Bao Huan, Dalal Kush, Cytrynbaum Eric, Duong Franck
From the Departments of Biochemistry and Molecular Biology and.
Mathematics, University of British Columbia, Vancouver, British Columbia V6T1Z3, Canada.
J Biol Chem. 2015 Oct 16;290(42):25452-60. doi: 10.1074/jbc.M115.671826. Epub 2015 Sep 3.
ATP-binding cassette (ABC) transporters have evolved an ATP-dependent alternating-access mechanism to transport substrates across membranes. Despite important progress, especially in their structural analysis, it is still unknown how the substrate stimulates ATP hydrolysis, the hallmark of ABC transporters. In this study, we measure the ATP turnover cycle of MalFGK2 in steady and pre-steady state conditions. We show that (i) the basal ATPase activity of MalFGK2 is very low because the cleavage of ATP is rate-limiting, (ii) the binding of open-state MalE to the transporter induces ATP cleavage but leaves release of Pi limiting, and (iii) the additional presence of maltose stimulates release of Pi, and therefore increases the overall ATP turnover cycle. We conclude that open-state MalE stabilizes MalFGK2 in the outward-facing conformation until maltose triggers return to the inward-facing state for substrate and Pi release. This concerted action explains why ATPase activity of MalFGK2 depends on maltose, and why MalE is essential for transport.
ATP结合盒（ABC）转运蛋白进化出一种依赖ATP的交替 access 机制，以跨膜转运底物。尽管取得了重要进展，尤其是在其结构分析方面，但底物如何刺激ATP水解（ABC转运蛋白的标志）仍然未知。在本研究中，我们测量了MalFGK2在稳态和预稳态条件下的ATP周转循环。我们表明：（i）MalFGK2的基础ATP酶活性非常低，因为ATP的裂解是限速步骤；（ii）开放状态的MalE与转运蛋白的结合诱导ATP裂解，但Pi的释放受到限制；（iii）麦芽糖的额外存在刺激Pi的释放，因此增加了整体ATP周转循环。我们得出结论，开放状态的MalE将MalFGK2稳定在向外的构象，直到麦芽糖触发其回到向内的构象以释放底物和Pi。这种协同作用解释了为什么MalFGK2的ATP酶活性依赖于麦芽糖，以及为什么MalE对转运至关重要。
