Characterization of the E506Q and H537A dysfunctional mutants in the E. coli ABC transporter MsbA.
Characterization of the E506Q and H537A dysfunctional mutants in the E. coli ABC transporter MsbA.
复制标题
大肠杆菌 ABC 转运蛋白 MsbA 中 E506Q 和 H537A 功能障碍突变体的表征。
DOI：
10.1021/bi101666p
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发表时间：
2011
期刊：
影响因子：
2.9
通讯作者：
Klug,CandiceS
中科院分区：
文献类型：
--
作者：
Schultz,KathrynM;Merten,JacquelineA;Klug,CandiceS
MSBA是ABC转运蛋白超家族的成员，该家族专门在革兰氏阴性细菌中发现，与细菌和人类耐药相关的蛋白质同源。E506Q和H537A突变已经被引入并用于ABC转运蛋白家族的其他成员的结晶，包括BMRA和ATPase结构域Malk、HlyB-NBD和MJ0796，但以前还没有在MSBA Liped A Exporter中进行详细研究或调查。我们利用一系列的生化和EPR波谱方法来表征这些核苷酸结合域突变对THE的局部和全球影响。ColiMsbA同源二聚体。在ANIN活体生长试验中缺乏细胞活性证实了MSBA中E506Q或H537A突变的存在创造了一种功能失调的蛋白质。为了进一步研究功能障碍的模式，使用了荧光ATP结合试验，结果表明两个突变蛋白都保持了与ATP结合的能力，但ATPase分析表明，每个突变都严重抑制了水解性。使用先前在核苷酸结合结构域中确定和表征的报告位点的EPR波谱数据以及ATP检测分析表明，随着时间的推移，两个突变体中确实发生了水解，但在H537A蛋白中更容易发生。鹿光谱表明，所研究的两种蛋白质都是以闭合二聚体的形式纯化的，这表明细胞内的事件可以诱导MSBA同源二聚体的稳定、闭合的构象，即使在没有核苷酸的情况下也不会重新打开。
MsbA is a member of the ABC transporter superfamily that is specifically found in Gram-negative bacteria and is homologous to proteins involved in both bacterial and human drug resistance. The E506Q and H537A mutations have been introduced and used for crystallization of other members of the ABC transporter protein family, including BmrA and the ATPase domains MalK, HlyB-NBD, and MJ0796, but have not been previously studied in detail or investigated in the MsbA lipid A exporter. We utilized an array of biochemical and EPR spectroscopy approaches to characterize the local and global effects of these nucleotide binding domain mutations on theE. coliMsbA homodimer. The lack of cell viability in anin vivogrowth assay confirms that the presence of the E506Q or H537A mutations within MsbA creates a dysfunctional protein. To further investigate the mode of dysfunction, a fluorescent ATP binding assay was used and showed that both mutant proteins maintain their ability to bind ATP, but ATPase assays indicate hydrolysis is severely inhibited by each mutation. EPR spectroscopy data using previously identified and characterized reporter sites within the nucleotide binding domain along with ATP detection assays show that hydrolysis does occur over time in both mutants, though more readily in the H537A protein. DEER spectroscopy demonstrates that both proteins studied are purified in a closed dimer conformation, indicating that events within the cell can induce a stable, closed conformation of the MsbA homodimer that does not reopen even in the absence of nucleotide.