Trapping intermediates in metal transfer reactions of the CusCBAF export pump of Escherichia coli

Chacón, Kelly N.; Perkins, Jonathan; Mathe, Zachary; Alwan, Katherine; Ho, Ethan N.; Ucisik, Melek N.; Merz, Kenneth M.; Blackburn, Ninian J.

doi:10.1038/s42003-018-0181-9

Trapping intermediates in metal transfer reactions of the CusCBAF export pump of Escherichia coli

Journal Article · Wed Nov 14 00:00:00 EST 2018 · Communications Biology

DOI:https://doi.org/10.1038/s42003-018-0181-9· OSTI ID:1624530

^[1]; Perkins, Jonathan ^[2]; Mathe, Zachary ^[2]; Alwan, Katherine ^[3]; Ho, Ethan N. ^[2]; Ucisik, Melek N. ^[4]; ^[5]; Blackburn, Ninian J. ^[3]

Reed College, Portland, OR (United States); DOE/OSTI
Reed College, Portland, OR (United States)
Oregon Health & Science University, Portland, OR (United States)
Baylor College of Medicine, Houston, TX (United States)
Michigan State Univ., East Lansing, MI (United States)

Escherichia coli 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.

View Accepted Manuscript (DOE)

Research Organization:: SLAC National Accelerator Laboratory, Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL)

Sponsoring Organization:: Murdock Trust Natural Sciences; National Institute of General Medical Sciences (NIGMS); National Institutes of Health (NIH); USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-76SF00515

OSTI ID:: 1624530

Journal Information:: Communications Biology, Journal Name: Communications Biology Journal Issue: 1 Vol. 1; ISSN 2399-3642

Publisher:: Springer NatureCopyright Statement

Country of Publication:: United States

Language:: English

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