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Structure and Function of the Antibiotic-Specific Channel

抗生素特异性通道的结构和功能

基本信息

批准号：
10044320
负责人：
NAKAE Taiji
金额：
$ 1.92万
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：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10044320/
关键词：
Antibiotics Drug resistance Efflux Pump Membrane protein Crystal X-ray diffraction Infection X-線結晶回析 X線回析透過性耐性

项目摘要

Pseudomonas aeruginosa is a major pathogen in the hospital. Infection of this organism is a large problem, since this bacterium exhibits resistance to multiple species of antibiotics that is largely due to a tight outer membrane barrier and the expression of xenobiotic efflux pump. A major efflux pump in this organism is the MexAB-OprM pump. Aim of this study is to elucidate structure and function of the pump subunits. (I) The MexA subunit is an inner membrane associated protein assumed to link the MexB and OprM subunits. We tried to crystalize native from to MexA in the presence of surfactant, polyethyleneglycol 2000, LiCl and obtained 100 to 150 μmィイD13ィエD1 crystals. Preliminary X-ray diffraction analysis showed about 4 AィイD4゜ィエD4 resolution. Deacylated MexA removing fatty acid moiety by replacing N-terminal cysteine with other amino acid was also crystallized in the absence of surfactant and the current resolution is about 3.8 ィイD4゜ィエD4A. (ii) The OprM protein was purified by tagging poly-histidine extension at the carboxyl terminal end and purifying by Nickel-Sepharose column. We obtained about 100〜150 μmィイD13ィエD1 crystals in the presence of octylhydroxyethylsulfoxide, NaCl, polyethylene glycol 2000. Present resolutions is about 4.8 AィイD4゜ィエD4. Obviously more refined crystallization conditions has to be set up. (iii) The MexB structure has been studied by the gene-fusion of a reporter gene, phoA, into the C-terminal truncated MexB protein. Expression of such hybrid proteins revealed that the MexB protein bears 12 transmembrane segments leaving N- and C- termini in the cytoplasm. The MexB protein had two large periplasmic domain containing 311 and 314 amino acid residues. The MexB protein showed two-fold symmetry having highly conserved N- and C-termini halves. These properties are unique to the MexB protein.

铜绿假单胞菌是医院的主要病原菌。这种细菌的感染是一个很大的问题，因为这种细菌对多种抗生素表现出抗药性，这在很大程度上是由于紧密的外膜屏障和异源外排泵的表达。这种生物体中的一个主要外排泵是MexAB-OprM泵。本研究的目的是阐明泵亚单位的结构和功能。(I)MexA亚基是一种内膜相关蛋白，被认为连接MexB和OprM亚基。我们尝试在表面活性剂聚乙二醇2000、氯化锂的存在下，将天然的甲氧基甲酸甲酯结晶，得到了100~150μmィイD13ィエD_1晶体。初步的X射线衍射分析表明，其分辨率约为4AィイD4゜ィエD4。在无表面活性剂存在的条件下，用其他氨基酸取代N-端半胱氨酸来去除脂肪酸的脱酰基-脱酰基-MexA也得到了结晶，目前的分离度约为3.8ィイD4゜ィエD4A。(Ii)通过标记羧基末端的多组氨酸延伸和镍-琼脂糖柱层析纯化OprM蛋白。在辛基羟乙基亚砜、氯化钠、聚乙二醇2 0 0 0存在下，得到了约10 0~15 0μmィイD13ィエD1晶体。目前的分辨率约为4.8AィイD4゜ィエD4。显然，必须建立更精细的结晶条件。(3)通过将报告基因PhoA融合到C端截短的MexB蛋白中，研究了MexB的结构。表达结果表明，MexB蛋白含有12个跨膜片段，N-末端和C-末端位于细胞质中。MexB蛋白有两个大的周质结构域，分别含有311和314个氨基酸残基。MexB蛋白具有高度保守的N-末端和C-末端两重对称性。这些特性是MexB蛋白所特有的。