Involvement of a Conserved Intramembrane Acidic Residue in Cholesterol Binding of Mitochondrial VDAC1 Revealed by Microsecond-Scale Simulations

Abstract

Voltage-dependent anion channel 1 (VDAC1), a β-barrel membrane protein abundant in the mitochondrial outer membrane, mediates the exchange of ions and metabolites between the mitochondrial matrix and the cytosol. Both experimental and previous computational studies suggest that a conserved, membrane-facing residue in VDAC1, glutamate 73 (E73), plays an essential role in the function of the channel, including its oligomerization and interaction with other cytosolic partner proteins. In addition, a recent photoaffinity labeling study identified E73 as a specific binding site for cholesterol, an endogenous membrane lipid that also regulates VDAC functions. However, how cholesterol interacts with E73 and the effects of cholesterol binding at E73 remain poorly understood. Here, we performed an extensive set of microsecond-long atomistic molecular dynamics simulations on both wildtype and mutant forms and in different protonation states (totaling 14 μs) to characterize cholesterol interactions with VDAC1. Analysis of lipid occupancy showed that cholesterol exhibited distinct preferred binding sites from phospholipids on VDAC1. Spontaneous binding and unbinding of cholesterol to E73 in its deprotonated state was captured, a process accompanied by local membrane deformation and water solvation at the E73 side-chain in the middle of the lipid bilayer. Compared to VDAC1 in a pure phospholipid environment, the presence of cholesterol reduces the fluctuations of the β-strands in proximity to E73. Based on our results, we propose a stabilizing role of cholesterol in regulating the VDAC1 dynamics in the region close to E73, which might further modulate VDAC1 function as an ion channel.