Electron Microscopy of VDAC Membrane Crystals Redux. Pore Shape, Size, and Location(s) of the N-Terminal Domain

Abstract

Atomic structures of mammalian VDAC, the mitochondrial outer membrane channel protein, have recently been determined by three groups using NMR and x-ray crystallography. The three structures are similar, showing a 19-strand β-barrel into which the N-terminal domain inserts. That this common protein fold was determined independently in three different labs strongly suggests that it is an easily accessible low-energy state of the protein. Nonetheless, concerns have been raised whether the atomic structures obtained using bacterially expressed proteins refolded in detergent micelles or lipid bicelles correspond to physiologically relevant states of this integral membrane protein. The definitive answer to this question will require detailed information about the topology of VDAC in its native mitochondrial outer membrane environment. Some insights can be provided by past electron microscopic (EM) studies of ordered arrays of fungal VDAC in isolated mitochondrial outer membranes. Projected density maps of the VDAC protein in frozen-hydrated membranes are consistent with a circular β-barrel having a diameter of 3.6+/-0.2 nm at the alpha-carbon backbone, in good agreement with the atomic models. The low resolution of the EM density maps may have precluded detection of the N-terminal domain within the pore lumen. However, antibodies against the N-terminal domain bound well both to isolated mitochondria and outer membranes, and the corresponding Fab fragments mapped to membrane regions adjacent to the pore. These data indicate that the N-terminus of VDAC in the mitochondrial outer membrane occurs outside the β-barrel at least some of the time. That the amphipathic N-terminal domain might move in and out of the lumen is suggested by changes in the pore detected by multivariate statistical analysis and difference imaging of VDAC arrays embedded in gold-glucose. (Supported by NSF grants and NIH/NCRR grant RR01219.)