UreE-UreG Complex Facilitates Nickel Transfer and Preactivates GTPase of UreG in Helicobacter pylori

Yang, Xinming; Li, Hongyan; Lai, Tsz-Pui; Sun, Hongzhe

doi:10.1074/jbc.m114.632364

Cited by 58 publications

(108 citation statements)

References 56 publications

(76 reference statements)

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“…Ni II or Zn II binding induce GTP-dependent dimerization of UreG [54,61] (Figure 2). The formation of (UreE) 2 –(UreG) 2 complex is a prerequisite for recruitment of GTP to UreG and Ni II transfer from UreE to UreG [53]. Although Zn II binding has been well documented for UreE, UreG and the (UreE) 2 –(UreG) 2 complex [54,61,64–66], the functional role of Zn II binding remains enigmatic.…”

Section: Metallochaperonesmentioning

confidence: 99%

“…Remarkably, these urease accessory proteins are also capable of receiving Ni II from the proteins that function in the maturation of [NiFe]-hydrogenase. For example, UreE and HypA are capable of forming a ternary UreE 2 –HypA complex [59] where HypA-derived Ni II can be trafficked to UreE, which is subsequently donated to UreG [53]. How Ni II moves from UreG 2 to the (UreFD) 2 complex in a GTP hydrolysis-dependent fashion and ultimately to the urease apo-enzyme is not yet known (see Figure 3).…”

Section: Metallochaperonesmentioning

confidence: 99%

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Metallochaperones and metalloregulation in bacteria

Capdevila

Edmonds

Giedroc

2017

Essays in Biochemistry

109

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Bacterial transition metal homoeostasis or simply ‘metallostasis’ describes the process by which cells control the intracellular availability of functionally required metal cofactors, from manganese (Mn) to zinc (Zn), avoiding bothmetal deprivation and toxicity. Metallostasis is an emerging aspect of the vertebrate host–pathogen interface that is defined by a ‘tug-of-war’ for biologically essential metals and provides the motivation for much recent work in this area. The host employs a number of strategies to starve the microbial pathogen of essential metals, while for others attempts to limit bacterial infections by leveraging highly competitive metals. Bacteria must be capable of adapting to these efforts to remodel the transition metal landscape and employ highly specialized metal sensing transcriptional regulators, termed metalloregulatory proteins, and metallochaperones, that allocate metals to specific destinations, to mediate this adaptive response. In this essay, we discuss recent progress in our understanding of the structural mechanisms and metal specificity of this adaptive response, focusing on energy-requiring metallochaperones that play roles in the metallocofactor active site assembly in metalloenzymes and metallosensors, which govern the systems-level response to metal limitation and intoxication.

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Section: Metallochaperonesmentioning

confidence: 99%

Section: Metallochaperonesmentioning

confidence: 99%

Metallochaperones and metalloregulation in bacteria

Capdevila

Edmonds

Giedroc

2017

Essays in Biochemistry

109

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“…27 The structure of dimeric Hp UreE 28 closely resembles that of Ka UreE and it forms a complex with Hp UreG. 29 Finally, H. pylori encodes a proton-gated urea permease (UreI). 30 Of great significance to urease activation, the crystal structure of ( Hp UreH:UreF:UreG) 2 is known 31 ( Hp UreH is homologous to Ka UreD), although the interaction surface of this complex with urease has not been determined.…”

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confidence: 99%

Mutational and Computational Evidence That a Nickel-Transfer Tunnel in UreD Is Used for Activation of Klebsiella aerogenes Urease

et al. 2015

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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-blocking 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.

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“…Additional studies have revealed a monomer-dimer equilibrium for UreG proteins from S. pasteurii [9], Helicobacter pylori ( Hp UreG) [10], Methanocaldococcus jannaschii ( Mj UreG) [11], Metallosphaera sedula ( Ms UreG) [11], and Glycine max (soybean; Gm UreG) [12], with the dimers stabilized by Zn coordination. All UreG samples examined bind Zn and Ni, but the number of equivalents bound and the measured affinities differ in each case [6, 7, 9–14]. The single Zn site in the nucleotide-free dimeric H. pylori protein is coordinated by two His and two Cys according to X-ray absorption spectroscopy (XAS) [15].…”

Section: Introductionmentioning

confidence: 99%

“…These results are consistent with Zn coordination to the symmetric Cys-Pro-His motifs in the Hp UreG dimer lacking nucleotide. Two equivalents of Ni bind to this nucleotide-free monomeric protein with weak affinity, but a single Ni binds per dimer with high affinity when GTP is present [13, 14]. Notably, the sites of Ni binding are not established for GTP-bound Hp UreG although the aforementioned motif is very likely to be involved.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

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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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UreE-UreG Complex Facilitates Nickel Transfer and Preactivates GTPase of UreG in Helicobacter pylori

Cited by 58 publications

References 56 publications

Metallochaperones and metalloregulation in bacteria

Metallochaperones and metalloregulation in bacteria

Mutational and Computational Evidence That a Nickel-Transfer Tunnel in UreD Is Used for Activation of Klebsiella aerogenes Urease

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

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