Timachi M Hadi, Hutter Cedric Aj, Hohl Michael, Assafa Tufa, Böhm Simon, Mittal Anshumali, Seeger Markus A, Bordignon Enrica
Faculty of Chemistry and Biochemistry, Ruhr-Universität Bochum, Bochum, Germany.
Department of Physics, Freie Universität Berlin, Berlin, Germany.
Elife. 2017 Jan 4;6:e20236. doi: 10.7554/eLife.20236.
ABC exporters pump substrates across the membrane by coupling ATP-driven movements of nucleotide binding domains (NBDs) to the transmembrane domains (TMDs), which switch between inward- and outward-facing (IF, OF) orientations. DEER measurements on the heterodimeric ABC exporter TM287/288 from , which contains a non-canonical ATP binding site, revealed that in the presence of nucleotides the transporter exists in an IF/OF equilibrium. While ATP binding was sufficient to partially populate the OF state, nucleotide trapping in the pre- or post-hydrolytic state was required for a pronounced conformational shift. At physiologically high temperatures and in the absence of nucleotides, the NBDs disengage asymmetrically while the conformation of the TMDs remains unchanged. Nucleotide binding at the degenerate ATP site prevents complete NBD separation, a molecular feature differentiating heterodimeric from homodimeric ABC exporters. Our data suggest hydrolysis-independent closure of the NBD dimer, which is further stabilized as the consensus site nucleotide is committed to hydrolysis.
ABC转运蛋白通过将ATP驱动的核苷酸结合结构域（NBD）的运动与跨膜结构域（TMD）偶联，将底物泵过膜，跨膜结构域在向内和向外朝向（IF，OF）方向之间切换。对来自的异源二聚体ABC转运蛋白TM287/288进行的双电子电子共振（DEER）测量显示，该转运蛋白含有一个非典型ATP结合位点，在核苷酸存在的情况下，转运蛋白处于IF/OF平衡状态。虽然ATP结合足以使部分转运蛋白处于OF状态，但需要在水解前或水解后状态捕获核苷酸才能发生明显的构象转变。在生理高温且不存在核苷酸的情况下，NBD不对称分离，而TMD的构象保持不变。在简并ATP位点结合核苷酸可防止NBD完全分离，这是异源二聚体ABC转运蛋白与同源二聚体ABC转运蛋白的分子特征差异。我们的数据表明NBD二聚体的关闭不依赖于水解，随着共有位点核苷酸进入水解状态，这种关闭会进一步稳定。
