The voltage-dependent anion channel (VDAC), also known as mitochondrial porin, is the most abundant protein in the mitochondrial outer membrane (MOM). VDAC is the channel known to guide the metabolic flux across the MOM and plays a key role in mitochondrially induced apoptosis. Here, we present the 3D structure of human VDAC1, which was solved conjointly by NMR spectroscopy and x-ray crystallography. Human VDAC1 (hVDAC1) adopts a ␤-barrel architecture composed of 19 ␤-strands with an ␣-helix located horizontally midway within the pore. Bioinformatic analysis indicates that this channel architecture is common to all VDAC proteins and is adopted by the general import pore TOM40 of mammals, which is also located in the MOM.T he outer membrane of mitochondria (MOM) contains three integral membrane protein families, two of which form channels as part of larger protein complexes (for review, see ref. 1). These two MOM complexes, the general import pore TOM and the SAM insertase, allow for the entire translocation and insertion of nearly all newly synthesized proteins destined to the mitochondrial organelle (2, 3). The third protein family of typically high abundance (Ϸ10,000 copies per mitochondrion) is termed voltage-dependent anion channels (VDACs), because of the voltage sensitivity of its open probability (4, 5). Together, this small number of protein families is sufficient for full communication between mitochondria with their cellular environment (1).The VDAC channel was initially described as being reminiscent of bacterial porins and primarily responsible for the exchange of chemical energy equivalents between the cytosol and the mitochondrion (4, 6). Indeed, a variety of structural features (like barrel geometry and dimension) known from the bacterial precursors are maintained (7,8). By contrast, a variety of functions have been ascribed to the VDAC isoforms among which the direct coupling of the mitochondrial matrix to the energy maintenance of the cytosol seems to be the most general function (9). The structure of VDAC is of interest because of a substantial body of evidence connecting VDAC to apoptosis. It is suggested that VDAC is a critical player in the release of apoptogenic factors from mitochondria of mammalian cells, and consequently several hypotheses describing the mechanism of mitochondria-mediated apoptosis involving VDAC have been proposed (for review, see ref. 10). Results and DiscussionStructure Determination of hVDAC1: Combining NMR Spectroscopy and X-Ray Crystallography. In a parallel structural biology approach, we set out to characterize the structure of hVDAC1, the major isoform of this channel in mammalian tissues, by a combination of NMR spectroscopy and x-ray crystallography. The idea behind this project was to gain complementary structural information to have a solid basis for future studies, e.g., analysis of protein heterocomplex formation by NMR and crystal structures as a basis for drug target design. Only information derived from both methods and the application of an iterative s...

The voltage-dependent anion channel (VDAC), located in the outer mitochondrial membrane, acts as a gatekeeper for the entry and exit of mitochondrial metabolites. Here we reveal functional dynamics of isoform one of VDAC (VDAC1) by a combination of solution NMR spectroscopy, Gaussian network model analysis, and molecular dynamics simulation. Micro-to millisecond dynamics are significantly increased for the N-terminal six β-strands of VDAC1 in micellar solution, in agreement with increased B-factors observed in the same region in the bicellar crystal structure of VDAC1. Molecular dynamics simulations reveal that a charge on the membrane-facing glutamic acid 73 (E73) accounts for the elevation of N-terminal protein dynamics as well as a thinning of the nearby membrane. Mutation or chemical modification of E73 strongly reduces the micro-to millisecond dynamics in solution. Because E73 is necessary for hexokinase-I-induced VDAC channel closure and inhibition of apoptosis, our results imply that microto millisecond dynamics in the N-terminal part of the barrel are essential for VDAC interaction and gating.membrane protein | molecular dynamics | NMR spectroscopy | protein-lipid interactions | structure D ynamics of proteins provide the essential link between structure and function. Membrane protein dynamics are gaining more attention due to an increasing number of high-resolution structures. A versatile experimental method for the study of membrane protein dynamics is NMR spectroscopy, providing atomic resolution information from nanoseconds to seconds (for an overview see ref. 1). In addition, when a three-dimensional structure is available, insight into fast dynamics of proteins, which occur on the nanosecond time scale, might be gained from molecular dynamics (MD) simulation and elastic network models (2, 3).NMR studies have revealed large conformational changes essential for the physiological function of α-helical membrane proteins, such as the interaction of phospholamban with effectors modulating heart muscle contractility (4, 5) and antimicrobial peptides adopting different conformations in membranes or solution (6, 7). Less well characterized are motions of outer membrane β-barrels. A pioneering solution NMR study revealed μs-ms interconversion of the catalytic loop of PagP between an excited and a nonexcited state, enabling both ligand binding and enzymatic catalysis (8). Nonenzymatic β-barrel channels exhibit more general dynamic features, such as loop flexibility and slow conformational exchange towards the extracellular barrel edges, altogether indicating relevance for substrate conductance and gating (9-11).The voltage-dependent anion channel (VDAC) is one of the few β-barrel membrane proteins, the structure of which has been determined to date (12-14). Serving as the major pass-through for metabolites between mitochondria and the cytosol (15), the highly abundant channel is regarded as a key regulator of cellular energy metabolism (16). Metabolite flow is regulated by a voltage-dependent conformational c...

