Conservation of fibrillarin residues important for methyltransferase function.

The residue mutated in this work (D238 in Xenopus laevis) is highlighted in red in both panels. In the atomic resolution structure of Archaeoglobus fulgidus fibrillarin-Nop5 complex bound to its cofactor and methyl donor S-adenosyl-_L-methionine (AdoMet), it was shown that Asp-133 (equivalent to Xenopus laevis D238) is situated within 3.5 Å of the thiomethyl carbon of the bound AdoMet, implying that it plays a role as a catalytic residue [48]. When this residue was mutated to an alanine, the methylation activity of the complex was indeed totally abolished in an in vitro methylation assay [49]. It has been suggested that Asp-133 in fibrillarin may act as a general base by deprotonating the 2’-OH group of the target RNA during catalysis. It has further been suggested that Asp-133 may also facilitate cofactor binding through favorable electrostatic interactions [48,49]. A, 3-D model of the catalytic pocket of human fibrillarin (based on PDB 2ipx). D238 (in red, Xenopus numbering) is directly adjacent to the AdoMet (stick representation) with the methyl group to be transferred from the cofactor to the RNA substrate represented in pink. B, Multiple alignment between fibrillarin proteins of different origins (HUMAN, Homo sapiens; XENLA; Xenopus laevis; YEAST, Saccharomyces cerevisiae; ARCFU, Archaeoglobus fulgidus; PYRFU, Pyrococcus furiosus; and METJA, Methanocaldococcus jannaschii). Residues highlighted in blue and red (K/D/K) are absolutely conserved and correspond to the catalytic triad. The D residue in this triad is the residue mutated in this work. Bold, residues important for SAM binding. Asterisks, residues identical across all six species examined. Sequences were aligned with CLUSTAL.

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