Structural Analysis of Membrane-associated Forms of Helicobacter pylori VacA Toxin.

Helicobacter pylori colonizes the stomach in about half of the human population, leading to an increased risk of peptic ulcer disease and gastric cancer. H. pylori secretes an 88 kDa VacA toxin that contributes to pathogenesis. VacA assembles into oligomeric complexes in solution and forms anion-selective channels in cell membranes. Cryo-electron microscopy (cryo-EM) analyses of VacA oligomers in solution provided insights into VacA oligomerization but failed to reveal the structure of the hydrophobic N-terminal region predicted to be a pore-forming domain. In this study, we incubated VacA with liposomes and used single particle cryo-EM to analyze detergent-extracted VacA oligomers. A 3D structure of detergent-solubilized VacA hexamers revealed the presence of six α-helices extending from the center of the oligomers, a feature not observed in previous studies of water-soluble VacA oligomers. Cryo-electron tomography analysis and 2D averages of VacA associated with liposomes confirmed that central regions of the membrane-associated VacA oligomers can insert into the lipid bilayer. However, insertion is heterogenous, with some membrane-associated oligomers appearing only partially inserted and others sitting on top of the bilayer. These studies indicate that VacA undergoes a conformational change when contacting the membrane and reveal an α-helical region positioned to extend into the membrane. Although the reported VacA 3D structure does not represent a selective anion channel, our combined single particle 3D analysis, cryo-electron tomography, and modeling allow us to propose a model for the structural organization of the VacA N-terminus in the context of a hexamer as it inserts into the membrane.

The Advanced Properties of Circularized MSP Nanodiscs Facilitate High-resolution NMR Studies of Membrane Proteins.

Membrane mimetics are essential for structural and functional studies of membrane proteins. A promising lipid-based system are phospholipid nanodiscs, where two copies of a so-called membrane scaffold protein (MSP) wrap around a patch of lipid bilayer. Consequently, the size of a nanodisc is determined by the length of the MSP. Furthermore, covalent MSP circularization was reported to improve nanodisc stability. However, a more detailed comparative analysis of the biophysical properties of circularized and linear MSP nanodiscs for their use in high-resolution NMR has not been conducted so far. Here, we analyze the membrane fluidity and temperature-dependent size variability of circularized and linear nanodiscs using a large set of analytical methods. We show that MSP circularization does not alter the membrane fluidity in nanodiscs. Further, we show that the phase transition temperature increases for circularized versions, while the cooperativity decreases. We demonstrate that circularized nanodiscs keep a constant size over a large temperature range, in contrast to their linear MSP counterparts. Due to this size stability, circularized nanodiscs are beneficial for high-resolution NMR studies of membrane proteins at elevated temperatures. Despite their slightly larger size as compared to linear nanodiscs, 3D NMR experiments of the voltage-dependent anion channel 1 (VDAC1) in circularized nanodiscs have a markedly improved spectral quality in comparison to VDAC1 incorporated into linear nanodiscs of a similar size. This study provides evidence that circularized MSP nanodiscs are a promising tool to facilitate high-resolution NMR studies of larger and challenging membrane proteins in a native lipid environment.

Homocysteine-Thiolactone Modulates Gating of Mitochondrial Voltage-Dependent Anion Channel (VDAC) and Protects It from Induced Oxidative Stress.

The gating of the Voltage-Dependent Anion Channel (VDAC) is linked to oxidative stress through increased generation of mitochondrial ROS with increasing mitochondrial membrane potential (ΔΨm). It has been already reported that H2O2 increases the single-channel conductance of VDAC on a bilayer lipid membrane. On the other hand, homocysteine (Hcy) has been reported to induce mitochondria-mediated cell death. It is argued that the thiol-form of homocysteine, HTL could be the plausible molecule responsible for the alteration in the function of proteins, such as VDAC. It is hypothesized that HTL interacts with VDAC that causes functional abnormalities. An investigation was undertaken to study the interaction of HTL with VDAC under H2O2 induced oxidative stress through biophysical and electrophysiological methods. Fluorescence spectroscopic studies indicate that HTL interacts with VDAC, but under induced oxidative stress the effect is prevented partially. Similarly, bilayer electrophysiology studies suggest that HTL shows a reduction in VDAC single-channel conductance, but the effects are partially prevented under an oxidative environment. Gly172 and His181 are predicted through bioinformatics tools to be the most plausible binding residues of HTL in Rat VDAC. The binding of HTL and H2O2 with VDAC appears to be cooperative as per our analysis of experimental data in the light of the Hill-Langmuir equation. The binding energies are estimated to be - 4.7 kcal mol-1 and - 2.8 kcal mol-1, respectively. The present in vitro studies suggest that when mitochondrial VDAC is under oxidative stress, the effects of amino acid metabolites like HTL are suppressed.

