Structural and thermodynamic evidence for a stabilizing role of Nop5p in S-adenosyl-L-methionine binding to fibrillarin

J Biol Chem. 2004 Oct 1;279(40):41822-9. doi: 10.1074/jbc.M406209200. Epub 2004 Jul 30.


In Archaea, fibrillarin and Nop5p form the core complex of box C/D small ribonucleoprotein particles, which are responsible for site-specific 2'-hydroxyl methylation of ribosomal and transfer RNAs. Fibrillarin has a conserved methyltransferase fold and employs S-adenosyl-l-methionine (AdoMet) as the cofactor in methyl transfer reactions. Comparison between recently determined crystal structures of free fibrillarin and fibrillarin-Nop5p-AdoMet tertiary complex revealed large conformational differences at the cofactor-binding site in fibrillarin. To identify the structural elements responsible for these large conformational differences, we refined a crystal structure of Archaeoglobus fulgidus fibrillarin-Nop5p binary complex at 3.5 A. This structure exhibited a pre-formed backbone geometry at the cofactor binding site similar to that when the cofactor is bound, suggesting that binding of Nop5p alone to fibrillarin is sufficient to stabilize the AdoMet-binding pocket. Calorimetry studies of cofactor binding to fibrillarin alone and to fibrillarin-Nop5p binary complex provided further support for this role of Nop5p. Mutagenesis and thermodynamic data showed that a cation-pi bridge formed between Tyr-89 of fibrillarin and Arg-169 of Nop5p, although dispensable for in vitro methylation activity, could partially account for the enhanced binding of cofactor to fibrillarin by Nop5p. Finally, assessment of cofactor-binding thermodynamics and catalytic activities of enzyme mutants identified three additional fibrillarin residues (Thr-70, Glu-88, and Asp-133) to be important for cofactor binding and for catalysis.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Archaeal Proteins / chemistry*
  • Archaeoglobales / chemistry
  • Binding Sites
  • Calorimetry
  • Chromosomal Proteins, Non-Histone / chemistry
  • Chromosomal Proteins, Non-Histone / metabolism*
  • Crystallography, X-Ray
  • Hydrogen Bonding
  • Methylation
  • Nuclear Proteins / chemistry*
  • Protein Binding
  • Ribonucleoproteins
  • S-Adenosylmethionine / chemistry
  • S-Adenosylmethionine / metabolism*
  • Thermodynamics*


  • Archaeal Proteins
  • Chromosomal Proteins, Non-Histone
  • Nuclear Proteins
  • Ribonucleoproteins
  • fibrillarin
  • S-Adenosylmethionine