title: Mutational and Computational Evidence That a Nickel-Transfer Tunnel in UreD Is Used for Activation of Klebsiella aerogenes Urease.
abstract: Nickel-containing urease from Klebsiella aerogenes requires four accessory proteins for proper active site metalation. The metallochaperone UreE delivers nickel to UreG, a GTPase that forms a UreD/UreF/UreG complex, which binds to urease apoprotein via UreD. Prior in silico analysis of the homologous, structurally characterized UreH/UreF/UreG complex from Helicobacter pylori identified a water tunnel originating at a likely nickel-binding motif in UreG, passing through UreF, and exiting UreH, suggestive of a role for the channel in providing the metal to urease apoprotein for its activation; however, no experimental support was reported for the significance of this tunnel. Here, specific variants were designed to disrupt a comparable 34.6 Å predicted internal tunnel, alternative channels, and surface sites for UreD. Cells producing a set of tunnel-disrupting variants of UreD exhibited greatly reduced urease specific activities, whereas other mutants had no appreciable effect on activity. Affinity pull-down studies of cell-free extracts from tunnel-disrupting mutant cultures showed no loss of UreD interactions with urease or UreF/UreG. The nickel contents of urease samples enriched from activity-deficient cultures were decreased, while zinc and iron incorporation increased. Molecular dynamics simulations revealed size restrictions in the internal channels of the UreD variants. These findings support the role of a molecular tunnel in UreD as a direct facilitator of nickel transfer into urease, illustrating a new paradigm in active site metallocenter assembly.
authors.affiliation: Department of Biochemistry and Molecular Biology, ‡Department of Chemistry, and §Department of Microbiology and Molecular Genetics, Michigan State University , East Lansing, Michigan 48824, United States.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Farrugia
authors.affiliation: Department of Biochemistry and Molecular Biology, ‡Department of Chemistry, and §Department of Microbiology and Molecular Genetics, Michigan State University , East Lansing, Michigan 48824, United States.
authors.forename: Beibei
authors.initials: B
authors.lastname: Wang
authors.affiliation: Department of Biochemistry and Molecular Biology, ‡Department of Chemistry, and §Department of Microbiology and Molecular Genetics, Michigan State University , East Lansing, Michigan 48824, United States.
authors.forename: Michael
authors.initials: M
authors.lastname: Feig
authors.affiliation: Department of Biochemistry and Molecular Biology, ‡Department of Chemistry, and §Department of Microbiology and Molecular Genetics, Michigan State University , East Lansing, Michigan 48824, United States.
authors.forename: Robert P
authors.initials: RP
authors.lastname: Hausinger
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021902:Enterobacter aerogenes / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004756:Enterobacteriaceae Infections / Q000382:microbiology; D004789:Enzyme Activation; D006801:Humans; D056004:Molecular Dynamics Simulation; D016296:Mutagenesis; D009154:Mutation; D009532:Nickel / Q000378:metabolism*; D011487:Protein Conformation; D014510:Urease / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C086767:UreD protein, Bacteria; D009532:Nickel; D014510:Urease