Meir Aviv, Natan Adi, Moskovitz Yoni, Ruthstein Sharon
Department of Chemistry, Faculty of Exact Science, Bar Ilan University, Ramat Gan, 5290002, Israel.
Metallomics. 2015 Jul;7(7):1163-72. doi: 10.1039/c5mt00053j. Epub 2015 May 5.
Copper plays a key role in all living organisms by serving as a cofactor for a large variety of proteins and enzymes involved in electron transfer, oxidase and oxygenase activities, and the detoxification of oxygen radicals. Due to its toxicity, a conserved homeostasis mechanism is required. In E. coli, the CusCFBA efflux system is a copper-regulating system and is responsible for transferring Cu(I) and Ag(I) out of the periplasm domain into the extracellular domain. Two of the components of this efflux system, the CusF metallochaperone and the N-terminal domain of CusB, have been thought to play significant roles in the function of this efflux system. Resolving the metal ion transport mechanism through this efflux system is vital for understanding metal- and multidrug-resistant microorganisms. This work explores one aspect of the E. coli resistance mechanism by observing the interaction between the N-terminal domain of CusB and the CusF protein, using electron paramagnetic resonance (EPR) spectroscopy, circular dichroism (CD), and chemical cross-linking. The data summarized here show that M36 and M38 of CusB are important residues for both the Cu(I) coordination to the CusB N-terminal domain and the interaction with CusF, and K32 is essential for the interaction with CusF. In contrast, the K29 residue is less consequential for the interaction with CusF, whereas M21 is mostly important for the proper interaction with CusF.
铜在所有生物中都起着关键作用，它作为多种蛋白质和酶的辅助因子，参与电子转移、氧化酶和加氧酶活性以及氧自由基的解毒过程。由于其毒性，需要一种保守的稳态机制。在大肠杆菌中，CusCFBA外排系统是一种铜调节系统，负责将周质结构域中的Cu(I)和Ag(I)转运到细胞外结构域。该外排系统的两个组成部分，即CusF金属伴侣蛋白和CusB的N端结构域，被认为在该外排系统的功能中发挥着重要作用。解析通过该外排系统的金属离子转运机制对于理解金属和多药耐药微生物至关重要。这项工作通过使用电子顺磁共振（EPR）光谱、圆二色性（CD）和化学交联技术，观察CusB的N端结构域与CusF蛋白之间的相互作用，探索了大肠杆菌抗性机制的一个方面。此处总结的数据表明，CusB的M36和M38对于Cu(I)与CusB N端结构域的配位以及与CusF的相互作用都是重要残基，而K32对于与CusF的相互作用至关重要。相比之下，K29残基对于与CusF的相互作用影响较小，而M21对于与CusF的正确相互作用最为重要。
