Crystallographic Study of the 2-thioribothymidine-synthetic Complex TtuA-TtuB From Thermus Thermophilus

Acta Crystallogr F Struct Biol Commun. 2016 Oct 1;72(Pt 10):777-781. doi: 10.1107/S2053230X16014242. Epub 2016 Sep 22.

Abstract

The ubiquitin-like protein TtuB is a sulfur carrier for the biosynthesis of 2-thioribothymidine (s2T) at position 54 in some thermophilic bacterial tRNAs. TtuB captures a S atom at its C-terminus as a thiocarboxylate and transfers it to tRNA by the transferase activity of TtuA. TtuB also functions to suppress s2T formation by forming a covalent bond with TtuA. To explore how TtuB interacts with TtuA and switches between these two different functions, high-resolution structure analysis of the TtuA-TtuB complex is required. In this study, the TtuA-TtuB complex from Thermus thermophilus was expressed, purified and crystallized. To mimic the thiocarboxylated TtuB, the C-terminal Gly residue was replaced with Cys (G65C) to obtain crystals of the TtuA-TtuB complex. A Zn-MAD data set was collected to a resolution of 2.5 Å. MAD analysis successfully determined eight Zn sites, and a partial structure model composed of four TtuA-TtuB complexes in the asymmetric unit was constructed.

Keywords: TtuA; TtuB; Zn-MAD; post-transcriptional modification; sulfur transfer; ubiquitin-like protein.

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Cations, Divalent
  • Cloning, Molecular
  • Crystallization
  • Crystallography, X-Ray
  • Cysteine / chemistry
  • Cysteine / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • Glycine / chemistry
  • Glycine / metabolism
  • Mutation
  • RNA, Transfer / chemistry*
  • RNA, Transfer / genetics
  • RNA, Transfer / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Thermus thermophilus / chemistry*
  • Thermus thermophilus / enzymology
  • Thiouridine / analogs & derivatives*
  • Thiouridine / chemistry
  • Thiouridine / metabolism
  • X-Ray Diffraction
  • Zinc / chemistry
  • Zinc / metabolism

Substances

  • Bacterial Proteins
  • Cations, Divalent
  • Recombinant Proteins
  • Thiouridine
  • 2-thioribothymidine
  • RNA, Transfer
  • Zinc
  • Cysteine
  • Glycine