Effects of Excluded Volume and Induced N-Terminal Conformational Change on Ion Translocation across VDAC

Abstract

The Voltage Dependent Anion Channel (VDAC) is a mitochondrial outer membrane protein that mediates transfer of ions and small metabolites. It also allows apoptotic factors like cytochrome C into the cytoplasm, thereby playing a crucial role in mediating programmed cell death (apoptosis). Previous studies have indicated that Nicotinamide adenine dinucleotide in its reduced form (NADH) but not its oxidized form (NAD+) reduces conduction through VDAC. However, there is no available non-conducting structure of VDAC, and the mechanism of modulation by NADH remains poorly understood. Here we use long, fully-atomistic molecular dynamics simulations to study NADH binding to VDAC and its effect on channel dynamics and ion translocation. Simulations of VDAC were conducted using an NMR structure determined in the presence of NADH (and provided by Dr. Sebastian Hiller), and with NADH bound as in that structure, deprotonated in silico to NAD+, or removed entirely. We observed dissociation of NAD+ within 100 ns, while NADH remained bound, suggesting that insensitivity of VDAC to NAD+ reflects a significantly lowered affinity of NAD+ for the VDAC pore. In VDAC with NADH entirely removed from the complex before simulation, the N-terminal loop dramatically changed its conformation over the course of the simulations, eventually approaching its conformation in structures experimentally determined in an apo-state. The present results are consistent with a mechanism in which NADH reduces conduction by partial pore block, while concurrently forcing a conformational change of the N-terminus. Potential direct contributions of the N-terminal loop to modulating conduction, such as reduction of the favorable ion density in the pore, are also discussed.