N-helix and Cysteines Inter-regulate Human Mitochondrial VDAC-2 Function and Biochemistry

Abstract

Human voltage-dependent anion channel-2 (hVDAC-2) functions primarily as the crucial anti-apoptotic protein in the outer mitochondrial membrane, and additionally as a gated bidirectional metabolite transporter. The N-terminal helix (NTH), involved in voltage sensing, bears an additional 11-residue extension (NTE) only in hVDAC-2. In this study, we assign a unique role for the NTE as influencing the chaperone-independent refolding kinetics and overall thermodynamic stability of hVDAC-2. Our electrophysiology data shows that the N-helix is crucial for channel activity, whereas NTE sensitizes this isoform to voltage gating. Additionally, hVDAC-2 possesses the highest cysteine content, possibly for regulating reactive oxygen species content. We identify interdependent contributions of the N-helix and cysteines to channel function, and the measured stability in micellar environments with differing physicochemical properties. The evolutionary demand for the NTE in the presence of cysteines clearly emerges from our biochemical and functional studies, providing insight into factors that functionally demarcate hVDAC-2 from the other VDACs.

VDAC2-specific cellular functions and the underlying structure

“…1B). Thus, the N terminal extension can only be important for some functions confined to mammals [44,45]. …”

“…VDAC commonly appears as a large channel that at low potentials (<30 mV) is mostly fully open (~4.5 nS conductance in 1 M KCl) and weakly anion selective, whereas at high potentials, switches to cationic selectivity and is closed to approximately half of the original conductance (classical “closed” states) [85]. In the case of VDAC2, several species including human, mouse and bovine have been studied [18,44,63,86–88]. H/mVDAC2 have been expressed in yeast, purified and characterized by insertion into lipid bilayers [18,86].…”

“…In hVDAC2 as in VDAC1, the N-terminal helix is necessary for voltage gating and it doesn't affect ion selectivity of the channel [63]. Furthermore, based on studies of recombinant h Vdac2 (Δ1–12) a role for the N-terminal in sensitization of the channel to voltage gating was proposed [44]. Overall, these studies support that VDAC2 displays fundamentally similar channel forming properties to VDAC1 in terms of conductance and voltage dependence though some differences appear at least upon insertion of the channel into lipid bilayers [63].…”

Control of human VDAC-2 scaffold dynamics by interfacial tryptophans is position specific

“…While it is involved in maintenance of cellular homeostasis and transport of metabolites across the OMM [17], hVDAC-2 mainly contributes to cell survival by binding and inhibiting the Bcl-2 family protein BAK [18]. It is a 19-stranded asymmetric barrel with an N-terminal solvent-exposed helix that docks within the barrel, and is important for voltage gating [19]. While we have a tentative mechanism for hVDAC-1 folding [20], experimental evidences for hVDAC-2 folding is still lacking.…”

The N-terminus of VDAC: Structure, mutational analysis, and a potential role in regulating barrel shape

“…The cysteine-less version of (hVDAC2Δ1−32) was detected mainly in the closed substate, suggesting that the cysteines in VDAC2 help stabilize the barrel wall in an open state, even in the absence of the N-terminal 32 residues. Those gating may be occurred through proposed elliptical barrel formation[61].…”