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 language | English |
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Article number | 101445 |
Journal | Journal of Biological Chemistry |
Volume | 298 |
Issue number | 1 |
DOIs | |
State | Published - 1 Jan 2022 |
Externally published | Yes |
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.
Funders | Funder number |
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Abraham E. Kazan Chair | |
Israel Binational Science Foundation Molecular and Cellular Biosciences | 1938715 |
Rappaport Institute for Biomedical research | |
National Science Foundation | |
U.S. Department of Energy | |
Office of Science | |
Basic Energy Sciences | DE-SC0016284 |
Ames Research Center | NNA04CC36G |
Argonne National Laboratory | DE-AC02-06CH11356 |
Tel Aviv University |