Solution structure and DNA binding of the catalytic domain of the large serine resolvase TnpX

Stephen James Headey, Andrew Sivakumaran, Vicki Adams, Dena Lyras, Julian Ian Rood, Martin Scanlon, Matthew Charles James Wilce

Research output: Contribution to journalArticleResearchpeer-review

1 Citation (Scopus)

Abstract

The transfer of antibiotic resistance between bacteria is mediated by mobile genetic elements such as plasmids and transposons. TnpX is a member of the large serine recombinase subgroup of site-specific recombinases and is responsible for the excision and insertion of mobile genetic elements that encode chloramphenicol resistance in the pathogens Clostridium perfringens and Clostridium difficile. TnpX consists of three structural domains: domain I contains the catalytic site, whereas domains II and III contain DNA-binding motifs. We have solved the solution structure of residues 1-120 of the catalytic domain I of TnpX. The TnpX catalytic domain shares the same overall fold as other serine recombinases; however, differences are evident in the identity of the proposed hydrogen donor and in the size, amino acid composition, conformation, and dynamics of the TnpX active site loops. To obtain the interaction surface of TnpX1-120, we titrated a DNA oligonucleotide containing the circular intermediate joint attCI recombination site into 15N-labeled TnpX1-120 and observed progressive nuclear magnetic resonance chemical shift perturbations using 15N HSQC spectra. Perturbations were largely confined to a region surrounding the catalytic serine and encompassed residues of the active site loops. Utilizing the perturbation map and the data-driven docking program, HADDOCK, we have generated a model of the DNA interaction complex for the TnpX catalytic domain.
Original languageEnglish
Pages (from-to)316-324
Number of pages9
JournalJournal of Molecular Recognition
Volume28
Issue number5
DOIs
Publication statusPublished - 2015

Keywords

  • attCI
  • Chloramphenicol resistance
  • NMR
  • Serine recombinase
  • Solution structure
  • TnpX
  • Transposon

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