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Solution Structure of the Integral Human Membrane Protein VDAC-1 ...

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Solution Structure of the Integral Human Membrane Protein VDAC-1 ...

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    1. Solution Structure of the Integral Human Membrane Protein VDAC-1 in Detergent Micelles Presented by Lisa Nguyen

    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 ß barrel Short a helix A parallel ß-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 )

    7. Bcl-2 Family of Protein Involved in apoptosis (Bcl-xL, Bax, and Bak) Bcl-xL Antiapoptosis Binding to VDAC -1 opens the channel Inhibit the release of apoptogenic proteins (cytochrome c)

    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-xL, for reduced ß–nicotinamide adenine dinucleotide (ß-NADH) and for cholesterol. TROSY an experiment in protein NMR spectroscopy that allows studies of large molecules or complexes. is a new approach for suppression of transverse relaxation in multidimensional NMR experiments, which is based on constructive use of interference between DD coupling and CSA dipole-dipole (DD) mechanism and the chemical shift anisotropy (CSA) mechanism NOESY - It uses the dipolar interaction of spins (the nuclear Overhauser effect, NOE) for correlation of protons The NOESY experiment correlates all protons which are close enough. It also correlates protons which are distant in the amino acid sequence but close in space due to tertiary structure. This is the most important information for the determination of protein structures. TROSY an experiment in protein NMR spectroscopy that allows studies of large molecules or complexes. is a new approach for suppression of transverse relaxation in multidimensional NMR experiments, which is based on constructive use of interference between DD coupling and CSA dipole-dipole (DD) mechanism and the chemical shift anisotropy (CSA) mechanism NOESY - It uses the dipolar interaction of spins (the nuclear Overhauser effect, NOE) for correlation of protons The NOESY experiment correlates all protons which are close enough. It also correlates protons which are distant in the amino acid sequence but close in space due to tertiary structure. This is the most important information for the determination of protein structures.

    9. Architecture of VDAC-1. (A) The amino acid sequence of VDAC-1 in one-letter code (36) is arranged according to the secondary and tertiary structure. Amino acids in squares denote ß sheet secondary structure as identified by secondary chemical shifts; all other amino acids are in circles. Red and blue lines denote experimentally observed NOE contacts between two amide protons and NOE contacts involving side chain atoms, respectively. Bold lines indicate strong NOEs typically observed between hydrogen-bonded residues in ß sheets. For clarity of the presentation, not all observed NOEs are shown. The 19th strand is duplicated at the right, next to strand 1, to allow for indicating the barrel-closure NOEs. The side chains of white and orange residues point toward the inside and the outside of the barrel, respectively. Dashed lines show probable contacts between protons with degenerate 1H chemical shifts. Gray residues could not be assigned so far. Every 10th amino acid is marked with a heavy outline, and corresponding residue numbers are indicated. (B) Strips from a 3D [1H,1H]-NOESY-15N-TROSY defining the barrel closure between parallel strands 1 and 19. Red lines show the interstrand contacts for the depicted residues, whereas the violet lines indicate the NOE contacts for the respective opposite residues. ppm, parts per million. (C) Strip from a3D [1H,1H]-NOESY-13C-HMQC (heteronuclear multiple-quantum coherence) taken at the position of a methyl group of Leu10. The assignments of the individual NOE signals are indicated on the left and exemplify the NOEs defining the location of the N-terminal helix in the barrel. The frequency axes ?1, ?2, and ?3 are indicated.Architecture of VDAC-1. (A) The amino acid sequence of VDAC-1 in one-letter code (36) is arranged according to the secondary and tertiary structure. Amino acids in squares denote ß sheet secondary structure as identified by secondary chemical shifts; all other amino acids are in circles. Red and blue lines denote experimentally observed NOE contacts between two amide protons and NOE contacts involving side chain atoms, respectively. Bold lines indicate strong NOEs typically observed between hydrogen-bonded residues in ß sheets. For clarity of the presentation, not all observed NOEs are shown. The 19th strand is duplicated at the right, next to strand 1, to allow for indicating the barrel-closure NOEs. The side chains of white and orange residues point toward the inside and the outside of the barrel, respectively. Dashed lines show probable contacts between protons with degenerate 1H chemical shifts. Gray residues could not be assigned so far. Every 10th amino acid is marked with a heavy outline, and corresponding residue numbers are indicated. (B) Strips from a 3D [1H,1H]-NOESY-15N-TROSY defining the barrel closure between parallel strands 1 and 19. Red lines show the interstrand contacts for the depicted residues, whereas the violet lines indicate the NOE contacts for the respective opposite residues. ppm, parts per million. (C) Strip from a3D [1H,1H]-NOESY-13C-HMQC (heteronuclear multiple-quantum coherence) taken at the position of a methyl group of Leu10. The assignments of the individual NOE signals are indicated on the left and exemplify the NOEs defining the location of the N-terminal helix in the barrel. The frequency axes ?1, ?2, and ?3 are indicated.

