induction.     

Molecular dynamics analysis of potent inhibitors of M2 proton channel against H1N1 swine influenza virus

The recent H1N1 (swine) influenza pandemic highlighted the urgent need of having effective anti‐viral strategies. In addition to neuraminidase inhibitors, there is another class of anti-viral drug known as M2 inhibitors that were, in the past, effective in treating seasonal influenza. However, due to the emergence of M2 inhibitor‐resistant influenza viruses, this class of drugs was not recommended for clinical usage in the latest influenza pandemic. In order to identify novel M2 inhibitors, we have performed molecular docking using a traditional Chinese medicine database (http://tcm.cmu.edu.tw/index.php). Docking and subsequent de novo designs gave 10 derivatives that have much better docking results than the control. Of these 10 derivatives, the top three, methyl isoferulate_1, genipin_1 and genipin_2, were selected for molecular dynamics simulation. During the simulation run, the top three derivatives all had stable interactions with M2 residues, Ser31 and Ala30. Methyl isoferulate_1 also has stable interaction to His37. Therefore, we recommend these three derivatives for further biomolecular experiments and clinical studies.     

The mechanism of proton translocation in respiratory complex I from molecular dynamics

Abstract

Respiratory complex I, the biggest enzyme of respiratory chain, plays a key role in energy production by the mitochondrial respiratory chain and has been implicated in many human neurodegenerative diseases. Recently, the crystal structure of respiratory complex I is reported. We perform 50?ns molecular dynamics simulations on the membrane domain of respiratory complex I under two hypothetical states (oxidized state and reduced state). We find that the density of water molecules in the trans-membrane domain under reduced state is bigger than that under oxidized state. The connecting elements (helix HL and β-hairpins-helix element) fluctuate stronger under reduced state than that under oxidized state, causing more internal water molecules and facilitating the proton conduction. The conformational changes of helix HL and the crucial charged residue Glu in TM5 play key roles in the mechanism of proton translocation. Our results illustrate the dynamic behavior and the potential mechanism of respiratory complex I, which provides the structural basis for drug design of respiratory complex I.     

The proton pump/leak mechanism of unconsciousness

There are few explanations which account for the manner in which the catastrophic physiological consequences of anaesthesia, cold narcosis or, for that matter, a short, sharp upper-cut, come about. Most studies terminate with the presentation of ever-better correlations between an end-point in a model system (dough consistency, rubber elasticity, bacterial, protozoal or animal mobility, liposome permeability, luciferase activity, etc.) and oil/water partition co-efficients or with some arbitrary biological end-point. From what is currently known about (a) the permeating pathways of non-electrolytes, ions and protons across membranes e.g. liposomes, (b) the effect of anaesthetics on such pathways and (c) the effect of temperature and pressure on both liposomes and whole animals, it is possible to develop a testable hypothesis. It is called the ‘proton pump-leak’ hypothesis and involves a number of linked biophysical and biochemical processes. (d) It assumes that a living animal or plant is in a steady-state regarding all concentration gradients; passive leaks across membranes are balanced by temperature, pressure, and energy dependent ion/ion and/or proton/ion pumps (enzyme), working within an aqueous phase. (e) Consciousness is dependent upon inter-neuronal communication via release of transmitter substances. (f) Transmitter substances, characteristically either weak bases or weak acids e.g. catecholamines, accumulate passively in vesicles rich in acid-buffer, held to a low pH by the activity of H⁺/K⁺ energy-driven pumps. Interference with this finely-balanced system either by changing the chemical potential of the hydrophobic (membrane) phase at NTP (with anaesthetics), or by changing the chemical potential of both hydrophobic and aqueous (pump) phases by hyperbaric, hyphotermic, or anoxic conditions imposed (inevitably) on the whole animal, would result in the resetting of the steady-state parameters.     

The gating mechanism of the bacterial mechanosensitive channel MscL revealed by molecular dynamics simulations

One of the ultimate goals of the study on mechanosensitive (MS) channels is to understand the biophysical mechanisms of how the MS channel protein senses forces and how the sensed force induces channel gating. The bacterial MS channel MscL is an ideal subject to reach this goal owing to its resolved 3D protein structure in the closed state on the atomic scale and large amounts of electrophysiological data on its gating kinetics. However, the structural basis of the dynamic process from the closed to open states in MscL is not fully understood. In this study, we performed molecular dynamics (MD) simulations on the initial process of MscL opening in response to a tension increase in the lipid bilayer. To identify the tension-sensing site(s) in the channel protein, we calculated interaction energy between membrane lipids and candidate amino acids (AAs) facing the lipids. We found that Phe78 has a conspicuous interaction with the lipids, suggesting that Phe78 is the primary tension sensor of MscL. Increased membrane tension by membrane stretch dragged radially the inner (TM1) and outer (TM2) helices of MscL at Phe78, and the force was transmitted to the pentagon-shaped gate that is formed by the crossing of the neighboring TM1 helices in the inner leaflet of the bilayer. The radial dragging force induced radial sliding of the crossing portions, leading to a gate expansion. Calculated energy for this expansion is comparable to an experimentally estimated energy difference between the closed and the first subconductance state, suggesting that our model simulates the initial step toward the full opening of MscL. The model also successfully mimicked the behaviors of a gain of function mutant (G22N) and a loss of function mutant (F78N), strongly supporting that our MD model did simulate some essential biophysical aspects of the mechano-gating in MscL.     

