Minyong Kim, My Tra Le, Lixin Fan, Courtney Campbell, Sambuddha Sen, Daiana A. Capdevila, Timothy L. Stemmler and David P. Giedroc*,
{"title":"Characterization of the Zinc Uptake Repressor (Zur) from Acinetobacter baumannii","authors":"Minyong Kim, My Tra Le, Lixin Fan, Courtney Campbell, Sambuddha Sen, Daiana A. Capdevila, Timothy L. Stemmler and David P. Giedroc*, ","doi":"10.1021/acs.biochem.3c00679","DOIUrl":null,"url":null,"abstract":"<p >Bacterial cells tightly regulate the intracellular concentrations of essential transition metal ions by deploying a panel of metal-regulated transcriptional repressors and activators that bind to operator-promoter regions upstream of regulated genes. Like other zinc uptake regulator (Zur) proteins, <i>Acinetobacter baumannii</i> Zur represses transcription of its regulon when Zn<sup>II</sup> is replete and binds more weakly to DNA when Zn<sup>II</sup> is limiting. Previous studies established that Zur proteins are homodimeric and harbor at least two metal sites per protomer or four per dimer. Cd<sup>II</sup> X-ray absorption spectroscopy (XAS) of the Cd<sub>2</sub>Zn<sub>2</sub> <i>Ab</i>Zur metalloderivative with Cd<sup>II</sup> bound to the allosteric sites reveals a S(N/O)<sub>3</sub> first coordination shell. Site-directed mutagenesis suggests that H89 and C100 from the N-terminal DNA binding domain and H107 and E122 from the C-terminal dimerization domain comprise the regulatory metal site. <i>K</i><sub>Zn</sub> for this allosteric site is 6.0 (±2.2) × 10<sup>12</sup> M<sup>–1</sup> with a functional “division of labor” among the four metal ligands. N-terminal domain ligands H89 and C100 contribute far more to <i>K</i><sub>Zn</sub> than H107 and E122, while C100S <i>Ab</i>Zur uniquely fails to bind to DNA tightly as measured by an <i>in vitro</i> transcription assay. The heterotropic allosteric coupling free energy, Δ<i>G</i><sub>c</sub>, is negative, consistent with a higher <i>K</i><sub>Zn</sub> for the <i>Ab</i>Zur-DNA complex and defining a bioavailable Zn<sup>II</sup> set-point of ≈6 × 10<sup>–14</sup> M. Small-angle X-ray scattering (SAXS) experiments reveal that only the wild-type Zn homodimer undergoes allosteric switching, while the C100S <i>Ab</i>Zur fails to switch. These data collectively suggest that switching to a high affinity DNA-binding conformation involves a rotation/translation of one protomer relative to the other in a way that is dependent on the integrity of C100. We place these findings in the context of other Zur proteins and Fur family repressors more broadly.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9000,"publicationDate":"2024-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochemistry Biochemistry","FirstCategoryId":"1","ListUrlMain":"https://pubs.acs.org/doi/10.1021/acs.biochem.3c00679","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Bacterial cells tightly regulate the intracellular concentrations of essential transition metal ions by deploying a panel of metal-regulated transcriptional repressors and activators that bind to operator-promoter regions upstream of regulated genes. Like other zinc uptake regulator (Zur) proteins, Acinetobacter baumannii Zur represses transcription of its regulon when ZnII is replete and binds more weakly to DNA when ZnII is limiting. Previous studies established that Zur proteins are homodimeric and harbor at least two metal sites per protomer or four per dimer. CdII X-ray absorption spectroscopy (XAS) of the Cd2Zn2AbZur metalloderivative with CdII bound to the allosteric sites reveals a S(N/O)3 first coordination shell. Site-directed mutagenesis suggests that H89 and C100 from the N-terminal DNA binding domain and H107 and E122 from the C-terminal dimerization domain comprise the regulatory metal site. KZn for this allosteric site is 6.0 (±2.2) × 1012 M–1 with a functional “division of labor” among the four metal ligands. N-terminal domain ligands H89 and C100 contribute far more to KZn than H107 and E122, while C100S AbZur uniquely fails to bind to DNA tightly as measured by an in vitro transcription assay. The heterotropic allosteric coupling free energy, ΔGc, is negative, consistent with a higher KZn for the AbZur-DNA complex and defining a bioavailable ZnII set-point of ≈6 × 10–14 M. Small-angle X-ray scattering (SAXS) experiments reveal that only the wild-type Zn homodimer undergoes allosteric switching, while the C100S AbZur fails to switch. These data collectively suggest that switching to a high affinity DNA-binding conformation involves a rotation/translation of one protomer relative to the other in a way that is dependent on the integrity of C100. We place these findings in the context of other Zur proteins and Fur family repressors more broadly.
期刊介绍:
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