Minyong Kim, My Tra Le, Lixin Fan, Courtney Campbell, Sambuddha Sen, Daiana A. Capdevila, Timothy L. Stemmler and David P. Giedroc*,
{"title":"鲍曼不动杆菌锌吸收抑制因子(Zur)的特征。","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":"{\"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. 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引用次数: 0
摘要
细菌细胞通过部署一组金属调控转录抑制因子和激活因子,与调控基因上游的运算子-启动子区域结合,严格调控细胞内必需过渡金属离子的浓度。与其他锌吸收调节蛋白(Zur)一样,鲍曼不动杆菌 Zur 在 ZnII 富集时抑制其调节子的转录,而在 ZnII 限制时与 DNA 的结合较弱。先前的研究证实,Zur 蛋白是同源二聚体,每个原体至少有两个金属位点,每个二聚体至少有四个金属位点。Cd2Zn2 AbZur 金属衍生物的 CdII X 射线吸收光谱(XAS)显示,CdII 与异构位点结合后,第一配位层为 S(N/O)3。定点突变表明,N 端 DNA 结合结构域的 H89 和 C100 以及 C 端二聚化结构域的 H107 和 E122 构成了调节金属位点。该异构位点的 KZn 为 6.0 (±2.2) × 1012 M-1,四种金属配体进行了功能性 "分工"。N 端结构域配体 H89 和 C100 对 KZn 的贡献远大于 H107 和 E122,而 C100S AbZur 独一无二地无法与 DNA 紧密结合(通过体外转录试验测定)。小角 X 射线散射(SAXS)实验显示,只有野生型 Zn 同源二聚体发生了异构转换,而 C100S AbZur 则没有发生转换。这些数据共同表明,向高亲和力 DNA 结合构象的转换涉及一个原体相对于另一个原体的旋转/平移,其方式取决于 C100 的完整性。我们将这些发现与其他 Zur 蛋白和更广泛的 Fur 家族抑制因子联系起来。
Characterization of the Zinc Uptake Repressor (Zur) from Acinetobacter baumannii
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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