Voltage Dependent Anion Channel 3 (VDAC3) protects mitochondria from oxidative stress.

Unraveling the role of VDAC3 within living cells is challenging and still requires a definitive answer. Unlike VDAC1 and VDAC2, the outer mitochondrial membrane porin 3 exhibits unique biophysical features that suggest unknown cellular functions. Electrophysiological studies on VDAC3 carrying selective cysteine mutations and mass spectrometry data about the redox state of such sulfur containing amino acids are consistent with a putative involvement of isoform 3 in mitochondrial ROS homeostasis. Here, we thoroughly examined this issue and provided for the first time direct evidence of the role of VDAC3 in cellular response to oxidative stress. Depletion of isoform 3 but not isoform 1 significantly exacerbated the cytotoxicity of redox cyclers such as menadione and paraquat, and respiratory complex I inhibitors like rotenone, promoting uncontrolled accumulation of mitochondrial free radicals. High-resolution respirometry of transiently transfected HAP1-ΔVDAC3 cells expressing the wild type or the cysteine-null mutant VDAC3 protein, unequivocally confirmed that VDAC3 cysteines are indispensable for protein ability to counteract ROS-induced oxidative stress.

Characterization of the voltage-dependent anion channel (VDAC) gene family in wheat (Triticum aestivum L.) and its potential mechanism in response to drought and salinity stresses.

Voltage-dependent anion channels (VDACs) are major transport proteins localized in the outer membrane of mitochondria and play critical roles in regulating plant growth and responding to stress. In this study, a total of 26 VDAC genes in common wheat (Triticum aestivum L.) were identified. TaVDACs that contained ß-barrel structures were classified into three groups with phylogenetic and sequence alignment. Additionally, the gene structure and protein conserved motif composition varied among diverse subfamilies but were relatively conserved within the same subfamily. The basic elements that were stress- and hormone-related, including TATA-box, CAAT-box, MBS, LTR, TC-rich repeats, ABRE, P-box and TATC-box, were predicted within the promoter region of TaVDAC genes. TaVDAC expression patterns differed among tissues, organs and abiotic stress conditions. Overexpression (OE) of TaVDAC1-B conferred high tolerance to salinity and less resistance to drought stress in Arabidopsis thaliana. TaVDAC1-B interacted with Nucleoredoxin-D1 (TaNRX-D1) protein. Furthermore, compared with WT lines, salinity stress further upregulated the level of AtNRX1 (homologous gene of TaNRX-D1 in Arabidopsis) expression and the activity of superoxide dismutase in TaVDAC1-B OE lines, which led to a decrease in superoxide radical accumulation; drought stress further downregulated AtNRX1 expression and superoxide dismutase activity in TaVDAC1-B OE lines, resulting in the accumulation of superoxide radicals. Our study not only presents comprehensive information for understanding the VDAC gene family in wheat but also proposes a potential mechanism in response to drought and salinity stress.

Voltage-activated complexation of α-synuclein with three diverse ß-barrel channels: VDAC, MspA, and α-hemolysin.

Voltage-activated complexation is the process by which a transmembrane potential drives complex formation between a membrane-embedded channel and a soluble or membrane-peripheral target protein. Metabolite and calcium flux across the mitochondrial outer membrane was shown to be regulated by voltage-activated complexation of the voltage-dependent anion channel (VDAC) and either dimeric tubulin or α-synuclein (αSyn). However, the roles played by VDAC's characteristic attributes-its anion selectivity and voltage gating behavior-have remained unclear. Here, we compare in vitro measurements of voltage-activated complexation of αSyn with three well-characterized ß-barrel channels-VDAC, MspA, and α-hemolysin-that differ widely in their organism of origin, structure, geometry, charge density distribution, and voltage gating behavior. The voltage dependences of the complexation dynamics for the different channels are observed to differ quantitatively but have similar qualitative features. In each case, energy landscape modeling describes the complexation dynamics in a manner consistent with the known properties of the individual channels, while voltage gating does not appear to play a role. The reaction free energy landscapes thus calculated reveal a non-trivial dependence of the αSyn/channel complex stability on the surface density of αSyn.