The gating mechanism of the bacterial mechanosensitive channel MscL revealed by molecular dynamics simulations: From tension sensing to channel opening

One of the ultimate goals of the study on mechanosensitive (MS) channels is to understand the biophysical mechanisms of how the MS channel protein senses forces and how the sensed force induces channel gating. The bacterial MS channel MscL is an ideal subject to reach this goal owing to its resolved 3D protein structure in the closed state on the atomic scale and large amounts of electrophysiological data on its gating kinetics. However, the structural basis of the dynamic process from the closed to open states in MscL is not fully understood. In this study, we performed molecular dynamics (MD) simulations on the initial process of MscL opening in response to a tension increase in the lipid bilayer. To identify the tension-sensing site(s) in the channel protein, we calculated interaction energy between membrane lipids and candidate amino acids (AAs) facing the lipids. We found that Phe78 has a conspicuous interaction with the lipids, suggesting that Phe78 is the primary tension sensor of MscL. Increased membrane tension by membrane stretch dragged radially the inner (TM1) and outer (TM2) helices of MscL at Phe78, and the force was transmitted to the pentagon-shaped gate that is formed by the crossing of the neighboring TM1 helices in the inner leaflet of the bilayer. The radial dragging force induced radial sliding of the crossing portions, leading to a gate expansion. Calculated energy for this expansion is comparable to an experimentally estimated energy difference between the closed and the first subconductance state, suggesting that our model simulates the initial step toward the full opening of MscL. The model also successfully mimicked the behaviors of a gain of function mutant (G22N) and a loss of function mutant (F78N), strongly supporting that our MD model did simulate some essential biophysical aspects of the mechano-gating in MscL.     

Origins of proton transport behavior from selectivity domain mutations of the aquaporin-1 channel

The permeation free-energy profile and maximum ion conductance of proton transport along the channel of three aquaporin-1 (AQP1) mutants (H180A/R195V, H180A, and R195V) are calculated via molecular dynamics simulations and Poisson-Nernst-Planck theory. The proton dynamics was described by the multistate empirical valence bond (MS-EVB) model. The results reveal three major contributions to the overall free-energy barrier for proton transport in AQP1: 1), the bipolar field, 2), the electrostatic repulsion due to the Arg-195 residue, and 3), the dehydration penalty due to the narrow channel pore. The double mutation (H180A/R195V) drastically drops the overall free-energy barrier by roughly 20 kcal/mol via simultaneously relaxing the direct electrostatic interaction (by R195V) and dehydration effect (by H180A).     

Towards the mechanism of proton pumping by the haem-copper oxidases

The haem-copper oxidases comprise a large family of enzymes that is widespread among aerobic organisms. These remarkable membrane-bound proteins catalyse the respiratory reduction of dioxygen to water, and conserve free energy from this reaction by operating as proton pumps. The mechanism of redox-dependent proton translocation has been elusive despite the availability of high resolution crystal structures from several oxidases. Here, we discuss some recent as well as some older results that may shed light on this mechanism. We conclude that proton-pumping is initiated by vectorial proton transfer from a conserved glutamic acid (Glu242 in the bovine enzyme) to a proton acceptor above the haem groups, and that this primary event is mechanistically coupled to electron transfer from haem a to the binuclear haem a₃/Cu_B centre. Subsequently, Glu242 is reprotonated from the negatively charged side of the membrane. Next this proton is transferred to the binuclear site to complete the chemistry, Glu242 is reprotonated once more, and the “prepumped” proton is ejected on the opposite side of the membrane. The different kinetics of electron-coupled proton transfer in different steps of the catalytic cycle may be related to differences in the driving force due to different E_m values of the electron acceptor in the binuclear site.     

