title: Structure-based analysis of VDAC1: N-terminus location, translocation, channel gating and association with anti-apoptotic proteins.
abstract: Structural studies place the VDAC1 (voltage-dependent anion channel 1) N-terminal region within the channel pore. Biochemical and functional studies, however, reveal that the N-terminal domain is cytoplasmically exposed. In the present study, the location and translocation of the VDAC1 N-terminal domain, and its role in voltage-gating and as a target for anti-apoptotic proteins, were addressed. Site-directed mutagenesis and cysteine residue substitution, together with a thiol-specific cross-linker, served to show that the VDAC1 N-terminal region exists in a dynamic equilibrium, located within the pore or exposed outside the β-barrel. Using a single cysteine-residue-bearing VDAC1, we demonstrate that the N-terminal region lies inside the pore. However, the same region can be exposed outside the pore, where it dimerizes with the N-terminal domain of a second VDAC1 molecule. When the N-terminal region α-helix structure was perturbed, intra-molecular cross-linking was abolished and dimerization was enhanced. This mutant also displays reduced voltage-gating and reduced binding to hexokinase, but not to the anti-apoptotic proteins Bcl-2 and Bcl-xL. Replacing glycine residues in the N-terminal domain GRS (glycine-rich sequence) yielded less intra-molecular cross-linked product but more dimerization, suggesting that GRS provides the flexibility needed for N-terminal translocation from the internal pore to the channel face. N-terminal mobility may thus contribute to channel gating and interaction with anti-apoptotic proteins.
authors.affiliation: Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.
authors.forename: Shay
authors.initials: S
authors.lastname: Geula
authors.forename: Danya
authors.initials: D
authors.lastname: Ben-Hail
authors.forename: Varda
authors.initials: V
authors.lastname: Shoshan-Barmatz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D008969:Molecular Sequence Data; D021381:Protein Transport / Q000235:genetics; D051381:Rats; D050995:Voltage-Dependent Anion Channel 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; C494762:VDAC1 protein, human; D050995:Voltage-Dependent Anion Channel 1; D003545:Cysteine