Mutagenesis and modeling to predict structural and functional characteristics of the Staphylococcus aureus MepA multidrug efflux pump.
Mutagenesis and modeling to predict structural and functional characteristics of the Staphylococcus aureus MepA multidrug efflux pump.
复制标题
用于预测金黄色葡萄球菌 MepA 多药外排泵的结构和功能特征的诱变和建模。
DOI：
10.1128/jb.01679-12
复制
发表时间：
2013
影响因子：
3.2
通讯作者：
Kaatz,GlennW
中科院分区：
文献类型：
--
作者：
Schindler,BryanD;Patel,Diixa;Seo,SusanM;Kaatz,GlennW
MepA是一种多药和毒素挤出（MATE）家族蛋白，是金黄色葡萄球菌基因组中编码的唯一MATE蛋白。MATE蛋白的结构数据仅限于一个高分辨率的例子，NorM霍乱弧菌。使用含有底物和利血平作为外排泵抑制剂的梯度板在MepA中产生置换突变。基于质粒的mepA的定点诱变被用来复制这些突变，以及在mepA过表达的临床菌株中鉴定的独特或低频突变，并诱变保守的酸性残基。这些变化对蛋白质功能的影响进行了定量的anorA破坏宿主菌株的敏感性测试与抑制剂和无，并通过确定熟练的乙锭流出。向上功能取代聚集在羧基一半的MepA，附近的蛋白质的细胞质面。重复应用相同的梯度板条件经常再现相同的取代突变，这表明个别残基需要与特定底物的相互作用。在螺旋4和5中鉴定了对蛋白质功能至关重要的酸性残基。在计算机建模中揭示了一个向外的分子，螺旋1，2，4，7，8和10与一个中心空腔接触，这可能代表底物易位途径。该空腔内的功能重要残基包括S81、A161、M291和A302。这些数据提供了一个关键的起点，了解如何MATE多药外排蛋白的功能，并将在完善晶体学数据时，他们是有用的。
MepA is a multidrug and toxin extrusion (MATE) family protein and the only MATE protein encoded within the Staphylococcus aureus genome. Structural data for MATE proteins are limited to a single high-resolution example, NorM ofVibrio cholerae. Substitution mutations were created in MepA using gradient plates containing both a substrate and reserpine as an efflux pump inhibitor. Site-directed mutagenesis of plasmid-basedmepAwas used to reproduce these mutations, as well as unique or low-frequency mutations identified inmepA-overexpressing clinical strains, and to mutagenize conserved acidic residues. The effect of these changes on protein function was quantitated in anorA-disrupted host strain by susceptibility testing with and without inhibitors and by determining the proficiency of ethidium efflux. Up-function substitutions clustered in the carboxy half of MepA, near the cytoplasmic face of the protein. Repeated application of the same gradient plate conditions frequently reproduced identical substitution mutations, suggesting that individual residues are required for interaction with specific substrates. Acidic residues critical to protein function were identified in helices 4 and 5.In silicomodeling revealed an outward-facing molecule, with helices 1, 2, 4, 7, 8, and 10 having contact with a central cavity that may represent a substrate translocation pathway. Functionally important residues within this cavity included S81, A161, M291, and A302. These data provide a critical starting point for understanding how MATE multidrug efflux proteins function and will be useful in refining crystallographic data when they are available.
厌恶性条件反射和学习
DOI：
--
发表时间：
1971
期刊：
影响因子：
--
作者：
F. Brush;A. Black
通讯作者：
A. Black