Structural determinants of proton blockage in aquaporins

Aquaporins are an important class of membrane channels selective for water and linear polyols but impermeable to ions, including protons. Recent computational studies have revealed that the relay of protons through the water-conduction pathway of aquaporin channels is opposed by a substantial free energy barrier peaking at the signature NPA motifs. Here, free-energy simulations and continuum electrostatic calculations are combined to examine the nature and the magnitude of the contribution of specific structural elements to proton blockage in the bacterial glycerol uptake facilitator, GlpF. Potential of mean-force profiles for both hop and turn steps of structural diffusion in the narrow pore are obtained for artificial variants of the GlpF channel in which coulombic interactions between the pore contents and conserved residues Asn68 and Asn203 at the NPA signature motifs, Arg206 at the selectivity filter, and the peptidic backbone of the two half-helices M3 and M7, which are arranged in head-to-head fashion around the NPA motifs, are turned off selectively. A comparison of these results with electrostatic energy profiles for the translocation of a probe cation throughout the water permeation pathway indicates that the free-energy profile for proton movement inside the narrow pore is dominated by static effects arising from the distribution of charged and polar groups of the channel, whereas dielectric effects contribute primarily to opposing the access of H+ to the pore mouths (desolvation penalty). The single most effective way to abolish the free-energy gradients opposing the movement of H+ around the NPA motif is to turn off the dipole moments of helices M3 and M7. Mutation of either of the two NPA Asn residues to Asp compensates for charge-dipole and dipole-dipole effects opposing the hop and turn steps of structural diffusion, respectively, and dramatically reduces the free energy barrier of proton translocation, suggesting that these single mutants could leak protons.     

Molecular dynamics study of aquaporin-1 water channel in a lipid bilayer

The aquaporin-1 water channel was modeled in a palmitoyl-oleoyl-phosphatidyl-choline lipid bilayer, by means of molecular dynamics simulations. Interaction of the protein with the membrane and inter-monomer interactions were analyzed. Structural features of the channel important for its biological function, including the Asn-Pro-Ala (NPA) motifs, and the diffusion of water molecules into the channels, were investigated. Simulations revealed the formation of single file water inside the channels for certain relative positions of the NPA motifs.     

Molecular mechanism of statin-mediated LOX-1 inhibition

Statins are largely used in clinics in the treatment of patients with cardiovascular diseases for their effect on lowering circulating cholesterol. Lectin-like oxidized low-density lipoprotein (LOX-1), the primary receptor for ox-LDL, plays a central role in the pathogenesis of atherosclerosis and cardiovascular disorders. We have recently shown that chronic exposure of cells to lovastatin disrupts LOX-1 receptor cluster distribution in plasma membranes, leading to a marked loss of LOX-1 function. Here we investigated the molecular mechanism of statin-mediated LOX-1 inhibition and we demonstrate that all tested statins are able to displace the binding of fluorescent ox-LDL to LOX-1 by a direct interaction with LOX-1 receptors in a cell-based binding assay. Molecular docking simulations confirm the interaction and indicate that statins completely fill the hydrophobic tunnel that crosses the C-type lectin-like (CTLD) recognition domain of LOX-1. Classical molecular dynamics simulation technique applied to the LOX-1 CTLD, considered in the entire receptor structure with or without a statin ligand inside the tunnel, indicates that the presence of a ligand largely increases the dimer stability. Electrophoretic separation and western blot confirm that different statins binding stabilize the dimer assembly of LOX-1 receptors in vivo. The simulative and experimental results allow us to propose a CTLD clamp motion, which enables the receptor-substrate coupling. These findings reveal a novel and significant functional effect of statins.     

Toward elucidating the heat activation mechanism of the TRPV1 channel gating by molecular dynamics simulation

As a key cellular sensor, the TRPV1 cation channel undergoes a gating transition from a closed state to an open state in response to various physical and chemical stimuli including noxious heat. Despite years of study, the heat activation mechanism of TRPV1 gating remains enigmatic at the molecular level. Toward elucidating the structural and energetic basis of TRPV1 gating, we have performed molecular dynamics (MD) simulations (with cumulative simulation time of 3 μs), starting from the high‐resolution closed and open structures of TRPV1 solved by cryo‐electron microscopy. In the closed‐state simulations at 30°C, we observed a stably closed channel constricted at the lower gate (near residue I679), while the upper gate (near residues G643 and M644) is dynamic and undergoes flickery opening/closing. In the open‐state simulations at 60°C, we found higher conformational variation consistent with a large entropy increase required for the heat activation, and both the lower and upper gates are dynamic with transient opening/closing. Through ensemble‐based structural analyses of the closed state versus the open state, we revealed pronounced closed‐to‐open conformational changes involving the membrane proximal domain (MPD) linker, the outer pore, and the TRP helix, which are accompanied by breaking/forming of a network of closed/open‐state specific hydrogen bonds. By comparing the closed‐state simulations at 30°C and 60°C, we observed heat‐activated conformational changes in the MPD linker, the outer pore, and the TRP helix that resemble the closed‐to‐open conformational changes, along with partial formation of the open‐state specific hydrogen bonds. Some of the residues involved in the above key hydrogen bonds were validated by previous mutational studies. Taken together, our MD simulations have offered rich structural and dynamic details beyond the static structures of TRPV1, and promising targets for future mutagenesis and functional studies of the TRPV1 channel. Proteins 2016; 84:1938–1949. © 2016 Wiley Periodicals, Inc.     

