4.5 Article

Structure-based analysis of VDAC1: N-terminus location, translocation, channel gating and association with anti-apoptotic proteins

Journal

BIOCHEMICAL JOURNAL
Volume 444, Issue -, Pages 475-485

Publisher

PORTLAND PRESS LTD
DOI: 10.1042/BJ20112079

Keywords

Bc1-2; Bc1-xL; channel conductance; glycine-rich sequence; hexokinase; voltage-dependent anion channel 1 (VDAC1)

Funding

  1. Israel Science Foundation [649/09]

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Structural studies place the VDACI (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 antiapoptotic 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 beta-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 alpha-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 Bel-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.

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