VDAC Studied by Solution NMR: Implications for the Native Structure

Abstract

The voltage-dependent anion channel (VDAC) is the main pathway for metabolites, small molecules and ions across the eukaryotic outer mitochondrial membrane. VDAC has been extensively studied for over thirty years and recently, high-resolution structures of VDAC were determined by X-ray and NMR methods (1-3). These studies used recombinant, refolded protein in membrane mimicking environments and thus the valid question arises how well the resulting atomic structure might resemble the “native” structure of VDAC in the mitochondrial outer membrane (4).Here, we describe implications from solution NMR experiments to this question. Recombinant human VDAC-1 is stably folded in LDAO detergent micelles. Well resolved NMR spectra, including four-dimensional NOESYs, yielded a consistent set of more than 1000 spatial spin-spin correlations that unambiguously define the three-dimensional structure of VDAC-1 (1). The protein forms a 19-stranded beta-barrel with 18 antiparallel and 1 parallel strand pairing. The N-terminal 25 residues are not part of the beta-barrel and solution NMR data link the dynamic properties of this segment to the well-known voltage gating process. The inner diameter of the VDAC-1 barrel is about 25 A, in consistence with published micrographs of native or native-like preparations. The entire outside perimeter of the barrel is hydrophobic and covered by detergent molecules, compatible with a membrane bilayer topology. NMR measurements also revealed interactions of VDAC-1 with beta-NADH and cholesterol, providing a functional connection to experiments on native states of the protein. Furthermore, we can link the micelle-bound state of VDAC structurally and functionally to preparations in phospholipid bilayers by comparing NMR spectra and electron micrographs.(1) Hiller et al. Science 321, 1206 (2008).(2) Bayrhuber et al. PNAS 105, 15370 (2008).(3) Ujwal et al. PNAS 105, 17742 (2008).(4) Colombini. Trends Biochem. Sci. 34, 382 (2009).