{"title":"肺炎克雷伯氏菌蛋白腺苷酸转移酶的表达、纯化和生物物理特征:经验和计算建模方法的系统整合。","authors":"Reabetswe Maake, Ikechukwu Achilonu","doi":"10.1007/s10930-024-10210-3","DOIUrl":null,"url":null,"abstract":"<div><p>Infections that are acquired due to a prolonged hospital stay and manifest 2 days following the admission of a patient to a health-care institution can be classified as hospital-acquired infections. <i>Klebsiella pneumoniae</i> (<i>K. pneumoniae</i>) has become a critical pathogen, posing serious concern globally due to the rising incidences of hypervirulent and carbapenem-resistant strains. Glutaredoxin is a redox protein that protects cells from oxidative stress as it associates with glutathione to reduce mixed disulfides. Protein adenylyltransferase (PrAT) is a pseudokinase with a proposed mechanism of transferring an AMP group from ATP to glutaredoxin. Inducing oxidative stress to the bacterium by inhibiting the activity of PrAT is a promising approach to combating its contribution to hospital-acquired infections. Thus, this study aims to overexpress, purify, and analyse the effects of ATP and Mg<sup>2+</sup> binding to <i>Klebsiella pneumoniae</i> PrAT (<i>Kp</i>PrAT). The pET expression system and nickel affinity chromatography were effective in expressing and purifying <i>Kp</i>PrAT. Far-UV CD spectroscopy demonstrates that the protein is predominantly α-helical, even in the presence of Mg<sup>2+</sup>. Extrinsic fluorescence spectroscopy with ANS indicates the presence of a hydrophobic pocket in the presence of ATP and Mg<sup>2+</sup>, while mant-ATP studies allude to the potential nucleotide binding ability of <i>Kp</i>PrAT. The presence of Mg<sup>2+</sup> increases the thermostability of the protein. Isothermal titration calorimetry provides insight into the binding affinity and thermodynamic parameters associated with the binding of ATP to <i>Kp</i>PrAT, with or without Mg<sup>2+</sup>. Conclusively, the presence of Mg<sup>2+</sup> induces a conformation in <i>Kp</i>PrAT that favours nucleotide binding.</p></div>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":"43 4","pages":"751 - 770"},"PeriodicalIF":1.9000,"publicationDate":"2024-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11345332/pdf/","citationCount":"0","resultStr":"{\"title\":\"Expression, Purification and Biophysical Characterisation of Klebsiella Pneumoniae Protein Adenylyltransferase: A Systematic Integration of Empirical and Computational Modelling Approaches\",\"authors\":\"Reabetswe Maake, Ikechukwu Achilonu\",\"doi\":\"10.1007/s10930-024-10210-3\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Infections that are acquired due to a prolonged hospital stay and manifest 2 days following the admission of a patient to a health-care institution can be classified as hospital-acquired infections. <i>Klebsiella pneumoniae</i> (<i>K. pneumoniae</i>) has become a critical pathogen, posing serious concern globally due to the rising incidences of hypervirulent and carbapenem-resistant strains. Glutaredoxin is a redox protein that protects cells from oxidative stress as it associates with glutathione to reduce mixed disulfides. Protein adenylyltransferase (PrAT) is a pseudokinase with a proposed mechanism of transferring an AMP group from ATP to glutaredoxin. Inducing oxidative stress to the bacterium by inhibiting the activity of PrAT is a promising approach to combating its contribution to hospital-acquired infections. Thus, this study aims to overexpress, purify, and analyse the effects of ATP and Mg<sup>2+</sup> binding to <i>Klebsiella pneumoniae</i> PrAT (<i>Kp</i>PrAT). The pET expression system and nickel affinity chromatography were effective in expressing and purifying <i>Kp</i>PrAT. Far-UV CD spectroscopy demonstrates that the protein is predominantly α-helical, even in the presence of Mg<sup>2+</sup>. Extrinsic fluorescence spectroscopy with ANS indicates the presence of a hydrophobic pocket in the presence of ATP and Mg<sup>2+</sup>, while mant-ATP studies allude to the potential nucleotide binding ability of <i>Kp</i>PrAT. The presence of Mg<sup>2+</sup> increases the thermostability of the protein. Isothermal titration calorimetry provides insight into the binding affinity and thermodynamic parameters associated with the binding of ATP to <i>Kp</i>PrAT, with or without Mg<sup>2+</sup>. Conclusively, the presence of Mg<sup>2+</sup> induces a conformation in <i>Kp</i>PrAT that favours nucleotide binding.