The Role of Voltage-Dependent Anion Channel in Mitochondrial Dysfunction and Human Disease.

The voltage-dependent anion channel (VDAC) is a ß-barrel membrane protein located in the outer mitochondrial membrane (OMM). VDAC has two conductance states: an open anion selective state, and a closed and slightly cation-selective state. VDAC conductance states play major roles in regulating permeability of ATP/ADP, regulation of calcium homeostasis, calcium flux within ER-mitochondria contact sites, and apoptotic signaling events. Three reported structures of VDAC provide information on the VDAC open state via X-ray crystallography and nuclear magnetic resonance (NMR). Together, these structures provide insight on how VDAC aids metabolite transport. The interaction partners of VDAC, together with the permeability of the pore, affect the molecular pathology of diseases including Parkinson's disease (PD), Friedreich's ataxia (FA), lupus, and cancer. To fully address the molecular role of VDAC in disease pathology, major questions must be answered on the structural conformers of VDAC. For example, further information is needed on the structure of the closed state, how binding partners or membrane potential could lead to the open/closed states, the function and mobility of the N-terminal α-helical domain of VDAC, and the physiological role of VDAC oligomers. This review covers our current understanding of the various states of VDAC, VDAC interaction partners, and the roles they play in mitochondrial regulation pertaining to human diseases.

Self-standing, conducting and capacitive biomimetic hybrid nanomembranes for selective molecular ion separation.

Hybrid free-standing biomimetic materials are developed by integrating the VDAC36 ß-barrel protein into robust and flexible three-layered polymer nanomembranes. The first and third layers are prepared by spin-coating a mixture of poly(lactic acid) (PLA) and poly(vinyl alcohol) (PVA). PVA nanofeatures are transformed into controlled nanoperforations by solvent-etching. The two nanoperforated PLA layers are separated by an electroactive layer, which is successfully electropolymerized by introducing a conducting sacrificial substrate under the first PLA nanosheet. Finally, the nanomaterial is consolidated by immobilizing the VDAC36 protein, active as an ion channel, into the nanoperforations of the upper layer. The integration of the protein causes a significant reduction of the material resistance, which decreases from 21.9 to 3.9 kΩ cm2. Electrochemical impedance spectroscopy studies using inorganic ions and molecular metabolites (i.e.l-lysine and ATP) not only reveal that the hybrid films behave as electrochemical supercapacitors but also indicate the most appropriate conditions to obtain selective responses against molecular ions as a function of their charge. The combination of polymers and proteins is promising for the development of new devices for engineering, biotechnological and biomedical applications.

Regulation of Mitochondrial Respiration by VDAC Is Enhanced by Membrane-Bound Inhibitors with Disordered Polyanionic C-Terminal Domains.

The voltage-dependent anion channel (VDAC) is the primary regulating pathway of water-soluble metabolites and ions across the mitochondrial outer membrane. When reconstituted into lipid membranes, VDAC responds to sufficiently large transmembrane potentials by transitioning to gated states in which ATP/ADP flux is reduced and calcium flux is increased. Two otherwise unrelated cytosolic proteins, tubulin, and α-synuclein (αSyn), dock with VDAC by a novel mechanism in which the transmembrane potential draws their disordered, polyanionic C-terminal domains into and through the VDAC channel, thus physically blocking the pore. For both tubulin and αSyn, the blocked state is observed at much lower transmembrane potentials than VDAC gated states, such that in the presence of these cytosolic docking proteins, VDAC's sensitivity to transmembrane potential is dramatically increased. Remarkably, the features of the VDAC gated states relevant for bioenergetics-reduced metabolite flux and increased calcium flux-are preserved in the blocked state induced by either docking protein. The ability of tubulin and αSyn to modulate mitochondrial potential and ATP production in vivo is now supported by many studies. The common physical origin of the interactions of both tubulin and αSyn with VDAC leads to a general model of a VDAC inhibitor, facilitates predictions of the effect of post-translational modifications of known inhibitors, and points the way toward the development of novel therapeutics targeting VDAC.

