title: Metal Selectivity of a Cd-, Co-, and Zn-Transporting P1B-type ATPase.
abstract: The P1B-ATPases, a family of transmembrane metal transporters important for transition metal homeostasis in all organisms, are subdivided into classes based on sequence conservation and metal specificity. The multifunctional P1B-4-ATPase CzcP is part of the cobalt, zinc, and cadmium resistance system from the metal-tolerant, model organism Cupriavidus metallidurans. Previous work revealed the presence of an unusual soluble metal-binding domain (MBD) at the CzcP N-terminus, but the nature, extent, and selectivity of the transmembrane metal-binding site (MBS) of CzcP have not been resolved. Using homology modeling, we show that four wholly conserved amino acids from the transmembrane (TM) domain (Met254, Ser474, Cys476, and His807) are logical candidates for the TM MBS, which may communicate with the MBD via interactions with the first TM helix. Metal-binding analyses indicate that wild-type (WT) CzcP has three MBSs, and data on N-terminally truncated (ΔMBD) CzcP suggest the presence of a single TM MBS. Electronic absorption and electron paramagnetic resonance spectroscopic analyses of ΔMBD CzcP and variant proteins thereof provide insight into the details of Co2+coordination by the TM MBS. These spectroscopic data, combined with in vitro functional studies of WT and variant CzcP proteins, show that the side chains of Met254, Cys476, and His807contribute to Cd2+, Co2+, and Zn2+binding and transport, whereas the side chain of Ser474appears to play a minimal role. By comparison to other P1B-4-ATPases, we suggest that an evolutionarily adapted flexibility in the TM region likely afforded CzcP the ability to transport Cd2+and Zn2+in addition to Co2+.
authors.affiliation: Departments of Molecular Biosciences and of Chemistry, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Aaron T
authors.initials: AT
authors.lastname: Smith
authors.affiliation: Departments of Molecular Biosciences and of Chemistry, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Matthew O
authors.initials: MO
authors.lastname: Ross
authors.affiliation: Departments of Molecular Biosciences and of Chemistry, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Brian M
authors.identifier: http://orcid.org/0000-0002-3100-0746
authors.initials: BM
authors.lastname: Hoffman
authors.affiliation: Departments of Molecular Biosciences and of Chemistry, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Amy C
authors.identifier: http://orcid.org/0000-0001-8472-4134
authors.initials: AC
authors.lastname: Rosenzweig
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D001692:Biological Transport; D002104:Cadmium / Q000737:chemistry* / Q000378:metabolism; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003035:Cobalt / Q000737:chemistry* / Q000378:metabolism; D049921:Cupriavidus / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D027682:Cation Transport Proteins; D002104:Cadmium; D003035:Cobalt; D000251:Adenosine Triphosphatases; D015032:Zinc