Do folding elements trade‐off with function in the human mitochondrial metabolite transporter?

Abstract

In humans, transmembrane β-barrels are found exclusively in the outer mitochondrial membrane (OMM). They comprise metabolite transporters, translocases, and chaperones, which together control cellular homeostasis. The metabolite transporters — known as voltage-dependent anion channels (VDACs) – are the most abundant OMM barrels. They are 19-stranded β-barrel structures that also form important pharmacological targets due to their roles in metabolite flux, steroidogenesis, gametogenesis, and ROS regulation. Despite long-standing efforts, molecular factors that regulate VDAC function and folding at the atomistic level are not known. Here, we map the folding nucleus and the assembly pathway of human VDAC isoform 2 in phosphocholine vesicles. We carry out a comprehensive measurement of hVDAC2 folding kinetics, and equilibrium thermodynamic contribution, of each of the 270 non-Ala residues of the 294-residue barrel. Interestingly, we obtain a two-state thermodynamic equilibrium that is attained through two detectable on-pathway early folding intermediates, which are assembled in milliseconds – seconds. These intermediates undergo slow conformational restructuring to form the folded VDAC2 barrel. We find that specific charged residues that form a part of these early intermediates also positively regulate the gating characteristics of VDAC2. This observation is unprecedented, as directed evolution of protein sequences in favor of function usually place a stability burden on its structure that additionally impedes protein assembly. Our findings provide molecular insight on the assembly pathway of human VDAC2, and directly link these folding elements with VDAC2 function as the metabolite transporter of human mitochondria.