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Autophosphorylation of Archaeoglobus fulgidus Rio2 and crystal structures of its nucleotide–metal ion complexes
Author(s) -
LaRondeLeBlanc Nicole,
Guszczynski Tad,
Copeland Terry,
Wlodawer Alexander
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2005.04702.x
Subject(s) - autophosphorylation , nucleotide , protein kinase domain , active site , serine , kinase , chemistry , biochemistry , crystallography , enzyme , biology , protein kinase a , mutant , gene
The highly conserved, atypical RIO serine protein kinases are found in all organisms, from archaea to man. In yeast, the kinase activity of Rio2 is necessary for the final processing step of maturing the 18S ribosomal rRNA. We have previously shown that the Rio2 protein from Archaeoglobus fulgidus contains both a small kinase domain and an N‐terminal winged helix domain. Previously solved structures using crystals soaked in nucleotides and Mg 2+ or Mn 2+ showed bound nucleotide but no ordered metal ions, leading us to the conclusion that they did not represent an active conformation of the enzyme. To determine the functional form of Rio2, we crystallized it after incubation with ATP or ADP and Mn 2+ . Co‐crystal structures of Rio2–ATP–Mn and Rio2–ADP–Mn were solved at 1.84 and 1.75 Å resolution, respectively. The γ‐phosphate of ATP is firmly positioned in a manner clearly distinct from its location in canonical serine kinases. Comparison of the Rio2–ATP–Mn complex with the Rio2 structure with no added nucleotides and with the ADP complex indicates that a flexible portion of the Rio2 molecule becomes ordered through direct interaction between His126 and the γ‐phosphate oxygen of ATP. Phosphopeptide mapping of the autophosphorylation site of Rio2 identified Ser128, within the flexible loop and directly adjacent to the part that becomes ordered in response to ATP, as the target. These results give us further information about the nature of the active site of Rio2 kinase and suggest a mechanism of regulation of its enzymatic activity.