Proton Detected Solid-State NMR of Membrane Proteins at 28 Tesla (1.2 GHz) and 100 kHz Magic-Angle Spinning.

The available magnetic field strength for high resolution NMR in persistent superconducting magnets has recently improved from 23.5 to 28 Tesla, increasing the proton resonance frequency from 1 to 1.2 GHz. For magic-angle spinning (MAS) NMR, this is expected to improve resolution, provided the sample preparation results in homogeneous broadening. We compare two-dimensional (2D) proton detected MAS NMR spectra of four membrane proteins at 950 and 1200 MHz. We find a consistent improvement in resolution that scales superlinearly with the increase in magnetic field for three of the four examples. In 3D and 4D spectra, which are now routinely acquired, this improvement indicates the ability to resolve at least 2 and 2.5 times as many signals, respectively.

Exploring lipid-dependent conformations of membrane-bound α-synuclein with the VDAC nanopore.

Regulation of VDAC by α-synuclein (αSyn) is a rich and instructive example of protein-protein interactions catalyzed by a lipid membrane surface. αSyn, a peripheral membrane protein involved in Parkinson's disease pathology, is known to bind to membranes in a transient manner. αSyn's negatively charged C-terminal domain is then available to be electromechanically trapped by the VDAC ß-barrel, a process that is observed in vitro as the reversible reduction of ion flow through a single voltage-biased VDAC nanopore. Binding of αSyn to the lipid bilayer is a prerequisite of the channel-protein interaction; surprisingly, however, we find that the strength of αSyn binding to the membrane does not correlate in any simple way with its efficiency of blocking VDAC, suggesting that the lipid-dependent conformations of the membrane-bound αSyn control the interaction. Quantitative models of the free energy landscape governing the capture and release processes allow us to discriminate between several αSyn (sub-) conformations on the membrane surface. These results, combined with known structural features of αSyn on anionic lipid membranes, point to a model in which the lipid composition determines the fraction of αSyn molecules for which the charged C terminal domain is constrained to be close, but not tightly bound, to the membrane surface and thus readily captured by the VDAC nanopore. We speculate that changes in the mitochondrial membrane lipid composition may be key regulators of the αSyn-VDAC interaction and consequently of VDAC-facilitated transport of ions and metabolites in and out of mitochondria and, i.e. mitochondrial metabolism.

Structure, gating and interactions of the voltage-dependent anion channel.

The voltage-dependent anion channel (VDAC) is one of the most highly abundant proteins found in the outer mitochondrial membrane, and was one of the earliest discovered. Here we review progress in understanding VDAC function with a focus on its structure, discussing various models proposed for voltage gating as well as potential drug targets to modulate the channel's function. In addition, we explore the sensitivity of VDAC structure to variations in the membrane environment, comparing DMPC-only, DMPC with cholesterol, and near-native lipid compositions, and use magic-angle spinning NMR spectroscopy to locate cholesterol on the outside of the ß-barrel. We find that the VDAC protein structure remains unchanged in different membrane compositions, including conditions with cholesterol.

Renaissance of VDAC: New Insights on a Protein Family at the Interface between Mitochondria and Cytosol.

It has become impossible to review all the existing literature on Voltage-Dependent Anion selective Channel (VDAC) in a single article. A real Renaissance of studies brings this protein to the center of decisive knowledge both for cell physiology and therapeutic application. This review, after highlighting the similarities between the cellular context and the study methods of the solute carriers present in the inner membrane and VDAC in the outer membrane of the mitochondria, will focus on the isoforms of VDAC and their biochemical characteristics. In particular, the possible reasons for their evolutionary onset will be discussed. The variations in their post-translational modifications and the differences between the regulatory regions of their genes, probably the key to understanding the current presence of these genes, will be described. Finally, the situation in the higher eukaryotes will be compared to that of yeast, a unicellular eukaryote, where there is only one active isoform and the role of VDAC in energy metabolism is better understood.

