权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Structure and function of the xenobiotic-antibiotic extrusion transporters

外源抗生素挤压转运蛋白的结构和功能

基本信息

批准号：
13142209
负责人：
NAKAE Taiji
金额：
$ 54.4万
依托单位：
Tokai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2005
项目状态：
已结题

项目摘要

Pseudomonas aeruginosa expresses several tripartite xenobiotic-antibiotic exporters that confer the multidrug resistance to the cells. To elucidate molecular and atomic mechanism of this important transporter function, we did the following studies. (i) Crystal structure of MexAB-OprM pump subunits : Structure of MexA revealed a long sickle shape protein monomer that consisted of 4 main domains. The distal a-domain and proximal disordered domains were assigned to interact with OprM and MexB, respectively. Structure of MexA was registered as a totally new protein structure. Thus, the protein was designated as the molecular clump. The OprM structure revealed that the trimeric assembly of this subunit formed the transmembrane 13-barrel domain and the long a-helical periplasmic cavity domain. Thus, it was assumed that the antibiotics must be discharged through this OprM cavity. However, the size of the cavity end at periplasmic a-barrel was totally occluded and that of outer membrane 13-bar … More rel was very small that is difficult to accommodate the passage of antibiotics. It was assumed that external energy has to be exerted to open these OprM ends, probably through MexB and MexA. (ii) The substrate-recognizing domain of MexB transporteer was determined by constructing the MexB-MexY hybrid protein. The result revealed that the hybrid protein consisting of transmembrane domain of MexY and periplasmic domain of MexB showed the MexB type substrate selectivity. Therefore, it was concluded that the transporters recognize their substrate by the extramembrane periplasmic domain. This result established a new concept of xenobiotic export that the xenobiotic substrates were trapped at the periplasmic space before reaching to the cytoplasmic membrane or cytoplasm and efficiently discharged to the external medium. This mechanism of xenobiotic export sounds reasonable in terms for selfprotection. Mechanism of the transporter protein expression was studied by the experiment measuring the direct binding of the repressor protein and the operator-promoter DNA. MexR and MexZ repressor interacted with the respective operator-promoter DNA and the binding site was specified. This study revealed the mechanism of the xenobiotic transporter expression. (iii) Assembly mechanism of the MexAB-OprM transporter was studied by tagging in only one of the subunit and by the co-purification of untagged subunits. It was revealed that MexA subunit plays a central role in the assembly of the transporter complex. This conclusion sounds plausible because the MexA subunit seems to be the molecular damp as revealed by the xTay crystallographic study of MexA mentioned above. (iv)To yield large amount of transporter protein amenable for aystallization experiment, high expression vectors were constructed. The yield of MexB and MexY proteins became 50 to 100 times higher than that from the wild-type cells. Less

铜绿假单胞菌表达几个三重外源性抗生素输出者，赋予细胞多药耐药性。为了阐明这一重要转运蛋白功能的分子和原子机制，我们进行了以下研究。(i)MexAB-OprM泵亚基的晶体结构：MexA的结构揭示了由4个主要结构域组成的长镰刀形蛋白单体。远端α-结构域和近端无序结构域分别与OprM和MexB相互作用。MexA的结构被注册为一个全新的蛋白质结构。因此，该蛋白质被命名为分子团。OprM结构显示该亚基的三聚体组装形成跨膜13桶结构域和长α-螺旋周质腔结构域。因此，假设抗生素必须通过该OprM腔排出。而在周质a桶处的空腔末端完全闭塞，外膜13巴 ...更多信息 Rel非常小，难以容纳抗生素通过。假设必须施加外部能量来打开这些OprM末端，可能是通过MexB和MexA。(ii)通过构建MexB-MexY杂合蛋白，确定了MexB转运蛋白的底物识别结构域。结果表明，由MexY跨膜结构域和MexB周质结构域组成的杂合蛋白具有MexB型底物选择性。因此，可以得出结论，转运蛋白通过膜外周质结构域识别其底物。这一结果建立了一个新的概念，外源性物质的输出，在到达细胞质膜或细胞质之前，被困在周质空间，并有效地排放到外部介质。从自我保护的角度来看，这种外源性物质输出的机制听起来是合理的。通过测量阻遏蛋白与操纵子-启动子DNA的直接结合实验，研究了转运蛋白的表达机制。MexR和MexZ阻遏物与各自的操纵子-启动子DNA相互作用，并指定了结合位点。本研究揭示了外源物质转运蛋白表达的机制。(iii)MexAB-OprM转运蛋白的组装机制通过仅在一个亚基中标记和通过共纯化未标记的亚基来研究。结果表明，MexA亚基在转运蛋白复合物的组装中起着核心作用。这一结论听起来似乎是合理的，因为MexA亚基似乎是分子阻尼，如上面提到的MexA的xTay晶体学研究所揭示的。（iv）构建了高效表达载体，以获得大量的转运蛋白，并进行了系统化实验。MexB和MexY蛋白质的产量比野生型细胞高50至100倍。少