Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
Advanced Photon Technology Division, RIKEN Harima Institute at SPring-8, Sayo-gun, Hyogo, Japan.
Protein Sci. 2021 May;30(5):1064-1071. doi: 10.1002/pro.4058. Epub 2021 Mar 13.
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.
CmABCB1 是人类 P-糖蛋白的同源物，通过在内向和外向状态之间的构象变化的反复循环，将各种底物排出。CmABCB1 的内向和外向结构的比较表明，跨膜域中的关键接头调节跨膜螺旋的倾斜。跨膜螺旋 1（TM1）在 TM1-3 接头处与 TM3 形成紧密的螺旋-螺旋接触。TM1-3 接头处甘氨酸 132 突变为缬氨酸（G132V），导致 ATP 酶活性增加 10 倍，但这种变化的机制尚不清楚。在这里，我们报告了 CmABCB1 G132V 突变体的外向状态的晶体结构，分辨率为 2.15 Å。我们观察到 TM1-3 接头周围区域的 G132V 与之前的外向状态之间的结构位移，以及细胞外门的明显扩张。我们假设缬氨酸取代引起的空间位阻将构象平衡推向外向状态，有利于核苷酸结合域的二聚体状态，从而增加 G132V 突变体的 ATP 酶活性。
