Antonia D. Duran, Eric M. Danhart, Xiao Ma, Alexandra B. Kuzmishin Nagy, Karin Musier-Forsyth, Mark P. Foster
{"title":"NMR-based solution structure of the Caulobacter crescentus ProXp-ala trans-editing enzyme","authors":"Antonia D. Duran, Eric M. Danhart, Xiao Ma, Alexandra B. Kuzmishin Nagy, Karin Musier-Forsyth, Mark P. Foster","doi":"10.1007/s12104-024-10193-3","DOIUrl":null,"url":null,"abstract":"<div><p>ProXp-ala is a key component of the translational machinery in all three Domains of life. This enzyme helps to maintain the fidelity of proline codon translation through aminoacyl-tRNA<sup>Pro</sup> proofreading. In the first step of tRNA aminoacylation, the cognate aminoacyl-tRNA synthetase (aaRS) binds and activates an amino acid in the enzyme’s synthetic active site. If a non-cognate amino acid passes this first selection step and is charged onto the tRNA, a distinct aaRS editing active site may recognize the mischarged tRNA and deacylate it. Alternatively, this editing reaction may be carried out by a separate enzyme that deacylates the mischarged tRNA <i>in trans</i>. ProXp-ala is responsible for editing Ala mischarged onto tRNA<sup>Pro</sup>. Since <i>trans</i>-editing domains such as ProXp-ala bind their substrates after release from the synthetase, they must recognize not only the mischarged amino acid, but also the specific tRNA. Previous studies showed that <i>Caulobacter crescentus</i> (<i>Cc</i>) ProXp-ala distinguishes tRNA<sup>Pro</sup> from tRNA<sup>Ala</sup>, in part, based on the unique tRNA<sup>Pro</sup> acceptor stem base pair C1:G72. Previous crystallographic and NMR data also revealed a role for conformational selection by the ProXp-ala α2 helix in Ala- versus Pro-tRNA<sup>Pro</sup> substrate discrimination. The α2 helix makes lattice contacts in the crystal, which left some uncertainty as to its position in solution. We report resonance assignments for the substrate-free <i>Cc</i> ProXp-ala and the NMR-derived three-dimensional structure of the protein. These data reveal the position of the α2 helix in solution, with implications for substrate binding and recognition.</p></div>","PeriodicalId":492,"journal":{"name":"Biomolecular NMR Assignments","volume":null,"pages":null},"PeriodicalIF":0.8000,"publicationDate":"2024-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11511748/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biomolecular NMR Assignments","FirstCategoryId":"99","ListUrlMain":"https://link.springer.com/article/10.1007/s12104-024-10193-3","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOPHYSICS","Score":null,"Total":0}
引用次数: 0
Abstract
ProXp-ala is a key component of the translational machinery in all three Domains of life. This enzyme helps to maintain the fidelity of proline codon translation through aminoacyl-tRNAPro proofreading. In the first step of tRNA aminoacylation, the cognate aminoacyl-tRNA synthetase (aaRS) binds and activates an amino acid in the enzyme’s synthetic active site. If a non-cognate amino acid passes this first selection step and is charged onto the tRNA, a distinct aaRS editing active site may recognize the mischarged tRNA and deacylate it. Alternatively, this editing reaction may be carried out by a separate enzyme that deacylates the mischarged tRNA in trans. ProXp-ala is responsible for editing Ala mischarged onto tRNAPro. Since trans-editing domains such as ProXp-ala bind their substrates after release from the synthetase, they must recognize not only the mischarged amino acid, but also the specific tRNA. Previous studies showed that Caulobacter crescentus (Cc) ProXp-ala distinguishes tRNAPro from tRNAAla, in part, based on the unique tRNAPro acceptor stem base pair C1:G72. Previous crystallographic and NMR data also revealed a role for conformational selection by the ProXp-ala α2 helix in Ala- versus Pro-tRNAPro substrate discrimination. The α2 helix makes lattice contacts in the crystal, which left some uncertainty as to its position in solution. We report resonance assignments for the substrate-free Cc ProXp-ala and the NMR-derived three-dimensional structure of the protein. These data reveal the position of the α2 helix in solution, with implications for substrate binding and recognition.
期刊介绍:
Biomolecular NMR Assignments provides a forum for publishing sequence-specific resonance assignments for proteins and nucleic acids as Assignment Notes. Chemical shifts for NMR-active nuclei in macromolecules contain detailed information on molecular conformation and properties.
Publication of resonance assignments in Biomolecular NMR Assignments ensures that these data are deposited into a public database at BioMagResBank (BMRB; http://www.bmrb.wisc.edu/), where they are available to other researchers. Coverage includes proteins and nucleic acids; Assignment Notes are processed for rapid online publication and are published in biannual online editions in June and December.