Abstract
Copper resistance has emerged as an important virulence determinant of microbial pathogens. In Streptococcus pneumoniae, copper resistance is mediated by the copper-responsive repressor CopY, CupA and the copper-effluxing P1B-type ATPase CopA. We show here that CupA is a previously uncharacterized cell membrane–anchored Cu(I) chaperone and that a Cu(I) binding–competent, membrane-localized CupA is obligatory for copper resistance. The crystal structures of the soluble domain of CupA and the N-terminal metal-binding domain (MBD) of CopA (CopAMBD) reveal isostructural cupredoxin-like folds that each harbor a binuclear Cu(I) cluster unprecedented in bacterial copper trafficking. NMR studies reveal unidirectional Cu(I) transfer from the low-affinity site on the soluble domain of CupA to the high-affinity site of CopAMBD. However, copper binding by CopAMBD is not essential for cellular copper resistance, consistent with a primary role of CupA in cytoplasmic Cu(I) sequestration and/or direct delivery to the transmembrane site of CopA for cellular efflux.
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Acknowledgements
The authors gratefully acknowledge support from the US National Institutes of Health (NIH; GM042569 to D.P.G., GM094472 to C.E.D. III, AI095814 to M.E.W. and GM069696 to M.J.M.), the Lilly Endowment (to M.E.W.) and an Indiana University Quantitative and Chemical Biology Training Fellowship (to J.P.L.). We thank L. Christiansen for assistance in strain construction, members of the Giedroc laboratory for help in acquiring the NMR data and H. Hu and N. Giri from of the Maroney laboratory for XAS data collection. Crystallographic data collection at the Advanced Light Source at the Lawrence Berkeley National Laboratory is supported by the US Department of Energy and Indiana University. The Stanford Synchrotron Radiation Lightsource Structural Molecular Biology Program is supported by the US Department of Energy, Office of Biological and Environmental Research and by the NIH National Center for Research Resources, Biomedical Technology Program. XAS data collected at the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory was supported by the US Department of Energy, Division of Materials Sciences and Division of Chemical Sciences. Beamline X3B at NSLS is supported by the Center for Synchrotron Biosciences (grant P30-EB-009998) from the National Institute of Biomedical Imaging and Bioengineering.
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Y.F. carried out all protein purification, Cu(I) binding experiments and NMR studies, and solved the crystallographic structures of sCupA and CopAMBD, the latter in collaboration with C.E.D. III., H.-C.T.T., K.E.B. and L.-T.S. K.M.K. constructed S. pneumoniae strains and carried out all cell culture and western blotting experiments under the direction of M.E.W. K.A.H. prepared samples for XAS and analyzed these spectra in collaboration with M.J.M., and J.P.L. made the ICP-MS measurements on cultures grown by K.E.B. D.P.G. conceived and directed the study, and wrote the manuscript.
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Fu, Y., Tsui, HC., Bruce, K. et al. A new structural paradigm in copper resistance in Streptococcus pneumoniae. Nat Chem Biol 9, 177–183 (2013). https://doi.org/10.1038/nchembio.1168
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DOI: https://doi.org/10.1038/nchembio.1168
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