TAILIEUCHUNG - Báo cáo khoa học: Autophosphorylation of Archaeoglobus fulgidus Rio2 and crystal structures of its nucleotide–metal ion complexes

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 Archaeo-globus 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 act-ive conformation of the enzyme | iFEBS Journal Autophosphorylation of Archaeoglobus fulgidus Rio2 and crystal structures of its nucleotide-metal ion complexes Nicole LaRonde-LeBlanc1 Tad Guszczynski2 Terry Copeland2 and Alexander Wlodawer1 1 Protein Structure Section Macromolecular Crystallography Laboratory NationalCancer Institute NCI-Frederick MD USA 2 Laboratory of Protein Dynamics and Signaling NationalCancer Institute NCI-Frederick MD USA Keywords autophosphorylation nucleotide complex protein kinase ribosome biogenesis Rio2 Correspondence A. Wlodawer NationalCancer Institute MCL Bldg. 536 Rm. 5 Frederick MD 21702-1201 USA Fax 1 301 846 6322 Tel 1 301 846 5036 E-mail wlodawer@ Received 9 February 2005 revised 1 April 2005 accepted 5 April 2005 doi 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 Archaeo-globus fulgidus contains both a small kinase domain and an N-terminal winged helix domain. Previously solved structures using crystals soaked in nucleotides and Mg2 or Mn2 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 Mn2 . Co-crystal structures of Rio2-ATP-Mn and Rio2-ADP-Mn were solved at and A resolution respectively. The y-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 y-phosphate oxygen of ATP. Phosphopeptide mapping of the .

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