The crystal structure of the CmABCB1 G132V mutant, which favors the outward-facing state, reveals the mechanism of the pivotal joint between TM1 and TM3.

Abstract

CmABCB1 is a homologue of human P-glycoprotein, which extrudes various substrates by iterative cycles of conformational changes between the inward- and outward-facing states. Comparison of the inward- and outward-facing structures of CmABCB1 suggested that pivotal joints in the transmembrane domain regulate the tilt of transmembrane helices. Transmembrane helix 1 (TM1) forms a tight helix-helix contact with TM3 at the TM1-3 joint. Mutation of Gly132 to valine at the TM1-3 joint, G132V, caused a 10-fold increase in ATPase activity, but the mechanism underlying this change remains unclear. Here, we report a crystal structure of the outward-facing state of the CmABCB1 G132V mutant at a 2.15 Å resolution. We observed structural displacements between the outward-facing states of G132V and the previous one at the region around the TM1-3 joint, and a significant expansion at the extracellular gate. We hypothesize that steric hindrance caused by the Val substitution shifted the conformational equilibrium toward the outward-facing state, favoring the dimeric state of the nucleotide-binding domains and thereby increasing the ATPase activity of the G132V mutant.