    11. 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 a-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.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 a-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.

    12. Hydrophobic surface of VDAC-1. (A) Result of a titration with the spin-labeled detergent 16-DSA. Residues with a relaxation enhancement ? > 20 s–1 mM–1 are green (30). These residues are in close contact to the hydrophobic interior of the micelle. Residues with ? = 20 s–1 mM–1 are white. Gray residues are unassigned. Residues 1 to 21 have been omitted; no interaction with the spin label was observed for these. (B and C) Surface plot of outer and inner surfaces of VDAC-1, respectively, with the side chains of the hydrophobic residues Leu, Val, Ile, Met, Phe, and Trp shown in yellow and all other residues in white.Hydrophobic surface of VDAC-1. (A) Result of a titration with the spin-labeled detergent 16-DSA. Residues with a relaxation enhancement ? > 20 s–1 mM–1 are green (30). These residues are in close contact to the hydrophobic interior of the micelle. Residues with ? = 20 s–1 mM–1 are white. Gray residues are unassigned. Residues 1 to 21 have been omitted; no interaction with the spin label was observed for these. (B and C) Surface plot of outer and inner surfaces of VDAC-1, respectively, with the side chains of the hydrophobic residues Leu, Val, Ile, Met, Phe, and Trp shown in yellow and all other residues in white.

    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 There’s evidence to support the formation of dimers, trimers, and tetramers.There’s evidence to support the formation of dimers, trimers, and tetramers.

    14. Cholesterol, ß-NADH and Bcl-xL 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 ß-NAD do not interact with a specific site of the VDAC-1 ß -NADH interacts with VDAC-1 at strands 17 and 18 ß -NADH favors the closure of VDAC, which limits metabolite flux across the outer membrane and inhibits mitochondrial function Bcl-xL binds to the VDAC-1 at strands 17 and 18 Bcl-xL provides protection against apoptosis through interaction with the VDAC-1

    15. Interactions of VDAC-1. In all three panels, the loop connecting strands 18 and 19 is indicated for orientation. (A) Residues with substantial chemical shift changes [?d(HN) > 0.05 ppm] caused by cholesterol binding are shown in yellow (fig. S12). The amino acids of VDAC-1 are shown as in Fig. 1A. (B) Amide resonances of VDAC-1 with substantial chemical shift changes (fig. S13) caused by ß-NADH are labeled magenta in this ribbon representation; all other residues are gray. (C) Residues involved in Bcl-xL binding (13) are marked red in this ribbon representation; all other residues are gray.Interactions of VDAC-1. In all three panels, the loop connecting strands 18 and 19 is indicated for orientation. (A) Residues with substantial chemical shift changes [?d(HN) > 0.05 ppm] caused by cholesterol binding are shown in yellow (fig. S12). The amino acids of VDAC-1 are shown as in Fig. 1A. (B) Amide resonances of VDAC-1 with substantial chemical shift changes (fig. S13) caused by ß-NADH are labeled magenta in this ribbon representation; all other residues are gray. (C) Residues involved in Bcl-xL binding (13) are marked red in this ribbon representation; all other residues are gray.

    16. Conclusion VDAC1 was shown to adopt a ß-barrel architecture comprising 19 ß-strands and an a-helix located within the pore. The a-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 a -helix acts as a voltage gating ß-NADH and Bcl-xL interacts with VDAC-1 in its open conformation

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