The proton gradient across the vacuo-lysosomal membrane of lutoids from the latex ofHevea brasiliensis. I. Further evidence for a proton-translocating ATPase on the vacuo-lysosomal membrane of intact lutoids

Summary Lutoids (vacuo-lysosomal particles) were isolated from the latex ofHevea brasiliensis. Using flow dialysis with¹⁴C-methylamine uptake as a pH probe and⁸⁶Rb rubidium+valinomycin distribution for estimations of transmembrane electrical potential, intact lutoids exhibited a pH of 1 unit (interior more acid) and a of –70 mV (interior negative), when suspended in an isotonic medium at physiological concentration of potassium (30mm) and pH 7.0, in the absence of ATP. In most cases, the Donnan potential was shown to fully account for pH in nonenergized lutoids. The addition of Mg-ATP (5mm) resulted in a marked acidification of the lutoidic internal space (0.7 to 1 pH unit) depending on the composition of the medium, and in a membrane depolarization by 60 mV (interior becoming less negative). The resulting electrochemical potential of protons ( ) increased by a hundred millivolts when lutoids were energized by ATP. These data strongly support an inward electrogenic proton translocating function for the ATPase of the vacuo-lysosomal membrane of lutoids. Results are discussed in terms of thein vivo maintenance of large lutoids/cytoplasm proton gradients, and of the rôle of these vacuo-lysosomes in the homeostasis of the cytoplasmic metabolism.     

The structural basis of water permeation and proton exclusion in aquaporins

Aquaporins (AQPs) represent a ubiquitous class of integral membrane proteins that play critical roles in cellular osmoregulations in microbes, plants and mammals. AQPs primarily function as water-conducting channels, whereas members of a sub-class of AQPs, termed aquaglyceroporins, are permeable to small neutral solutes such as glycerol. While AQPs facilitate transmembrane permeation of water and/or small neutral solutes, they preclude the conduction of protons. Consequently, openings of AQP channels allow rapid water diffusion down an osmotic gradient without dissipating electrochemical potentials. Molecular structures of AQPs portray unique features that define the two central functions of AQP channels: effective water permeation and strict proton exclusion. This review describes AQP structures known to date and discusses the mechanisms underlying water permeation, proton exclusion and water permeability regulation.     

Potential role of the glial water channel aquaporin-4 in epilepsy

Recent studies have implicated glial cells in novel physiological roles in the CNS, such as modulation of synaptic transmission, so it is possible that glial cells might have a functional role in the hyperexcitability that is characteristic of epilepsy. Indeed, alterations in distinct astrocyte membrane channels, receptors and transporters have all been associated with the epileptic state. This paper focuses on the potential roles of the glial water channel aquaporin-4 (AQP4) in modulating brain excitability and in epilepsy. We review studies of seizure phenotypes, K(+) homeostasis and extracellular space physiology of mice that lack AQP4 (AQP4(-/-) mice) and discuss the human studies demonstrating alterations of AQP4 in specimens of human epilepsy tissue. We conclude with new studies of AQP4 regulation by seizures and discuss its potential role in the development of epilepsy (epileptogenesis). Although many questions remain unanswered, the available data indicate that AQP4 and its molecular partners might represent important new therapeutic targets.     

Interaction of Piscidin-1 with zwitterionic versus anionic membranes: a comparative molecular dynamics study

Plasma membrane of each micro-organism has a unique set of lipid composition as a consequence of the environmental adaptation or a response to exposure to antimicrobial peptides (AMPs) as antibiotic agents. Understanding the relationship between lipid composition and action of antimicrobial peptides or considering how different lipid bilayers respond to AMPs may help us design more effective peptide drugs in the future. In this contribution, we intend to elucidate how two currently used membrane models, namely palmitoyl-oleoyl-phosphtidylglycerol (POPG) and 1-palmitoyl-oleoyl-glycero-phosphocholine (POPC), respond to antimicrobial peptide Piscidin-1 (Pis-1).The computed density profile of the peptide as it moves from the bulk solvent toward the membrane core suggests that Pis-1 penetrates into the POPG bilayer less than the POPC membrane. Furthermore, we showed that the two model membranes used in this study have different behavior in the presence of Pis-1. Hence, we suggest that membrane composition could be an important factor in determining lytic ability of peptide drugs to kill a unique bacterial species.

An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:37