Abstract
Nickel-dependent ureases are activated by a multiprotein complex that includes the GTPase UreG. Prior studies showed that nucleotide-free UreG from Klebsiella aerogenes is monomeric and binds one nickel or zinc ion with near-equivalent affinity using an undefined binding site, whereas nucleotide-free UreG from Helicobacter pylori selectively binds one zinc ion per dimer via a universally conserved Cys-Pro-His motif in each protomer. Iodoacetamide-treated K. aerogenes UreG was nearly unaffected in nickel binding compared to non-treated sample, suggesting the absence of thiolate ligands to the metal. X-ray absorption spectroscopy of nickel-bound UreG showed the metal possessed four-coordinate geometry with all O/N donor ligands including one imidazole, thus confirming the absence of thiolate ligation. The nickel site in Strep-tag II-modified protein possessed six-coordinate geometry, again with all O/N donor ligands, but now including two or three imidazoles. An identical site was noted for the Strep-tag II-modified H74A variant, substituted in the Cys-Pro-His motif, ruling out coordination by this His residue. These results are consistent with metal binding to both His6 and a His residue of the fusion peptide in Strep-tagged K. aerogenes UreG. We conclude that the nickel- and zinc-binding site in nucleotide-free K. aerogenes UreG is distinct from that of nucleotide-free H. pylori UreG and does not involve the Cys-Pro-His motif. Further, we show the Strep-tag II can perturb metal coordination of this protein.
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Abbreviations
- EXAFS:
-
Extended X-ray absorption fine structure
- LMCT:
-
Ligand-to-metal charge transfer
- XANES:
-
X-ray absorption near-edge spectroscopy
- XAS:
-
X-ray absorption spectroscopy
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Acknowledgements
These studies were supported by the National Institutes of Health (Grant DK045686, RPH, and GM069696, MJM). XAS data collection at the National Synchrotron Light Source at Brookhaven National Laboratory was supported by the U.S. Department of Energy, Division of Materials Science and Division of Chemical Sciences. Beamline X3B at NSLS was supported by the NIH Grant P30-EB-009998 from the National Institute of Biomedical Imaging and Bioengineering.
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Martin-Diaconescu, V., Joseph, C.A., Boer, J.L. et al. Non-thiolate ligation of nickel by nucleotide-free UreG of Klebsiella aerogenes . J Biol Inorg Chem 22, 497–503 (2017). https://doi.org/10.1007/s00775-016-1429-9
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DOI: https://doi.org/10.1007/s00775-016-1429-9