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

Rose C. Hadley, Daniel Zhitnitsky, Nurit Livnat-Levanon, Gal Masrati, Elena Vigonsky, Jessica Rose, Nir Ben-Tal, Amy C. Rosenzweig, Oded Lewinson

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2 Scopus citations

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.

Original languageEnglish
Article number101445
JournalJournal of Biological Chemistry
Volume298
Issue number1
DOIs
StatePublished - 1 Jan 2022
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2021 THE AUTHORS.

Funding

Funding and additional information—This work was supported by the National Science Foundation and Israel Binational Science Foundation Molecular and Cellular Biosciences grant 1938715 (to A. C. R., O. L., and N. B.-T.) and US Department of Energy (DOE) Basic Energy Sciences grant DE-SC0016284 (to A. C. R.). The research of N. B.-T. is supported in part by the Abraham E. Kazan Chair in Structural Biology, Tel Aviv University. Acknowledgments—Research in the Lewinson laboratory is supported in part by the Rappaport Institute for Biomedical research. Proteomic LC–MS–MS analysis was performed at the Smoler Proteomics Center, Technion, Israel. ICP–MS analysis was performed at the Quantitative Bio-element Imaging Center at Northwestern University, supported by NASA Ames Research Center grant NNA04CC36G, or at the Fredy & Nadine Herrmann Institute of Earth Sciences at the Hebrew University in Jerusalem. SEC–MALS analysis was performed by the Northwestern Keck Biophysics Facility. The LS-CAT beamlines of the Advanced Photon Source are supported by a US DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract DE-AC02-06CH11356. We thank Zdzislaw Wawrzak for assistance with crystallographic data analysis.

FundersFunder number
Abraham E. Kazan Chair
Israel Binational Science Foundation Molecular and Cellular Biosciences1938715
Rappaport Institute for Biomedical research
National Science Foundation
U.S. Department of Energy
Office of Science
Basic Energy SciencesDE-SC0016284
Ames Research CenterNNA04CC36G
Argonne National LaboratoryDE-AC02-06CH11356
Tel Aviv University

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