The voltage-dependent anion channel (VDAC) regulates the flux of metabolites and ions across the outer mitochondrial membrane. Regulation of ion flow involves conformational transitions in VDAC, but the nature of these changes has not been resolved to date. By combining single-molecule force spectroscopy with nuclear magnetic resonance spectroscopy we show that the β barrel of human VDAC embedded into a membrane is highly flexible. Its mechanical flexibility exceeds by up to one order of magnitude that determined for β strands of other membrane proteins and is largest in the N-terminal part of the β barrel. Interaction with Ca(2+), a key regulator of metabolism and apoptosis, considerably decreases the barrel's conformational variability and kinetic free energy in the membrane. The combined data suggest that physiological VDAC function depends on the molecular plasticity of its channel.

The voltage-dependent anion channel 1 (VDAC-1) is an important protein of the outer mitochondrial membrane that transports energy metabolites and is involved in apoptosis. The available structures of VDAC proteins show a wide β-stranded barrel pore, with its N-terminal α-helix (N-α) bound to its interior. Electrophysiology experiments revealed that voltage, its polarity, and membrane composition modulate VDAC currents. Experiments with VDAC-1 mutants identified amino acids that regulate the gating process. However, the mechanisms for how these factors regulate VDAC-1, and which changes they trigger in the channel, are still unknown. In this study, molecular dynamics simulations and single-channel experiments of VDAC-1 show agreement for the current-voltage relationships of an "open" channel and they also show several subconducting transient states that are more cation selective in the simulations. We observed voltage-dependent asymmetric distortions of the VDAC-1 barrel and the displacement of particular charged amino acids. We constructed conformational models of the protein voltage response and the pore changes that consistently explain the protein conformations observed at opposite voltage polarities, either in phosphatidylethanolamine or phosphatidylcholine membranes. The submicrosecond VDAC-1 voltage response shows intrinsic structural changes that explain the role of key gating amino acids and support some of the current gating hypotheses. These voltage-dependent protein changes include asymmetric barrel distortion, its interaction with the membrane, and significant displacement of N-α amino acids.

A large-scale comparison of essential dynamics (ED) modes from molecular dynamic simulations and normal modes from coarse-grained normal mode methods (CGNM) was performed on a dataset of 335 proteins. As CGNM methods, the elastic network model (ENM) and the rigid cluster normal mode analysis (RCNMA) were used. Low-frequency normal modes from ENM correlate very well with ED modes in terms of directions of motions and relative amplitudes of motions. Notably, a similar performance was found if normal modes from RCNMA were used, despite a higher level of coarse graining. On average, the space spanned by the first quarter of ENM modes describes 84% of the space spanned by the five ED modes. Furthermore, no prominent differences for ED and CGNM modes among different protein structure classes (CATH classification) were found. This demonstrates the general potential of CGNM approaches for describing intrinsic motions of proteins with little computational cost. For selected cases, CGNM modes were found to be more robust among proteins that have the same topology or are of the same homologous superfamily than ED modes. In view of recent evidence regarding evolutionary conservation of vibrational dynamics, this suggests that ED modes, in some cases, might not be representative of the underlying dynamics that are characteristic of a whole family, probably due to insufficient sampling of some of the family members by MD.