Mapping the VDAC1 Oligomerization Contact Sites and N-Terminus Translocation

Abstract

Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel. Accumulating evidence implicates the voltage-dependent anion channel (VDAC) as functioning in mitochondria-mediated apoptosis involving cytochrome c release, leading to caspases activation and apoptosis. The mechanisms regulating cytochrome c release and the molecular architecture of the cytochrome c conducting channel remain unknown. Previously, we demonstrated that apoptosis induction was accompanied by VDAC oligomerization, as revealed by cross-linking and directly monitored in living cells using Bioluminescence Resonance Energy Transfer technology. Moreover, apoptosis inhibitors inhibited VDAC oligomerization and a correlation between the levels of VDAC oligomerization and apoptosis was observed. Here, we combined site-directed mutagenesis with chemical cross-linking to reveal the contact sites between VDAC1 molecules in dimers and higher oligomers. Replacing hydrophobic amino acids with charged amino acids in b-strands 1,2 and 19, but not 14, interfered with VDAC1 oligomerization and apoptosis induction. Cysteine cross-linking results, from introducing cysteine at a defined position in cysteine-less VDAC1 and applying the cysteine-specific cross-linker, BMOE, supported the close vicinity of b-strands 1,2 and 19 in VDAC1 dimer. Moreover, the results suggest that VDAC1 exists as a dimer that undergoes conformational changes upon apoptosis induction to assemble into a higher oligomeric state. Additionally we demonstrated that the N-terminal region of VDAC1 lies inside the pore, but could also move and interact with the N-terminus from a second molecule to form a dimer. Our results suggest that the glycine rich sequence 21-GYGFG-25 is involved in the N-terminus translocation from the internal pore to the channel face. These results provide structural insight into cellular VDAC1's oligomeric state and its N-terminal region location and translocation.