Chacón Kelly N, Perkins Jonathan, Mathe Zachary, Alwan Katherine, Ho Ethan N, Ucisik Melek N, Merz Kenneth M, Blackburn Ninian J
Department of Chemistry and Biochemistry, Reed College, Portland, OR, 97202, USA.
Department of Biochemistry and Molecular Biology, School of Medicine, Oregon Health & Science University, Portland, OR, 97239, USA.
Commun Biol. 2018 Nov 14;1:192. doi: 10.1038/s42003-018-0181-9. eCollection 2018.
CusCBAF represents an important class of bacterial efflux pump exhibiting selectivity towards Cu(I) and Ag(I). The complex is comprised of three proteins: the CusA transmembrane pump, the CusB soluble adaptor protein, and the CusC outer-membrane pore, and additionally requires the periplasmic metallochaperone CusF. Here we used spectroscopic and kinetic tools to probe the mechanism of copper transfer between CusF and CusB using selenomethionine labeling of the metal-binding Met residues coupled to RFQ-XAS at the Se and Cu edges. The results indicate fast formation of a protein-protein complex followed by slower intra-complex metal transfer. An intermediate coordinated by ligands from each protein forms in 100 ms. Stopped-flow fluorescence of the capping CusF-W44 tryptophan that is quenched by metal transfer also supports this mechanism. The rate constants validate a process in which shared-ligand complex formation assists protein association, providing a driving force that raises the rate into the diffusion-limited regime.
CusCBAF代表一类重要的细菌外排泵，对Cu(I)和Ag(I)具有选择性。该复合物由三种蛋白质组成：CusA跨膜泵、CusB可溶性衔接蛋白和CusC外膜孔，此外还需要周质金属伴侣CusF。在这里，我们使用光谱学和动力学工具，通过对金属结合Met残基进行硒代蛋氨酸标记，并结合在Se和Cu边缘的RFQ-XAS，来探究CusF和CusB之间铜转移的机制。结果表明，蛋白质-蛋白质复合物快速形成，随后复合物内的金属转移较慢。由每种蛋白质的配体配位的中间体在100毫秒内形成。被金属转移淬灭的封端CusF-W44色氨酸的停流荧光也支持这一机制。速率常数验证了一个过程，即共享配体复合物的形成有助于蛋白质缔合，提供一种驱动力，使速率提高到扩散限制区域。
