Chin-Chuan Wei , Amena Abdul Razzak , Hadis Ghasemi , Rahil Khedri , Alexandria Fraase
{"title":"Ca2+ 结合使二聚体双氧化酶的截短 EF-手结构域转变为单体","authors":"Chin-Chuan Wei , Amena Abdul Razzak , Hadis Ghasemi , Rahil Khedri , Alexandria Fraase","doi":"10.1016/j.bpc.2024.107271","DOIUrl":null,"url":null,"abstract":"<div><p>Hydrogen peroxide, produced by Dual Oxidase (Duox), is essential for thyroid hormone synthesis. Duox activation involves Ca<sup>2+</sup> binding to its EF-hand Domain (EFD), which contains two EF-hands (EFs). In this study, we characterized a truncated EFD using spectrometry, calorimetry, electrophoretic mobility, and gel filtration to obtain its Ca<sup>2+</sup> binding thermodynamic and kinetics, as well as to assess the associated conformational changes. Our results revealed that its 2nd EF-hand (EF2) exhibits a strong exothermic Ca<sup>2+</sup> binding (K<sub>a</sub> = 10<sup>7</sup> M<sup>−1</sup>) while EF1 shows a weaker binding (K<sub>a</sub> = 10<sup>5</sup> M<sup>−1</sup>), resulting in the burial of its negatively charged residues. The Ca<sup>2+</sup> binding to EFD results in a stable structure with a melting temperature shifting from 67 to 99 °C and induces a structural transition from a dimeric to monomeric form. EF2 appears to play a role in dimer formation in its apo form, while the hydrophobic exposure of Ca<sup>2+</sup>-bound-EF1 is crucial for dimer formation in its holo form. The result is consistent with structures obtained from Cryo-EM, indicating that a stable structure of EFD with hydrophobic patches upon Ca<sup>2+</sup> binding is vital for its Duox's domain-domain interaction for electron transfer.</p></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":"312 ","pages":"Article 107271"},"PeriodicalIF":3.3000,"publicationDate":"2024-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0301462224001005/pdfft?md5=5e25f10a33b0c823dc273ce71a965548&pid=1-s2.0-S0301462224001005-main.pdf","citationCount":"0","resultStr":"{\"title\":\"Ca2+ binding shifts dimeric dual oxidase's truncated EF-hand domain to monomer\",\"authors\":\"Chin-Chuan Wei , Amena Abdul Razzak , Hadis Ghasemi , Rahil Khedri , Alexandria Fraase\",\"doi\":\"10.1016/j.bpc.2024.107271\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Hydrogen peroxide, produced by Dual Oxidase (Duox), is essential for thyroid hormone synthesis. Duox activation involves Ca<sup>2+</sup> binding to its EF-hand Domain (EFD), which contains two EF-hands (EFs). In this study, we characterized a truncated EFD using spectrometry, calorimetry, electrophoretic mobility, and gel filtration to obtain its Ca<sup>2+</sup> binding thermodynamic and kinetics, as well as to assess the associated conformational changes. Our results revealed that its 2nd EF-hand (EF2) exhibits a strong exothermic Ca<sup>2+</sup> binding (K<sub>a</sub> = 10<sup>7</sup> M<sup>−1</sup>) while EF1 shows a weaker binding (K<sub>a</sub> = 10<sup>5</sup> M<sup>−1</sup>), resulting in the burial of its negatively charged residues. The Ca<sup>2+</sup> binding to EFD results in a stable structure with a melting temperature shifting from 67 to 99 °C and induces a structural transition from a dimeric to monomeric form. EF2 appears to play a role in dimer formation in its apo form, while the hydrophobic exposure of Ca<sup>2+</sup>-bound-EF1 is crucial for dimer formation in its holo form. The result is consistent with structures obtained from Cryo-EM, indicating that a stable structure of EFD with hydrophobic patches upon Ca<sup>2+</sup> binding is vital for its Duox's domain-domain interaction for electron transfer.</p></div>\",\"PeriodicalId\":8979,\"journal\":{\"name\":\"Biophysical chemistry\",\"volume\":\"312 \",\"pages\":\"Article 107271\"},\"PeriodicalIF\":3.3000,\"publicationDate\":\"2024-05-24\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.sciencedirect.com/science/article/pii/S0301462224001005/pdfft?md5=5e25f10a33b0c823dc273ce71a965548&pid=1-s2.0-S0301462224001005-main.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biophysical chemistry\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0301462224001005\",\"RegionNum\":3,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biophysical chemistry","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0301462224001005","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
Hydrogen peroxide, produced by Dual Oxidase (Duox), is essential for thyroid hormone synthesis. Duox activation involves Ca2+ binding to its EF-hand Domain (EFD), which contains two EF-hands (EFs). In this study, we characterized a truncated EFD using spectrometry, calorimetry, electrophoretic mobility, and gel filtration to obtain its Ca2+ binding thermodynamic and kinetics, as well as to assess the associated conformational changes. Our results revealed that its 2nd EF-hand (EF2) exhibits a strong exothermic Ca2+ binding (Ka = 107 M−1) while EF1 shows a weaker binding (Ka = 105 M−1), resulting in the burial of its negatively charged residues. The Ca2+ binding to EFD results in a stable structure with a melting temperature shifting from 67 to 99 °C and induces a structural transition from a dimeric to monomeric form. EF2 appears to play a role in dimer formation in its apo form, while the hydrophobic exposure of Ca2+-bound-EF1 is crucial for dimer formation in its holo form. The result is consistent with structures obtained from Cryo-EM, indicating that a stable structure of EFD with hydrophobic patches upon Ca2+ binding is vital for its Duox's domain-domain interaction for electron transfer.
期刊介绍:
Biophysical Chemistry publishes original work and reviews in the areas of chemistry and physics directly impacting biological phenomena. Quantitative analysis of the properties of biological macromolecules, biologically active molecules, macromolecular assemblies and cell components in terms of kinetics, thermodynamics, spatio-temporal organization, NMR and X-ray structural biology, as well as single-molecule detection represent a major focus of the journal. Theoretical and computational treatments of biomacromolecular systems, macromolecular interactions, regulatory control and systems biology are also of interest to the journal.