title: Non-thiolate ligation of nickel by nucleotide-free UreG of Klebsiella aerogenes.
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.
authors.affiliation: Department of Chemistry, University of Massachusetts, Amherst, MA, 01003, USA.|Group de Química Bioinorgànica, Supramolecular i Catàlisi (QBIS-CAT), Institut de Química Computacional i Catàlisi (IQCC), Departament de Química, Universitat de Girona, Campus Montilivi, 17071, Girona, Spain.
authors.forename: Vlad
authors.initials: V
authors.lastname: Martin-Diaconescu
authors.affiliation: Department of Chemistry, University of Massachusetts, Amherst, MA, 01003, USA.|Department of Chemistry and Biochemistry, University of California Santa Barbara, Santa Barbara, CA, USA.
authors.forename: Crisjoe A
authors.initials: CA
authors.lastname: Joseph
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, 48824, USA.|Department of Chemistry and Biochemistry, Calvin College, Grand Rapids, MI, USA.
authors.forename: Jodi L
authors.initials: JL
authors.lastname: Boer
authors.affiliation: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, 48824, USA.
authors.forename: Scott B
authors.initials: SB
authors.lastname: Mulrooney
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, 48824, USA. hausinge@msu.edu.|Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, 48824, USA. hausinge@msu.edu.
authors.forename: Robert P
authors.initials: RP
authors.lastname: Hausinger
authors.affiliation: Department of Chemistry, University of Massachusetts, Amherst, MA, 01003, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Maroney
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry*; D007709:Klebsiella / Q000737:chemistry*; D009532:Nickel / Q000737:chemistry*; D028044:Phosphate-Binding Proteins; D015032:Zinc / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D028044:Phosphate-Binding Proteins; C082270:ureG protein, Bacteria; D009532:Nickel; D015032:Zinc
keywords: Metallocenter assembly; Nickel binding; Urease; X-ray absorption spectroscopy