Kidney-disease-associated variants of Apolipoprotein L1 show gain of function in cation channel activity.

Variants in Apolipoprotein L1 (ApoL1) are known to be responsible for increased risk of some progressive kidney diseases among people of African ancestry. ApoL1 is an amphitropic protein that can insert into phospholipid membranes and confer anion- or cation-selective permeability to phospholipid membranes depending on pH. Whether these activities differ among the variants or whether they contribute to disease pathogenesis is unknown. We used assays of voltage-driven ion flux from phospholipid vesicles and of stable membrane association to assess differences among ApoL1 isoforms. There is a significant (approximately twofold) increase in the cation-selective ion permease activity of the two kidney-disease-associated variants compared with the reference protein. In contrast, we find no difference in the anion-selective permease activity at low pH among the isoforms. Compared with the reference sequence, the two disease-associated variants show increased stable association with phospholipid vesicles under conditions that support the cation permease activity, suggesting that the increased activity may be due to more efficient membrane association and insertion. There is no difference in membrane association among isoforms under optimal conditions for the anion permease activity. These data support a model in which enhanced cation permeability may contribute to the progressive kidney diseases associated with high-risk ApoL1 alleles.

MicroED structure of lipid-embedded mammalian mitochondrial voltage-dependent anion channel.

A structure of the murine voltage-dependent anion channel (VDAC) was determined by microcrystal electron diffraction (MicroED). Microcrystals of an essential mutant of VDAC grew in a viscous bicelle suspension, making it unsuitable for conventional X-ray crystallography. Thin, plate-like crystals were identified using scanning-electron microscopy (SEM). Crystals were milled into thin lamellae using a focused-ion beam (FIB). MicroED data were collected from three crystal lamellae and merged for completeness. The refined structure revealed unmodeled densities between protein monomers, indicative of lipids that likely mediate contacts between the proteins in the crystal. This body of work demonstrates the effectiveness of milling membrane protein microcrystals grown in viscous media using a focused ion beam for subsequent structure determination by MicroED. This approach is well suited for samples that are intractable by X-ray crystallography. To our knowledge, the presented structure is a previously undescribed mutant of the membrane protein VDAC, crystallized in a lipid bicelle matrix and solved by MicroED.

Evolutionary selection of a 19-stranded mitochondrial ß-barrel scaffold bears structural and functional significance.

Transmembrane ß-barrels of eukaryotic outer mitochondrial membranes (OMMs) are major channels of communication between the cytosol and mitochondria and are indispensable for cellular homeostasis. A structurally intriguing exception to all known transmembrane ß-barrels is the unique odd-stranded, i.e. 19-stranded, structures found solely in the OMM. The molecular origins of this 19-stranded structure and its associated functional significance are unclear. In humans, the most abundant OMM transporter is the voltage-dependent anion channel. Here, using the human voltage-dependent anion channel as our template scaffold, we designed and engineered odd- and even-stranded structures of smaller (V216, V217, V218) and larger (V220, V221) barrel diameters. Determination of the structure, dynamics, and energetics of these engineered structures in bilayer membranes reveals that the 19-stranded barrel surprisingly holds modest to low stability in a lipid-dependent manner. However, we demonstrate that this structurally metastable protein possesses superior voltage-gated channel regulation, efficient mitochondrial targeting, and in vivo cell survival, with lipid-modulated stability, all of which supersede the occurrence of a metastable 19-stranded scaffold. We propose that the unique structural adaptation of these transmembrane transporters exclusively in mitochondria bears strong evolutionary basis and is functionally significant for homeostasis.

Regulation of Single-Channel Conductance of Voltage-Dependent Anion Channel by Mercuric Chloride in a Planar Lipid Bilayer.

