Channel conformational plasticity of mitochondrial VDAC2 controls its interaction kinetics with cytosolic proteins

Abstract

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 it regulates the BAK/BAX-dependent apoptosis pathways. Yet, the biophysical underpinnings of VDAC2 functions remain limited. In this study we reevaluate VDAC2’s properties, utilizing recombinant human VDAC2 WT and its three mutants, to understand the biophysical and structural basis that distinguishes VDAC2 from the other isoforms using single-molecule electrophysiology and solution NMR. We found that contrary to VDAC1 and VDAC3, which are characterized by a unique open state, VDAC2 displays dynamic switching between multiple high-conductive anion-selective open states. We employed α-synuclein—a known potent cytosolic regulator of VDAC1 and VDAC3—as a sensitive molecular probe, demonstrating that it induces characteristic blockage events in all open substates of VDAC2 but with up to 10 times different on-rates and blockage times. Despite profound changes in interaction kinetics, the substates exhibit the same equilibrium constant, which takes into account the blockage time, thus resulting in the same affinity of the α-synuclein-VDAC2 interaction. This striking observation suggests that once the α-synuclein molecule is captured, its physical state and interactions within the pore are conserved for all substates. These results imply that the α-synuclein molecule senses a structural change in the channel prior to its final capture within the pore. We propose that this conformational flexibility may allow VDAC2 to recognize a larger number of binding partners. This data could tentatively explain the physiological significance of VDAC2: its ability to dynamically adapt to metabolic cell conditions and to change the rates of interaction with its multiple protein partners.