Abstract
The P1B-ATPases, which couple cation transport across membranes to ATP hydrolysis, are central to metal homeostasis in all organisms. An important feature of P1B-ATPases is the presence of soluble metal binding domains (MBDs) that regulate transport activity. Only one type of MBD has been characterized extensively, but bioinformatics analyses indicate that a diversity of MBDs may exist in nature. Here we report the biochemical, structural and functional characterization of a new MBD from the Cupriavidus metallidurans P1B-4-ATPase CzcP (CzcP MBD). The CzcP MBD binds two Cd2+, Co2+ or Zn2+ ions in distinct and unique sites and adopts an unexpected fold consisting of two fused ferredoxin-like domains. Both in vitro and in vivo activity assays using full-length CzcP, truncated CzcP and several variants indicate a regulatory role for the MBD and distinct functions for the two metal binding sites. Taken together, these findings elucidate a previously unknown MBD and suggest new regulatory mechanisms for metal transport by P1B-ATPases.
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Acknowledgements
This work was supported by US National Institutes of Health grants GM58518 (A.C.R.), DK068139 (T.L.S.), F32GM105339 (A.T.S.) and T32HL120822 (D.B.). Sequence searches used both database and analysis functions of the Universal Protein Resource (UniProt) Knowledgebase and Reference Clusters (http://www.uniprot.org/) and the US National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Portions of this research were carried out at the National Synchrotron Light Source (NSLS). NSLS, located at Brookhaven National Laboratory, is supported by the US Department of Energy, Division of Materials Sciences and Division of Chemical Sciences under contract no. DE-AC02-98CH10886.
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A.T.S. and D.B. conducted all of the experiments, and A.C.R. and T.L.S. directed the research. A.T.S., D.B., T.L.S. and A.C.R. wrote the manuscript.
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Smith, A., Barupala, D., Stemmler, T. et al. A new metal binding domain involved in cadmium, cobalt and zinc transport. Nat Chem Biol 11, 678–684 (2015). https://doi.org/10.1038/nchembio.1863
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DOI: https://doi.org/10.1038/nchembio.1863
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