The existence of mercury in various forms, e.g., elemental, organic, and inorganic has been known for decades. In any of these forms, it is poisonous to metabolism. In this, an investigation about the effect of the inorganic form of mercury, i.e., mercuric chloride (HgCl2) to the mitochondrial voltage-dependent anion channel (VDAC), has been done after isolation from the cardiac and brain tissues of Wistar rats. In vitro electrophysiology experiments were performed in Cardiolipin planar lipid bilayer membrane (BLM) to study the change in the conductance, selectivity, and gating charge of VDAC post HgCl2 treatment. A reduction in mean conductance of VDAC from 4.3 ± 0.18 to 1.66 ± 0.11 nS was observed. Further, the Gating charge calculated before (± 3.5) and after HgCl2 treatment (± 2.3) showed significant difference. Later, VDAC's behavior was studied at different concentrations of HgCl2 ranging from 0.1 µM to 1 mM. The Inhibitory concentration (IC50) was calculated from the linear regression plot. The IC50 was found to be 488.1 µM. In the asymmetrical HgCl2 (5:1), a permeability ratio of cation to anion was found to be 4.2. It is interpreted that VDAC functioning is affected due to the application of 4 mM HgCl2 and a reduction in the conductance, gating charge, and permeability of VDAC was detected. The results provide clues to HgCl2-induced toxicity mediated through VDAC in the Cardiolipin BLM.

Cryo-EM structure of the volume-regulated anion channel LRRC8D isoform identifies features important for substrate permeation.

Members of the leucine-rich repeat-containing 8 (LRRC8) protein family, composed of the five LRRC8A-E isoforms, are pore-forming components of the volume-regulated anion channel (VRAC). LRRC8A and at least one of the other LRRC8 isoforms assemble into heteromers to generate VRAC transport activities. Despite the availability of the LRRC8A structures, the structural basis of how LRRC8 isoforms other than LRRC8A contribute to the functional diversity of VRAC has remained elusive. Here, we present the structure of the human LRRC8D isoform, which enables the permeation of organic substrates through VRAC. The LRRC8D homo-hexamer structure displays a two-fold symmetric arrangement, and together with a structure-based electrophysiological analysis, revealed two key features. The pore constriction on the extracellular side is wider than that in the LRRC8A structures, which may explain the increased permeability of organic substrates. Furthermore, an N-terminal helix protrudes into the pore from the intracellular side and may be critical for gating.

The inverse relation between mitochondrial transmembrane potential and proteins α-helix in neuronal-like cells under static magnetic field and the role of VDAC.

In this study, a correlation between cell channel α-helices displacement and the mitochondrial transmembrane potential after exposure of 3, 7, 15 and 24 h of neuronal-like cells to a uniform magnetic field at the intensity of 2 mT was shown. Fourier Transform Infrared (FTIR) Spectroscopy and fluorescence techniques were used to analyze the secondary structure of protein content and mitochondrial transmembrane potential, respectively. The main result of this study was represented by a significant inverse relation between the mitochondrial transmembrane potential and the intensity of the Amide I band that can be associated with time exposure. Given that mitochondrial transmembrane potential should be related to the gating state of voltage-dependent anion channel (VDAC) in mitochondrial membrane, this result could have a relevant role in medicine. Indeed, VDAC's irregular behavior can be associated with several varieties of mitochondria-associated pathologies and various forms of cancer and neurodegeneration.

A size-modified poisson-boltzmann ion channel model in a solvent of multiple ionic species: Application to voltage-dependent anion channel.

We present a new size-modified Poisson-Boltzmann ion channel (SMPBIC) model and use it to calculate the electrostatic potential, ionic concentrations, and electrostatic solvation free energy for a voltage-dependent anion channel (VDAC) on a biological membrane in a solution mixture of multiple ionic species. In particular, the new SMPBIC model adopts a membrane surface charge density and a natural Neumann boundary condition to reflect the charge effect of the membrane on the electrostatics of VDAC. To avoid the singularity difficulties caused by the atomic charges of VDAC, the new SMPBIC model is split into three submodels such that the solution of one of the submodels is obtained analytically and contains all the singularity points of the SMPBIC model. The other two submodels are then solved numerically much more efficiently than the original SMPBIC model. As an application of this SMPBIC submodel partitioning scheme, we derive a new formula for computing the electrostatic solvation free energy. Numerical results for a human VDAC isoform 1 (hVDAC1) in three different salt solutions, each with up to five different ionic species, confirm the significant effects of membrane surface charges on both the electrostatics and ionic concentrations. The results also show that the new SMPBIC model can describe well the anion selectivity property of hVDAC1, and that the new electrostatic solvation free energy formula can significantly improve the accuracy of the currently used formula. © 2019 Wiley Periodicals, Inc.