Orientation of Dimeric Tubulin on Lipid Membranes Studied using Neutron Reflectometry

Abstract

Dimeric tubulin has emerged as an important regulatory factor of the permeability of voltage-dependent anion channel (VDAC) in the mitochondrial outer membrane, with implications for mitochondrial energetics as well as the Warburg effect observed in cancer cells. Previously, single-channel studies revealed that the on-rate of the VDAC-tubulin interaction is strongly dependent on the lipid environment. To understand the orientation of this abundant cytosolic water-soluble protein when bound to lipid membranes, we have employed neutron reflectometry (NR) of tubulin on a tethered bilayer lipid membrane (tBLM) platform. NR is particularly well-suited to study the profile of high-molecular weight proteins associated with membranes because contrast can be generated with mixtures of light and heavy water, and because of the variation in the cross-section of neutron scattering from the various light elements composing biological materials. The NR data are consistent with two models of tubulin binding, in which the tubulin heterodimer is bound to the membrane surface by either its alpha- or beta-subunit, but not both simultaneously. This result is consistent with coarse-grained molecular dynamics (MD) simulations of the interaction of the alpha subunit of tubulin with membranes of different compositions. The binding surfaces derived from the NR results are highly conserved and correspond to the dimer-dimer interface in microtubule protofilaments. We discuss the implications of the orientation of membrane-bound dimeric tubulin for the mechanism by which tubulin regulates VDAC, including the accessibility of the negatively charged C-terminal tails for VDAC pore blockage and the possibility that the two subunits have different regulatory roles.