Solution Structure of the Integral Human Membrane Protein VDAC-1 in Detergent Micelles Presented by Lisa Nguyen Sebastian Hiller,1 Robert G. Garces,1* Thomas J. Malia,1*† Vladislav Y. Orekhov,1,3 Marco Colombini,2 Gerhard Wagner1‡

Introduction Mitochondria Production of cellular energy via ATP synthase and ETC Other Metabolic Activities – Regulation of the membrane potential – Apoptosis – Cellular proliferation and differentiation – Heme Synthesis

Voltage-dependent anion channel (VDAC) Integral membrane protein Mitochondrial porin Three isoforms: VDAC-1, VDAC-2, VDAC-3 Diffusion of small hydrophilic molecules Conserved across eukaryotes (30% similarity between yeast and human)

Structure and Functions of VDAC-1 19-stranded β barrel Short α helix A parallel β-strand pairing N- and C- terminus are on opposite side Open conformation at low membrane potential ~10mV Closed conformation at ~30mV VDAC mediates traffic of small molecules Involved in apoptotic pathways (via interaction with Bcl-2 family ) Ujwal R et al. PNAS 2008;105:

Bcl-2 Family of Protein Involved in apoptosis (Bcl-x L, Bax, and Bak) Bcl-x L – Antiapoptosis – Binding to VDAC -1 opens the channel – Inhibit the release of apoptogenic proteins (cytochrome c) Defects in Bcl-2 lead to Cancer Cardiovascular diseases Neurological diseases

Studies Overview 3D solution structure of VDAC-1 reconstituted in detergent micelles LDAO in solution Nuclear magnetic resonance (NMR) – TROSY (Transverse Relaxation- Optimized Spectroscopy) – NOESY (Nuclear Overhauser Effect Spectroscopy) NMR measurements revealed the binding sites of VDAC-1 for the Bcl-2 protein Bcl-x L, for reduced β–nicotinamide adenine dinucleotide (β-NADH) and for cholesterol.

Architecture of VDAC-1. S Hiller et al. Science 2008;321: Published by AAAS

NMR solution structure of VDAC-1 in LDAO micelles. S Hiller et al. Science 2008;321: Published by AAAS

Proposed model of the transition from open to closed state. Ujwal R et al. PNAS 2008;105: ©2008 by National Academy of Sciences

Hydrophobic surface of VDAC-1. S Hiller et al. Science 2008;321: Published by AAAS

Limitation of the experiment Cannot tell which opening of the channel faces into the cytosol and which faces into mitochondria The exact structure and localization of the N-terminal segment cannot be determined due to unassigned regions of the N-terminus The data excludes formation of stable oligomers in the LDAO micelles

Cholesterol, β-NADH and Bcl-x L In the presence of cholesterol, recombinant VDAC-1 can form voltage- gated channels in phospholipid bilayers similar to those of the native protein ATP and β-NAD do not interact with a specific site of the VDAC-1 β -NADH interacts with VDAC-1 at strands 17 and 18 β -NADH favors the closure of VDAC, which limits metabolite flux across the outer membrane and inhibits mitochondrial function Bcl-x L binds to the VDAC-1 at strands 17 and 18 – Bcl-x L provides protection against apoptosis through interaction with the VDAC-1

Interactions of VDAC-1. S Hiller et al. Science 2008;321: Published by AAAS

Conclusion VDAC1 was shown to adopt a β- barrel architecture comprising 19 β-strands and an α-helix located within the pore. The α-helix of the N-terminal segment is oriented against the interior wall, causing a partial narrowing at the center of the pore. Studies of VDAC-1 structures suggest that the hydrophilic N- terminus of the protein is nestled within the pore N-terminal α -helix acts as a voltage gating β-NADH and Bcl-x L interacts with VDAC-1 in its open conformation

Table S1. NMR structure statistics NOE distance restraintsAllHN-HNHN-Meth.Meth.-Meth. Intraresidual690 0 Sequential Medium range (2 ≤ │i-j│ ≤ 4) Long range (│i-j│>4) Total Hydrogen bond restraints139 Dihedral angle restraints (φ and ψ)2 x 158 Ramachandran plot Most favored region77.1% Additionally allowed region21.6% generously allowed region0.8% Disallowed region0.4% Deviations from idealized geometry Bond lengths (Å)0.005 Bond angles (°)0.7 Average pairwise r.m.s.d. (backbone, Å) All residues except 1–253.6 All residues except 1–25 and loops a 2.9