Structural and thermodynamic evidence for a stabilizing role of Nop5p in S-adenosyl-L-methionine binding to fibrillarin.
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.
Department of Chemistry and Biochemistry, Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306, USA.
Mohamed
M
Aittaleb
Thomas
T
Visone
Marcia O
MO
Fenley
Hong
H
Li
D019843:Archaeal Proteins / Q000737:chemistry*; D019608:Archaeoglobales / Q000737:chemistry; D001665:Binding Sites; D002151:Calorimetry; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D008745:Methylation; D009687:Nuclear Proteins / Q000737:chemistry*; D011485:Protein Binding; D012261:Ribonucleoproteins; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics
D019843:Archaeal Proteins; D002868:Chromosomal Proteins, Non-Histone; D009687:Nuclear Proteins; D012261:Ribonucleoproteins; C047458:fibrillarin; D012436:S-Adenosylmethionine