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Structure and function of the xenobiotic-multidrug efflux pump

外源多药外排泵的结构和功能

基本信息

批准号：
12470066
负责人：
NAKAE Taiji
金额：
$ 8.19万
依托单位：
Tokai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

Psudomonas aeruginosa exhibits resistant to structurally and functionally dissimilar antibiotics , which is mainly due to the expression of the multidrug efflux proteins. To solve the problems, we studied structure and function of the efflux pump and obtained the following results. 1. Membrane topology of MexA and OprM: (l) MexA is a lipoprotein anchoring the inner membrane and exposed most of the polypeptide moiety to the periplasmic aqueous phase : (2) OprM anchored the outer membrane via fatty acids exposing protein moiety to the periplasmic space. 2. Role of charged amino acid residues of MexB TMS has been analyzed and obtained the results that Asp407 and Asp408 in the TMS-4 and a Lys939 in TMS-10 form a salt bridge and a free Asp residue may be bound with proton. These amino acid residues are likely to form the proton pathway, which supply energy to the transporter. 3. To access the regulation of the mexR-mexA-MexB-oprM operon, physical interaction of MexR with MexOP-DNA was tested and found that MexR binds to the mexR proximal site of MexOP-DNA and regulates transcription of both mexAB-oprM and mexR. 4. Assembly process of MexEF-OprN transporter has been studied. The transporter appeared to be assembled by the following sequence. In the presence of functional intact MexF, MexE have partial proteolytic processing that is essential to assemble OprN into the complex. Presence of the impaired MexF or deletion of MexF caused proteolytic degradation of OprN and only a partial processing of MexE. 5. The substrate-recognition domain was analyzed by the domain-swapping experiments between MexB and MexY. The results showed that the transporters recognized the substrate at large periplasmic domains and directly transport the substrate from periplasmic space. A new active transport model has been proposed.

铜绿假单胞菌对结构和功能上不同的抗生素表现出耐药性，这主要是由于多药外排蛋白的表达。为解决这一问题，我们对射流泵的结构和功能进行了研究，并取得了以下成果。1. MexA和OprM的膜拓扑结构：（1）MexA是锚定内膜并将大部分多肽部分暴露于周质水相的脂蛋白;（2）OprM通过脂肪酸锚定外膜，将蛋白部分暴露于周质空间。2.分析了MexB TMS中带电氨基酸残基的作用，发现TMS-4中的Asp 407和Asp 408与TMS-10中的Lys 939形成盐桥，游离的Asp残基可能与质子结合。这些氨基酸残基很可能形成质子通路，为转运蛋白提供能量。3.为了获得mexR-mexA-MexB-oprM操纵子的调节，测试了MexR与MexOP-DNA的物理相互作用，发现MexR结合到MexOP-DNA的mexR近端位点并调节mexAB-oprM和mexR的转录。4.研究了MexEF-OprN转运蛋白的组装过程。转运蛋白似乎是按以下顺序组装的。在功能完整的MexF的存在下，MexE具有部分蛋白水解加工，这对于将OprN组装成复合物是必需的。受损的MexF或MexF的缺失的存在下，导致OprN的蛋白水解降解和MexE的只有部分加工。5.通过MexB和MexY之间的结构域交换实验分析底物识别结构域。结果表明，转运蛋白在大的周质区域识别底物，并直接从周质空间转运底物。提出了一种新的主动输运模型。