The crystal structure of mouse VDAC1 at 2.3 Å resolution reveals mechanistic insights into metabolite gating

Abstract

The voltage-dependent anion channel (VDAC) constitutes the major pathway for the entry and exit of metabolites across the outer membrane of the mitochondria and can serve as a scaffold for molecules that modulate the organelle. We report the crystal structure of a ␤-barrel eukaryotic membrane protein, the murine VDAC1 (mVDAC1) at 2.3 Å resolution, revealing a high-resolution image of its architecture formed by 19 ␤-strands. Unlike the recent NMR structure of human VDAC1, the position of the voltagesensing N-terminal segment is clearly resolved. The ␣-helix of the N-terminal segment is oriented against the interior wall, causing a partial narrowing at the center of the pore. This segment is ideally positioned to regulate the conductance of ions and metabolites passing through the VDAC pore.␤-barrel ͉ mitochondria ͉ outer membrane protein ͉ ATP flux A ll eukaryotic cells require efficient exchange of metabolites between the cytoplasm and the mitochondria. This exchange is mediated by the most abundant protein in the outer mitochondrial membrane, the voltage-dependent anion channel (VDAC), which facilitates movement of ions and metabolites between the cytoplasm and the intermembrane space of the mitochondria. VDAC was first discovered in 1976 (1) and has since been extensively studied by a number of biochemical and biophysical techniques demonstrating its conserved properties of voltage gating and ion selectivity and its ability to act as a scaffold for modulator proteins from both sides of the outer membrane (2, 3).Single-channel conductance experiments on VDAC1 at low membrane potential (10 mV) show a high conductance indicative of a large pore, often referred to as the open state of the channel (2). As voltage is increased (Ͼ30 mV) in either a positive or negative direction, a lower conductance, ostensibly the closed state, is obtained. Endogenous potentials caused by chemical gradients across the outer membrane [Donnan potentials (4)] may thus be sufficient to regulate this channel. Although the nature of either of these states is unknown in the absence of structural data, transition between them presumably involves conformational changes constituting a gating action that hinders the passage of metabolites such as adenine nucleotides. Furthermore, this transition is associated with altered ion selectivity because the channel shifts from weakly anion selective to weakly cation selective as it moves from open to closed. This complex gating behavior has driven numerous investigations that have provided sometimes contradictory findings; however, the role of VDAC to regulate metabolite traffic across the outer membrane is firmly established.As the major pathway into and out of mitochondria, VDAC mediates an intimate dichotomy between metabolism and death in all cells (5). Mitochondrial-dependent cell death involves numerous proteins [including hexokinase (6) and the Bcl-2 family of proteins (7), in particular] that alternatively promote or prevent mitochondrial dysfunction through interaction with, and potentially m...

Swapping of the N‐terminus of VDAC1 with VDAC3 restores full activity of the channel and confers anti‐aging features to the cell

“…The N-terminal end of VDAC contains amphipathic a-helix elements [19]. This structure represents an addition to the b-barrel pore structure and was proposed to have functionally relevant properties [20][21][22][23][24][25][26]35].…”

Probing the orientation of yeast VDAC1 in vivo

“…VDAC1 structure and FLAG-epitope positions. (A) Three-dimensional model of hsVDAC1 drawn with Pymol [DeLano Scientific] using PDB coordinates 2K4T from [11] (see also 2JK4 [12] and 3EMN [13]) indicating FLAG-epitope locations used in the current study (1)(2)(3)(4)(5). FLAG3, which contradicts the structure, is highlighted (Ã).…”

“…The remaining epitopes reported in the only other study of orientation in intact mitochondria [20] are shown by the recent structures to be mostly buried in membrane strands and therefore difficult to interpret. In the published structures [11][12][13] the N-terminal segment first exits the membrane on the trans side [11,12] before folding through the lumen to almost reach the cis side [13]. The FLAG1, which is 11-20 residues from the N-terminus, will most likely have a trans exposure (see Fig.…”

“…It has also been suggested to play a role in apoptosis [5] although this remains controversial [6]. Despite its importance, the structure of VDAC1 was unclear [7][8][9][10] until recently published high resolution structures of human and mouse VDAC1 revealed a 19 stranded b-barrel capable of dimerization and dynamic fluctuation [11][12][13] (Fig. 1).…”

“…Therefore, this work is based upon the hypothesis that VDAC1 adopts a similar structure in yeast and human mitochondria. Antibody binding to FLAG-epitopes (DYKDDDDK) indicates their surface exposure [15,16] and FLAG was placed in selected regions of VDAC and probed by immunoprecipitation (IP) [11][12][13]17]. …”