1. 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‡

2. 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

3. 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)

4. 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;105:17742-17747

7. 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

8. 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.

9. Architecture of VDAC-1. S Hiller et al. Science 2008;321:1206-1210 Published by AAAS

10. NMR solution structure of VDAC-1 in LDAO micelles. S Hiller et al. Science 2008;321:1206-1210 Published by AAAS

11. Proposed model of the transition from open to closed state. Ujwal R et al. PNAS 2008;105:17742-17747 ©2008 by National Academy of Sciences

12. Hydrophobic surface of VDAC-1. S Hiller et al. Science 2008;321:1206-1210 Published by AAAS

13. 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

14. 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

15. Interactions of VDAC-1. S Hiller et al. Science 2008;321:1206-1210 Published by AAAS

16. 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

17. Table S1. NMR structure statistics NOE distance restraintsAllHN-HNHN-Meth.Meth.-Meth. Intraresidual690 0 Sequential199129691 Medium range (2 ≤ │i-j│ ≤ 4)72283410 Long range (│i-j│>4)2721318556 Total61228825767 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