Linking Folding Landscape with Function in the Human Mitochondrial VDAC2

Abstract

Transmembrane β-barrels in humans are exclusive to the mitochondrial outer membrane (OMM). They include translocases, porins, and chaperones, which together regulate cellular homeostasis. The most abundant OMM proteins are the voltage-dependent anion channels (VDACs), which are 19-stranded multifunctional metabolite flux porins that are also important pharmacological targets. In humans, VDAC2 is anti-apoptotic and functions additionally in metabolite flux, steroidogenesis, gametogenesis, and ROS regulation, making this isoform indispensable for the cell. Despite long-standing efforts, molecular elements regulating VDAC function and folding are not known. Here, we map the folding nucleus and the assembly pathway of human VDAC2 in phosphocholine vesicles. We carry out a comprehensive measurement of folding kinetics and equilibrium thermodynamic contribution of each of the 270 non-Ala residues in the 294-residue barrel. Interestingly, we obtain a two-state thermodynamic equilibrium that is achieved through two detectable on-pathway early folding intermediates, which drive barrel assembly. Detailed analysis of the transition state structure reveals that specific strands at the N-terminal and central region of the barrel assemble first, which is followed by structuring of the C-terminal region. Residues causing frustration in the folding (Φ-value < 0) are more populated towards the C-terminal strands. Furthermore, specific charged residues that regulate the gating characteristics of human VDAC2 are surprisingly a part of the folding nucleus. The only interesting exception is D169, which is functionally important in vivo for cell survival, but establishes non-native contacts during VDAC2 folding. Relating our findings with the proposed mechanism of chaperone–assisted folding in the OMM provides molecular insight that links the assembly pathway of human VDAC2 with function.