Dynamics and interactions of the voltage-dependent anion channel 1 studied by NMR spectroscopy

Abstract

The voltage-dependent anion channel (VDAC), the most abundant protein in the outer mitochondrial membrane, acts as a gatekeeper for the entry and exit of mitochondrial metabolites and is involved in mitochondrial apoptosis. Recent determination of its high-resolution structure by three independent groups revealed a 19-stranded β-barrel with a differently arranged N-terminal α-helix inside the pore.In this thesis, the NMR resonance assignment of isoform one of human VDAC (VDAC1) in solution is increased. In addition, this study provides evidence for a kinked α-helical structure of the N-terminus, which is compatible with the crystal structure of VDAC1, although other structures cannot be excluded. Furthermore, this study reveals functional dynamics of VDAC1 by a combination of solution NMR spectroscopy, Gaussian network model (GNM) analysis, and molecular dynamics (MD) simulations.Low signal intensities indicate conformational exchange in the second part of the Nterminal α-helix and the linker connecting the α-helix to the first β-strand. In addition, mutation of arginine 15 in the second α-helical part affects the stability of the α-helix and the overall barrel in a complex manner. Micro-to millisecond dynamics are significantly increased in the N-terminal α-helix, the linker, and the N-terminal six βstrands of VDAC1 in micellar solution. In addition, hydrogen bonds are instable in the N-terminal three β-strands. In agreement, the N-terminal β-strands exhibit increased Bfactors in the crystal structure of VDAC1 and intrinsic instability predicted by the GNM analysis. Mutation or chemical modification of the membrane facing glutamic acid 73 (E73) strongly reduces the micro-to millisecond dynamics in solution. MD simulations reveal that a charge on E73 accounts for the elevation of N-terminal protein dynamics as well as a thinning of the nearby membrane. Since E73 is necessary for hexokinase-Iinduced VDAC1 channel closure and inhibition of apoptosis, these results imply that micro-to millisecond dynamics in the N-terminal part of the β-barrel are essential for VDAC1 interaction and gating. Moreover, the data suggest that dynamics in the α-helix are connected with these processes. III Furthermore, this study reveals two binding sites for the pore's most important transport substrate ATP. The location of the ATP binding sites, one of them comprising the N-terminal α-helix, the linker, and nearby β-strands, suggests controlled metabolite flux and ligand-induced stabilization of the open state of the VDAC1 pore. Finally, Ca 2+ is found to interact with two distinct N-terminal and C-terminal regions in the β-barrel. These regions overlap with VDAC1 oligomerization sites and dynamic regions, suggesting a connection between Ca 2+ interaction, gating, and oligomerization. Zusammenfassung Der spannungsabhängige Anionenkanal (engl. "voltage-dependent anion channel", VDAC), das häufigste Protein in der äußeren Mitochondrienmembran, dient als wichtiger Kontrollpunkt für den Ein-und Austritt von mitochondrialen Metaboliten und ...