</p></div>\",\"PeriodicalId\":793,\"journal\":{\"name\":\"The Protein Journal\",\"volume\":\"43 4\",\"pages\":\"751 - 770\"},\"PeriodicalIF\":1.9000,\"publicationDate\":\"2024-07-09\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11345332/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"The Protein Journal\",\"FirstCategoryId\":\"2\",\"ListUrlMain\":\"https://link.springer.com/article/10.1007/s10930-024-10210-3\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"The Protein Journal","FirstCategoryId":"2","ListUrlMain":"https://link.springer.com/article/10.1007/s10930-024-10210-3","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
摘要
因长期住院而感染并在患者入住医疗机构两天后表现出来的感染可归类为医院获得性感染。肺炎克雷伯氏菌(K. pneumoniae)已成为一种严重的病原体,由于对碳青霉烯类耐药菌株的高化脓性发病率不断上升,已引起全球的严重关注。谷胱甘肽是一种氧化还原蛋白,能保护细胞免受氧化应激,因为它能与谷胱甘肽结合还原混合二硫化物。蛋白腺苷基转移酶(PrAT)是一种假激酶,其作用机制是将 AMP 基团从 ATP 转移到 Glutaredoxin。通过抑制 PrAT 的活性来诱导细菌产生氧化应激是一种很有前景的方法,可有效防止医院感染。因此,本研究旨在过表达、纯化和分析 ATP 与 Mg2+ 结合对肺炎克雷伯菌 PrAT(KpPrAT)的影响。pET 表达系统和镍亲和层析能有效表达和纯化 KpPrAT。远紫外 CD 光谱显示,即使在 Mg2+ 存在的情况下,该蛋白质也主要是 α-螺旋形的。用 ANS 进行的外荧光光谱分析表明,在 ATP 和 Mg2+ 存在的情况下,KpPrAT 存在一个疏水口袋,而 mant-ATP 研究则暗示了 KpPrAT 潜在的核苷酸结合能力。Mg2+ 的存在增加了蛋白质的热稳定性。通过等温滴定量热法,我们可以深入了解 ATP 与 KpPrAT(无论有无 Mg2+)结合的亲和力和热力学参数。结论是,Mg2+ 的存在会诱导 KpPrAT 形成有利于核苷酸结合的构象。
Expression, Purification and Biophysical Characterisation of Klebsiella Pneumoniae Protein Adenylyltransferase: A Systematic Integration of Empirical and Computational Modelling Approaches
Infections that are acquired due to a prolonged hospital stay and manifest 2 days following the admission of a patient to a health-care institution can be classified as hospital-acquired infections. Klebsiella pneumoniae (K. pneumoniae) has become a critical pathogen, posing serious concern globally due to the rising incidences of hypervirulent and carbapenem-resistant strains. Glutaredoxin is a redox protein that protects cells from oxidative stress as it associates with glutathione to reduce mixed disulfides. Protein adenylyltransferase (PrAT) is a pseudokinase with a proposed mechanism of transferring an AMP group from ATP to glutaredoxin. Inducing oxidative stress to the bacterium by inhibiting the activity of PrAT is a promising approach to combating its contribution to hospital-acquired infections. Thus, this study aims to overexpress, purify, and analyse the effects of ATP and Mg2+ binding to Klebsiella pneumoniae PrAT (KpPrAT). The pET expression system and nickel affinity chromatography were effective in expressing and purifying KpPrAT. Far-UV CD spectroscopy demonstrates that the protein is predominantly α-helical, even in the presence of Mg2+. Extrinsic fluorescence spectroscopy with ANS indicates the presence of a hydrophobic pocket in the presence of ATP and Mg2+, while mant-ATP studies allude to the potential nucleotide binding ability of KpPrAT. The presence of Mg2+ increases the thermostability of the protein. Isothermal titration calorimetry provides insight into the binding affinity and thermodynamic parameters associated with the binding of ATP to KpPrAT, with or without Mg2+. Conclusively, the presence of Mg2+ induces a conformation in KpPrAT that favours nucleotide binding.
期刊介绍:
The Protein Journal (formerly the Journal of Protein Chemistry) publishes original research work on all aspects of proteins and peptides. These include studies concerned with covalent or three-dimensional structure determination (X-ray, NMR, cryoEM, EPR/ESR, optical methods, etc.), computational aspects of protein structure and function, protein folding and misfolding, assembly, genetics, evolution, proteomics, molecular biology, protein engineering, protein nanotechnology, protein purification and analysis and peptide synthesis, as well as the elucidation and interpretation of the molecular bases of biological activities of proteins and peptides. We accept original research papers, reviews, mini-reviews, hypotheses, opinion papers, and letters to the editor.