Clifton Matthew C, Simon Michael J, Erramilli Satchal K, Zhang Huide, Zaitseva Jelena, Hermodson Mark A, Stauffacher Cynthia V
From the Department of Biological Sciences and the Purdue Center for Cancer Research and.
the Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
J Biol Chem. 2015 Feb 27;290(9):5555-65. doi: 10.1074/jbc.M114.621573. Epub 2014 Dec 22.
Bacterial ATP-binding cassette (ABC) importers are primary active transporters that are critical for nutrient uptake. Based on structural and functional studies, ABC importers can be divided into two distinct classes, type I and type II. Type I importers follow a strict alternating access mechanism that is driven by the presence of the substrate. Type II importers accept substrates in a nucleotide-free state, with hydrolysis driving an inward facing conformation. The ribose transporter in Escherichia coli is a tripartite complex consisting of a cytoplasmic ATP-binding cassette protein, RbsA, with fused nucleotide binding domains; a transmembrane domain homodimer, RbsC2; and a periplasmic substrate binding protein, RbsB. To investigate the transport mechanism of the complex RbsABC2, we probed intersubunit interactions by varying the presence of the substrate ribose and the hydrolysis cofactors, ATP/ADP and Mg(2+). We were able to purify a full complex, RbsABC2, in the presence of stable, transition state mimics (ATP, Mg(2+), and VO4); a RbsAC complex in the presence of ADP and Mg(2+); and a heretofore unobserved RbsBC complex in the absence of cofactors. The presence of excess ribose also destabilized complex formation between RbsB and RbsC. These observations suggest that RbsABC2 shares functional traits with both type I and type II importers, as well as possessing unique features, and employs a distinct mechanism relative to other ABC transporters.
细菌ATP结合盒（ABC）转运体是对营养物质摄取至关重要的初级主动转运蛋白。基于结构和功能研究，ABC转运体可分为两个不同的类别，即I型和II型。I型转运体遵循由底物存在驱动的严格交替访问机制。II型转运体以无核苷酸状态接受底物，水解驱动向内的构象。大肠杆菌中的核糖转运体是一种三聚体复合物，由具有融合核苷酸结合结构域的胞质ATP结合盒蛋白RbsA、跨膜结构域同型二聚体RbsC2和周质底物结合蛋白RbsB组成。为了研究复合物RbsABC2的转运机制，我们通过改变底物核糖以及水解辅因子ATP/ADP和Mg(2+)的存在来探测亚基间的相互作用。我们能够在存在稳定的过渡态模拟物（ATP、Mg(2+)和VO4）的情况下纯化出完整的复合物RbsABC2；在存在ADP和Mg(2+)的情况下纯化出RbsAC复合物；以及在不存在辅因子的情况下纯化出一种此前未观察到的RbsBC复合物。过量核糖的存在也会破坏RbsB和RbsC之间复合物的形成。这些观察结果表明，RbsABC2兼具I型和II型转运体的功能特征，同时拥有独特特性，并且相对于其他ABC转运蛋白采用了不同的机制。
