Crystal structure of a protein repair methyltransferase from Pyrococcus furiosus with its L-isoaspartyl peptide substrate

Protein L-isoaspartyl (D-aspartyl) methyltransferases (EC 2.1.1.77) are fou nd in almost all organisms. These enzymes catalyze the S-adenosylmethionine (AdoMet)-dependent methylation of isomerized and racemized aspartyl residu es in age-damaged proteins as part of an essential protein repair process. Here, we report crystal structures of the repair methyltransferase at resol utions up to 1.2 Angstrom from the hyperthermophilic archaeon Pyrococcus fu riosus. Refined structures include binary complexes with the active cofacto r AdoMet, its reaction product S-adenosylhomocysteine (AdoHcy), and adenosi ne. The enzyme places the methyl-donating cofactor in a deep, electrostatic ally negative pocket that is shielded from solvent. Across the multiple cry stal structures visualized, the presence or absence of the methyl group on the cofactor correlates with a significant conformational change in the enz yme in a loop bordering the active site, suggesting a role for motion in ca talysis or cofactor exchange. We also report the structure of a ternary com plex of the enzyme with adenosine and the methyl-accepting polypeptide subs trate VYP(L-isoAsp)HA at 2.1 Angstrom. The substrate binds in a narrow acti ve site cleft with three of its residues in an extended conformation, sugge sting that damaged proteins may be locally denatured during the repair proc ess in cells. Manual and computer-based docking studies on different isomer s help explain how the enzyme uses steric effects to make the critical dist inction between normal L-aspartyl and age-damaged L-isoaspartyl and D-aspar tyl residues. (C) 2001 Academic Press.