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The copper-linked Escherichia coli AZY operon: Structure, metal binding, and a possible physiological role in copper delivery.

ABSTRACT: The Escherichia coli yobA-yebZ-yebY (AZY) operon encodes the proteins YobA, YebZ, and YebY. YobA and YebZ are homologs of the CopC periplasmic copper-binding protein and the CopD putative copper importer, respectively, whereas YebY belongs to the uncharacterized Domain of Unknown Function 2511 family. Despite numerous studies of E. coli copper homeostasis and the existence of the AZY operon in a range of bacteria, the operon's proteins and their functional roles have not been explored. In this study, we present the first biochemical and functional studies of the AZY proteins. Biochemical characterization and structural modeling indicate that YobA binds a single Cu²⁺ ion with high affinity. Bioinformatics analysis shows that YebY is widespread and encoded either in AZY operons or in other genetic contexts unrelated to copper homeostasis. We also determined the 1.8 Å resolution crystal structure of E. coli YebY, which closely resembles that of the lantibiotic self-resistance protein MlbQ. Two strictly conserved cysteine residues form a disulfide bond, consistent with the observed periplasmic localization of YebY. Upon treatment with reductants, YebY binds Cu⁺ and Cu²⁺ with low affinity, as demonstrated by metal-binding analysis and tryptophan fluorescence. Finally, genetic manipulations show that the AZY operon is not involved in copper tolerance or antioxidant defense. Instead, YebY and YobA are required for the activity of the copper-related NADH dehydrogenase II. These results are consistent with a potential role of the AZY operon in copper delivery to membrane proteins.

SUBMITTER: Hadley RC

PROVIDER: S-EPMC8689200 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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The copper-linked Escherichia coli AZY operon: Structure, metal binding, and a possible physiological role in copper delivery.

Hadley Rose C RC Zhitnitsky Daniel D Livnat-Levanon Nurit N Masrati Gal G Vigonsky Elena E Rose Jessica J Ben-Tal Nir N Rosenzweig Amy C AC Lewinson Oded O

The Journal of biological chemistry 20211123 1

The Escherichia coli yobA-yebZ-yebY (AZY) operon encodes the proteins YobA, YebZ, and YebY. YobA and YebZ are homologs of the CopC periplasmic copper-binding protein and the CopD putative copper importer, respectively, whereas YebY belongs to the uncharacterized Domain of Unknown Function 2511 family. Despite numerous studies of E. coli copper homeostasis and the existence of the AZY operon in a range of bacteria, the operon's proteins and their functional roles have not been explored. In this s ...[more]

PMID: 34822841

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Project description:In Escherichia coli and other bacteria, nickel uptake is regulated by the transcription factor NikR. Nickel binding at high-affinity sites in E. coli NikR (EcNikR) facilitates EcNikR binding to the nik operon, where it then suppresses transcription of genes encoding the nickel uptake transporter, NikABCDE. A structure of the EcNikR-DNA complex suggests that a second metal-binding site is also present when NikR binds to the nik operon. Moreover, this co-crystal structure raises the question of what metal occupies the second site under physiological conditions: K(+), which is present in the crystal structure, or Ni(2+), which has been proposed to bind to low- as well as high-affinity sites on EcNikR. To determine which ion is preferred at the second metal-binding site and the physical basis for any preference of one ion over another in both the second metal-binding site and the high-affinity sites, we conducted a series of detailed molecular simulations on the EcNikR structure. Simulations that place Ni(2+) at high-affinity sites lead to stable trajectories with realistic ion-ligand distances and geometries, while simulations that place K(+) at these sites lead to conformational changes in the protein that are likely unfavorable for ion binding. By contrast, simulations on the second metal site in the EcNikR-DNA complex lead to stable trajectories with realistic geometries regardless of whether K(+) or Ni(2+) occupies this site. Electrostatic binding free energy calculations, however, suggest that EcNikR binding to DNA is more favorable when the second metal-binding site contains K(+). An analysis of the energetic contributions to the electrostatic binding free energy suggests that, while the interaction between EcNikR and DNA is more favorable when the second site contains Ni(2+), the large desolvation penalty associated with moving Ni(2+) from solution to the relatively buried second site offsets this favorable interaction term. Additional free energy simulations that account for both electrostatic and non-electrostatic effects argue that EcNikR binding to DNA is most favorable when the second site contains a monovalent ion the size of K(+). Taken together, these data suggest that the EcNikR structure is most stable when Ni(2+) occupies high-affinity sites and that EcNikR binding to DNA is more favorable when the second site contains K(+).

Dataset Information

The copper-linked Escherichia coli AZY operon: Structure, metal binding, and a possible physiological role in copper delivery.

Publications

The copper-linked Escherichia coli AZY operon: Structure, metal binding, and a possible physiological role in copper delivery.

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