Su Chih-Chia, Li Ming, Gu Ruoyu, Takatsuka Yumiko, McDermott Gerry, Nikaido Hiroshi, Yu Edward W
Department of Molecular and Cell Biology, 16 Barker Hall, University of California, Berkeley, CA 94720-3202, USA.
J Bacteriol. 2006 Oct;188(20):7290-6. doi: 10.1128/JB.00684-06.
We previously reported the X-ray structures of wild-type Escherichia coli AcrB, a proton motive force-dependent multidrug efflux pump, and its N109A mutant. These structures presumably reflect the resting state of AcrB, which can bind drugs. After ligand binding, a proton may bind to an acidic residue(s) in the transmembrane domain, i.e., Asp407 or Asp408, within the putative network of electrostatically interacting residues, which also include Lys940 and Thr978, and this may initiate a series of conformational changes that result in drug expulsion. Herein we report the X-ray structures of four AcrB mutants, the D407A, D408A, K940A, and T978A mutants, in which the structure of this tight electrostatic network is expected to become disrupted. These mutant proteins revealed remarkably similar conformations, which show striking differences from the previously known conformations of the wild-type protein. For example, the loop containing Phe386 and Phe388, which play a major role in the initial binding of substrates in the central cavity, becomes prominently extended into the center of the cavity, such that binding of large substrate molecules may become difficult. We believe that this new conformation may mimic, at least partially, one of the transient conformations of the transporter during the transport cycle.
我们之前报道过野生型大肠杆菌AcrB（一种质子动力依赖型多药外排泵）及其N109A突变体的X射线结构。这些结构可能反映了能够结合药物的AcrB的静止状态。配体结合后，质子可能会与跨膜结构域中的酸性残基（即Asp407或Asp408）结合，在也包括Lys940和Thr978的静电相互作用残基的假定网络内，这可能引发一系列构象变化，导致药物排出。在此我们报道四个AcrB突变体（D407A、D408A、K940A和T978A突变体）的X射线结构，在这些突变体中，这个紧密的静电网络结构预计会被破坏。这些突变蛋白显示出非常相似的构象，与野生型蛋白先前已知的构象有显著差异。例如，包含Phe386和Phe388的环在中央腔中底物的初始结合中起主要作用，它显著地延伸到腔内中心，使得大的底物分子可能难以结合。我们认为这种新构象可能至少部分模拟了转运蛋白在转运循环中的一种瞬时构象。
