The crystal structure of mouse VDAC1 at 2.3 A 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 beta-barrel eukaryotic membrane protein, the murine VDAC1 (mVDAC1) at 2.3 A resolution, revealing a high-resolution image of its architecture formed by 19 beta-strands. Unlike the recent NMR structure of human VDAC1, the position of the voltage-sensing N-terminal segment is clearly resolved. The alpha-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.

Fig. 1.

Secondary structure schematic of mVDAC1. The mVDAC1 protein structure is colored from the N terminus in blue to the C terminus in red. The β-strands and α-helix are depicted as arrows and cylinders, respectively, and are colored by using single-letter amino acid code. β-Strand residues facing the inside of the pore are shown with a black border. Residues in red form the hinge region.

Fig. 2.

Overall structure of mVDAC1. (A) Cartoon representation of mVDAC1 viewed parallel to the plane of the membrane. The mVDAC1 protein structure is colored from the N terminus in blue to the C terminus in red. (B) Cross-section view of A rotated 90° clockwise. β-Strands 3–7 are removed to illustrate positioning of the N-terminal segment. (C) Cross-section view of A with β-strands 19 and 1–4 removed. The interior surface of the mVDAC1 channel (cyan), created using the program HOLLOW (http://hollow.sourceforge.net), illustrates the contour of the pore. Dimensions at the entrance and along the narrowest point in the center of the pore are displayed. (D) Cartoon representation of mVDAC1 viewed perpendicular to the membrane plane (same coloring as in A).

Fig. 3.

Charge distribution and electrostatic potential of the mVDAC1 pore. (A) Cartoon representation of mVDAC1 (viewed perpendicular to the membrane plane) showing symmetrical charge distribution along β-strands 1–8 and the N-terminal α-helix. Residues with positive (Arg and Lys) and negative (Glu and Asp) charges are colored blue and red, respectively. (B) Same as in A without the α-helix, depicting the reduced number of charged residues on β-strands 9–19 (located behind the α-helix). (C and D) Electrostatic potential, calculated by using the nonlinear Poisson–Boltzmann equation, is displayed on the surface-accessible surface at a contour level of ± 2 KT. Positive and negative charges are colored blue and red, respectively. mVDAC1 is viewed perpendicular to the membrane plane with the helix present (C) or absent (D). Note that the presence of the N-terminal segment (A and C) engenders a high positive charge density, whereas in its absence the wall of the β-barrel harbors a reduced charge density (B and D).

Fig. 4.

The soluble N-terminal segment of mVDAC1. (A) The hydrogen-bonding pattern of the N-terminal segment facilitates its orientation against the interior wall of the pore. Only residues involved in bonding between the β-barrel wall and the soluble N-terminal domain are indicated. (B) Blow-up view of the N-terminal α-helix showing bridging bidentate hydrogen bond between Arg-15 and the carbonyl oxygens of Ala-8 and Leu-10.

Fig. 5.

Proposed model of the transition from open to closed state. The structure of mVDAC1 obtained from this study is colored cyan and displayed as viewed perpendicular to the membrane. The region predicted to interact with NADH (21) is colored yellow and indicated by a dashed circle. The solid circle surrounds the proposed hinge region (Gly-21–Tyr-22–Gly-23–Phe-24–Gly-25). The α-helix in red represents the proposed model of a closed state. The model was made by 10° rotation along the horizontal axis of the protein at the hinge region and would produce a conformation sufficient to perturb metabolite flux. As detailed in Discussion, the structure indicates that this hypothetical occluded state could be stabilized by interactions of the soluble N-terminal domain with the opposite wall of the pore.