Peripheral Binding of Hexokinase-2 to the Rim of VDAC1 Mediated by the Mitochondrial Outer Membrane

Abstract

Complex formation between hexokinase-2 (HK2) and the mitochondrial channel VDAC1 plays a crucial role in regulating cell death, however, structural details of this complex remain elusive. While previous protein-protein docking studies proposed direct insertion of HK2 into the VDAC1 pore, several experimental studies suggest that this process might be facilitated by an initial insertion of HK2 into the mitochondrial outer membrane. Therefore, in order to systematically model the complex of membrane-embedded VDAC1 with membrane-bound HK2, we adopted a hybrid approach combining molecular dynamics (MD) and Brownian dynamics (BD) simulations, first describing membrane binding of HK2 with MD, and then formation of its complex with VDAC1 with BD. Spontaneous insertion of HK2 N-terminal helix was consistently captured in multiple independent MD simulations. The positioning (insertion and orientation) of HK2 in the membrane were then used as restraints in 100 independent BD simulations (totaling 2 milliseconds), where binding of HK2 to a membrane-embedded VDAC1 was studied. Clustering analysis identified VDAC1 outer rim as the most probable HK2 binding site. The complex was further refined with MD simulations, during which additional salt-bridges formed to strengthen the complex. A major feature in the complex is the blockade of VDAC1's permeation pathway by HK2, a result supported by electrophysiological measurements. The HK2-VDAC1 interface is highlighted by the presence of a putative GSK3β phosphorylation site, S215, whose phosphorylation disrupts the most probable binding site of HK2 in VDAC1. Consistent with the simulation results, electrophysiological experiments show that HK2 blocks ion conduction in wildtype VDAC1 but not in the phosphomimetic S215E mutant. Furthermore, the S215E mutation reduces cell survival rate under hypoxia-reoxygenation stress, possibly due to reduced HK2 binding. Together, our results provide a novel, experimentally validated model for membrane-bound HK2-VDAC1 complex.