Nickel Binding and [NiFe]-Hydrogenase Maturation by the Metallochaperone SlyD with a Single Metal-Binding Site in Escherichia coli

Kaluarachchi, Harini; Altenstein, Matthias; Sugumar, Sonia; Balbach, Jochen; Zamble, Deborah B.; Haupt, Caroline

doi:10.1016/j.jmb.2012.01.037

Cited by 25 publications

(43 citation statements)

References 38 publications

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“…20–23 This trafficking system consists of an importer, NikABCDE, 24, 25 chaperones ( e.g . HypA for hydrogenase-3), 26–28 accessory proteins involved in hydrogenase metallocenter assembly (HypB and SlyD), 29, 30 and an exporter, RcnAB. 31, 32 In addition, there are two homotetrameric transcriptional regulators: Ec NikR, 33, 34 which controls the expression of the Ni(II) importer NikABCDE by increasing the affinity for DNA in the Ni(II)-bound form, and Ec RcnR, 35, 36 which controls the expression of the Ni(II) and Co(II) exporter RcnAB by binding to DNA in the apo-form and decreasing the affinity for DNA when bound to either Ni(II) or Co(II).…”

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Glutamate Ligation in the Ni(II)- and Co(II)-Responsive Escherichia coli Transcriptional Regulator, RcnR

et al. 2017

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Escherichia coli RcnR (resistance to cobalt and nickel regulator, EcRcnR), is a metal-responsive repressor of the genes encoding the Ni(II) and Co(II) exporter proteins RcnAB by binding to PrcnAB. DNA-binding affinity is weakened when the cognate ions Ni(II) or Co(II) bind to EcRcnR in a six-coordinate site that features a (N/O)5S ligand donor-atom set in distinct sites: while both metal ions are bound by the N-terminus, Cys35, and His64, Co(II) is additionally bound by His3. On the other hand, the non-cognate Zn(II) and Cu(I) ions feature a lower coordination number, have a solvent-accessible binding site, and coordinate protein ligands that do not include the N-terminal amine. A molecular model of apo-EcRcnR suggested potential roles for Glu34 and Glu63 in binding Ni(II) and Co(II) to EcRcnR. The roles of Glu34 and Glu63 in metal binding, metal selectivity and function were therefore investigated using a structure/function approach. X-ray absorption spectroscopy (XAS) was used to assess the structural changes in the Ni(II), Co(II), and Zn(II) binding sites of Glu → Ala and Glu → Cys variants at both positions. The effect of these structural alterations on the regulation of PrcnA by EcRcnR in response to metal binding was explored using LacZ reporter assays. These combined studies indicate that while Glu63 is a ligand for both metal ions, Glu34 is a ligand for Co(II) but possibly not for Ni(II). The Glu34 variants affect the structure of the cognate metal sites, but they have no effect on the transcriptional response. In contrast, the Glu63 variants affect both the structure and transcriptional response, although they do not completely abolish the function of EcRcnR. The structure of the Zn(II) site is not significantly perturbed by any of the Glu variations. The spectroscopic and functional data obtained on the mutants were used to calculate models of the metal site structures of EcRcnR bound to Ni(II), Co(II) and Zn(II). The results are interpreted in terms of a switch mechanism, in which a subset of the metal binding ligands is responsible for the allosteric response required for DNA release.

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Glutamate Ligation in the Ni(II)- and Co(II)-Responsive Escherichia coli Transcriptional Regulator, RcnR

et al. 2017

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“…It is commonly believed that HypA and HypB are involved in the nickel insertion into the large subunit of hydrogenase (19 -21) via the formation of HypAHypB complex (20,25). In addition, SlyD, a protein that binds Ni 2ϩ at its C terminus (28), might also participate in hydrogenase maturation through interaction with HypB (29,30). The IF (insert-in-flap) domain of SlyD is responsible for the SlyDHypB interaction, facilitating nickel transfer from the C terminus of SlyD to HypB in H. pylori (31), but stimulates nickel release from HypB in Escherichia coli (29).…”

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

Yang

Lai

et al. 2015

Journal of Biological Chemistry

107

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“…Biochemical experiments revealed that SlyD can act as such a trigger, and experiments with a truncated form of SlyD suggested that acceleration of metal release from HypB is a key component of the role of SlyD during hydrogenase maturation. 40, 43, 58 However, the selectivity of this process was not known. To examine the metal selectivity of the impact of SlyD on HypB, nickel and zinc release from HAS of HypB to PAR, which serves as a reporter for the amount of metal released from the protein, 58 in the absence or presence of SlyD was monitored by electronic absorption spectroscopy.…”

Section: Resultsmentioning

confidence: 99%

“…Additional analysis of the SlyD-HypB complex suggested that the role of SlyD during hydrogenase biosynthesis is to stimulate nickel release from the HypB HAS in a process that depends on complex formation between the two proteins as well as a full-length MBD on SlyD. 40, 42, 43 However, it is not understood how this activity is achieved or if SlyD stimulates the release of metals other than nickel from HypB.…”

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confidence: 99%

High-affinity metal binding by the Escherichia coli [NiFe]-hydrogenase accessory protein HypB is selectively modulated by SlyD

2017

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[NiFe]-hydrogenase, which catalyzes the reversible conversion between hydrogen gas and protons, is a vital component of the metabolism of many pathogens. Maturation of [NiFe]-hydrogenase requires selective nickel insertion that is completed, in part, by the metallochaperones SlyD and HypB. Escherichia coli HypB binds nickel with sub-picomolar affinity, and the formation of the HypB-SlyD complex activates nickel release from the high-affinity site (HAS) of HypB. In this study, the metal selectivity of this process was investigated. Biochemical experiments revealed that the HAS of full length HypB can bind stoichiometric zinc. Moreover, in contrast to the acceleration of metal release observed with nickel-loaded HypB, SlyD blocks the release of zinc from the HypB HAS. X-ray absorption spectroscopy (XAS) demonstrated that SlyD does not impact the primary coordination sphere of nickel or zinc bound to the HAS of HypB. Instead, computational modeling and X-ray absorption spectroscopy of HypB loaded with nickel or zinc indicated that zinc binds to HypB with a different coordination sphere than nickel. The data suggested that Glu9, which is not a nickel ligand, directly coordinates zinc. These results were confirmed through the characterization of E9A-HypB, which afforded weakened zinc affinity compared to wild-type HypB but similar nickel affinity. This mutant HypB fully supports the production of [NiFe]-hydrogenase in E. coli. Altogether, these results are consistent with the model that the HAS of HypB functions as a nickel site during [NiFe]-hydrogenase enzyme maturation and that the metal selectivity is controlled by activation of metal release by SlyD.

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Nickel Binding and [NiFe]-Hydrogenase Maturation by the Metallochaperone SlyD with a Single Metal-Binding Site in Escherichia coli

Cited by 25 publications

References 38 publications

Glutamate Ligation in the Ni(II)- and Co(II)-Responsive Escherichia coli Transcriptional Regulator, RcnR

Glutamate Ligation in the Ni(II)- and Co(II)-Responsive Escherichia coli Transcriptional Regulator, RcnR

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

High-affinity metal binding by the Escherichia coli [NiFe]-hydrogenase accessory protein HypB is selectively modulated by SlyD

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