A High-Affinity Metal-Binding Peptide from <i>Escherichia coli</i> HypB

Chung, Kim C. Chan; Cao, Li; Dias, Alistair V.; Pickering, Ingrid J.; George, Graham N.; Zamble, Deborah B.

doi:10.1021/ja8055003

Cited by 37 publications

(28 citation statements)

References 18 publications

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“…S6). 24, 25 The XAS data of Ni(II)-(HypB + SlyD146) were very similar to those of HypB alone (Fig. S7), indicating that complex formation with SlyD146 does not alter the HAS of HypB, consistent with the results of the metal release assays.…”

Section: Resultssupporting

confidence: 75%

“…S8). 59, 60 For consistency with previously published fits, 24, 25 and between nickel and zinc fits, nitrogen was used since EXAFS fitting analysis cannot discriminate between nitrogen and oxygen. The EXAFS data fit well to both S 3 /N and S 2 /N 2 coordination (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The B9 peptide (CTTCGCGEG) is comprised of the 9 N-terminal amino acids of the fully matured HypB with the N-terminal methionine removed (residues 2–10), and contains all the nickel ligands of the HAS of HypB. 24, 32 …”

Section: Resultsmentioning

confidence: 99%

“…23, 24 The G-domain metal site of HypB uses different sets of ligands to coordinate nickel versus zinc, 25–27 and occupancy of this site modulates GTP hydrolysis, a property that is essential for hydrogenase maturation. 27–29 Furthermore, the G-domain site rapidly and selectively transfers nickel, but not zinc, to HypA, 21, 22 the accessory protein that is thought to serve as an adapter for nickel delivery to the large hydrogenase subunit.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Resultssupporting

confidence: 75%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

2017

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“…One such example is the N-terminal high affinity site in E. coli HypB (absent in H. pylori HypB), which binds Ni(II) through a CXXCGC motif at the N-terminus, coordinating the N-terminal amine and the three S-Cys in a planar geometry. 45 Another such example is S. coelicolor nickel superoxide dismutase (NiSOD), which binds Ni(II) through the N-terminal amine of His1, the backbone amide of Cys2, and the sidechains of Cys2 and Cys6, with the sidechain imidazole of His1 also bound in the Ni(III) state. 46–48 Both the E. coli HypB N-terminal motif and NiSOD Ni binding sites have been referred to as “ATCUN-like” in literature due to the binding of the N-terminal amine and planar geometry.…”

Section: Discussionmentioning

confidence: 99%

Nickel Ligation of the N-Terminal Amine of HypA Is Required for Urease Maturation in Helicobacter pylori

Johnson

Merrell

et al. 2017

Biochemistry

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The human pathogen Helicobacter pylori requires nickel for colonization of the acidic environment of the stomach. HypA, a Ni metallochaperone that is typically associated with hydrogenase maturation, is also required for urease maturation and acid survival of H. pylori. There are two proposed Ni site structures for HypA; one is a paramagnetic 6-coordinate site characterized by X-ray absorption spectroscopy (XAS) in unmodified HypA while another is a diamagnetic four-coordinate planar site characterized by solution NMR in an N-terminally modified HypA construct. To determine the role of the N-terminal amine in Ni binding of HypA, an N-terminal extension variant, L2*-HypA, where a leucine residue was inserted into the second position of the amino acid sequence in the proposed Ni-binding motif, was characterized in vitro and in vivo. Structural characterization of the Ni site using x-ray absorption spectroscopy (XAS) showed a coordination change from 6-coordinate in WT-HypA to 5-coordinate pyramidal in L2*-HypA, which was accompanied by the loss of two N/O-donor protein ligands and the addition of an exogenous bromide ligand from the buffer. The magnetic properties of the Ni-sites in WT- compared to L2*-HypA confirmed that a spin-state change from high- to low-spin accompanied this change in structure. The L2*-HypA H. pylori strain was shown to be acid sensitive and deficient in urease activity in vivo. In vitro characterization showed that L2*-HypA did not disrupt the HypA-UreE interaction essential for urease maturation, but was at least 20-fold weaker in Ni-binding as compared to WT. Characterization of the L2*-HypA variant clearly demonstrates that the N-terminal amine of HypA is involved in proper Ni coordination and is necessary for urease activity and acid survival.

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Nickel Metallomics: General Themes Guiding Nickel Homeostasis

Sydor

Zamble

2012

Metal Ions in Life Sciences

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The nickel metallome describes the distribution and speciation of nickel within the cells of organisms that utilize this element. This distribution is a consequence of nickel homeostasis, which includes import, storage, and export of nickel, incorporation into metalloenzymes, and the modulation of these and associated cellular systems through nickel-regulated transcription. In this chapter, we review the current knowledge of the most common nickel proteins in prokaryotic organisms with a focus on their coordination environments. Several underlying themes emerge upon review of these nickel systems, which illustrate the common principles applied by nature to shape the nickel metallome of the cell.

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A High-Affinity Metal-Binding Peptide from Escherichia coli HypB

Cited by 37 publications

References 18 publications

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

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

Nickel Ligation of the N-Terminal Amine of HypA Is Required for Urease Maturation in Helicobacter pylori

Nickel Metallomics: General Themes Guiding Nickel Homeostasis

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