Conformational plasticity of mitochondrial VDAC2 controls the kinetics of its interaction with cytosolic proteins

Abstract The Voltage Dependent Anion Channel (VDAC) is the major conduit of water-soluble metabolites and small ions into and out of the mitochondria. In mammals, VDAC exists in three isoforms, VDAC1, VDAC2, and VDAC3, each characterized by distinct tissue-dependent distribution and physiological role. VDAC2 is the most notable among the three isoforms because its knockout results in embryonic lethality and regulates the BAK/BAX-dependent apoptosis pathways. Yet, understanding of the biophysical underpinnings of VDAC2 functions remains limited. In this study, we reevaluate VDAC2’s properties, utilizing recombinant human VDAC2 WT and its three mutants – cysteine-less VDAC2, VDAC2 with truncated first 11 residues, and E84A - to explore the biophysical basis that distinguishes VDAC2 from the other isoforms using single-molecule electrophysiology. We found that contrary to VDAC1 and VDAC3, which are characterized by a unique open state, VDAC2 displays dynamic switching between a few high-conductive anion-selective substates. We employed α-synuclein (αSyn) – a known potent cytosolic regulator of VDAC1 and VDAC3 – as a sensitive molecular probe to show that it induces characteristic blockage events in all open substates of VDAC2 but with up to ten-fold different on-rates and blockage times. A substate with higher conductance always corresponds to a higher on-rate of the αSyn-VDAC2 interaction but proportionally lower blockage times. This gives the same equilibrium constant for all substates, thus resulting in the same affinity of the αSyn-VDAC2 interaction. The pronounced difference is limited to the kinetic parameters, suggesting that once the αSyn molecule is captured, its physical state and free energy are the same for all substates. These striking results imply that the αSyn molecule senses the dynamic structural variations within the channel prior to its final capture by the pore. We propose that the discovered conformational flexibility may allow VDAC2 to recognize a larger number of binding partners, thus explaining the physiological significance of this isoform, namely, its ability to adapt to mitochondrial metabolic conditions in cells dynamically.