Non-thiolate ligation of nickel by nucleotide-free UreG of Klebsiella aerogenes

  • Vlad Martin-Diaconescu
  • Crisjoe A. Joseph
  • Jodi L. Boer
  • Scott B. Mulrooney
  • Robert P. Hausinger
  • Michael J. Maroney
Original Paper

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.

Keywords

Urease Metallocenter assembly Nickel binding X-ray absorption spectroscopy 

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

Supplementary material

775_2016_1429_MOESM1_ESM.pdf (1.4 mb)
Supplementary material 1 (PDF 1387 kb)

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Copyright information

© SBIC 2016

Authors and Affiliations

  • Vlad Martin-Diaconescu
    • 1
    • 4
  • Crisjoe A. Joseph
    • 1
    • 5
  • Jodi L. Boer
    • 2
    • 6
  • Scott B. Mulrooney
    • 3
  • Robert P. Hausinger
    • 2
    • 3
  • Michael J. Maroney
    • 1
  1. 1.Department of ChemistryUniversity of MassachusettsAmherstUSA
  2. 2.Department of Biochemistry and Molecular BiologyMichigan State UniversityEast LansingUSA
  3. 3.Department of Microbiology and Molecular GeneticsMichigan State UniversityEast LansingUSA
  4. 4.Group de Química Bioinorgànica, Supramolecular i Catàlisi (QBIS-CAT), Institut de Química Computacional i Catàlisi (IQCC), Departament de QuímicaUniversitat de GironaGironaSpain
  5. 5.Department of Chemistry and BiochemistryUniversity of California Santa BarbaraSanta BarbaraUSA
  6. 6.Department of Chemistry and BiochemistryCalvin CollegeGrand RapidsUSA

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