The crystal structure and small-angle X-ray analysis of CsdL/TcdA reveal a new tRNA binding motif in the MoeB/E1 superfamily

PLoS One. 2015 Apr 21;10(4):e0118606. doi: 10.1371/journal.pone.0118606. eCollection 2015.

Abstract

Cyclic N6-threonylcarbamoyladenosine ('cyclic t6A', ct(6)A) is a non-thiolated hypermodification found in transfer RNAs (tRNAs) in bacteria, protists, fungi and plants. In bacteria and yeast cells ct(6)A has been shown to enhance translation fidelity and efficiency of ANN codons by improving the faithful discrimination of aminoacylated tRNAs by the ribosome. To further the understanding of ct(6)A biology we have determined the high-resolution crystal structures of CsdL/TcdA in complex with AMP and ATP, an E1-like activating enzyme from Escherichia coli, which catalyzes the ATP-dependent dehydration of t6A to form ct(6)A. CsdL/TcdA is a dimer whose structural integrity and dimer interface depend critically on strongly bound K+ and Na+ cations. By using biochemical assays and small-angle X-ray scattering we show that CsdL/TcdA can associate with tRNA with a 1:1 stoichiometry and with the proper position and orientation for the cyclization of t6A. Furthermore, we show by nuclear magnetic resonance that CsdL/TcdA engages in transient interactions with CsdA and CsdE, which, in the latter case, involve catalytically important residues. These short-lived interactions may underpin the precise channeling of sulfur atoms from cysteine to CsdL/TcdA as previously characterized. In summary, the combination of structural, biophysical and biochemical methods applied to CsdL/TcdA has afforded a more thorough understanding of how the structure of this E1-like enzyme has been fine tuned to accomplish ct(6)A synthesis on tRNAs while providing support for the notion that CsdA and CsdE are able to functionally interact with CsdL/TcdA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Amino Acid Sequence
  • Crystallography, X-Ray
  • Escherichia coli / chemistry
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / metabolism*
  • Models, Molecular
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Nucleotidyltransferases / chemistry*
  • Nucleotidyltransferases / metabolism*
  • Protein Conformation
  • Protein Structure, Tertiary
  • RNA, Transfer / metabolism*
  • Sequence Homology, Amino Acid
  • Ubiquitin-Activating Enzymes / chemistry*
  • Ubiquitin-Activating Enzymes / metabolism*

Substances

  • CsdL protein, E coli
  • Escherichia coli Proteins
  • Adenosine Triphosphate
  • RNA, Transfer
  • MoeB protein, E coli
  • Nucleotidyltransferases
  • Ubiquitin-Activating Enzymes

Associated data

  • PDB/4D79
  • PDB/4D7A

Grant support

Ministerio de Economía y Competitividad (ES) (grants PET2008_0101, BIO2009-11184 and BFU2010-22260-C02-02 to MCV, BFU2008-02372/BMC, CONSOLIDER CSD 2006-23 and BFU2011-22588 to MC, CTQ2012-32035 to JJB), Generalitat de Catalunya (ES) (grant SGR2009-1309 to MC), the European Commission (Framework Programme 7 (FP7) projects ComplexINC No. 279039 to MCV and SILVER-GA No. 260644 to MC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.