Enzymatic methylation of L-isoaspartyl residues derived from aspartyl residues in affinity-purified calmodulin. The role of conformational flexibility in spontaneous isoaspartyl formation.
We have investigated the formation of D-aspartyl and L-isoaspartyl (beta-aspartyl) residues and their subsequent methylation in bovine brain calmodulin by the type II protein carboxyl methyltransferase. Based on the results of studies with unstructured peptides and denatured proteins, it has been proposed that the major sites of carboxyl methylation in calmodulin are at L-isoaspartyl residues that originate from two Asn-Gly sequences. To test this hypothesis, we directly identified the sites of methylation in affinity-purified preparations of calmodulin by peptide mapping using the proteases trypsin, endoproteinase Lys-C, clostripain, chymotrypsin, and Staphylococcus aureus V8 protease. We found, however, that the major high-affinity sites of methylation originate from aspartyl residues at position 2 and at positions 78 and/or 80. The methylatable residue in the first case was shown to be L-isoaspartate by comparison of the properties of a synthetic peptide corresponding to the N-terminal 13 residues substituted with an L-iso-Asp residue at position 2. The second methylatable residue, probably derived from Asp78, also appears to be an L-isoaspartyl residue. These sites appear to be readily accessible to the methyltransferase and are present in relatively flexible regions of calmodulin that may allow the spontaneous degradation reactions to occur that generate L-isoaspartyl residues via succinimide intermediates. Interestingly, the four calcium binding regions, each containing 3-4 aspartyl and asparaginyl residues (including the two Asn-Gly sequences), do not appear to contribute to the high-affinity methyl acceptor sites, even when calcium is removed prior to the methylation reaction. We propose that methylatable residues do not form at these sites because of the inflexibility of these regions when calcium is bound.
Ota
I M
IM
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
Clarke
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001921:Brain / Q000378:metabolism; D002147:Calmodulin / Q000302:isolation & purification; D002417:Cattle; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D008745:Methylation; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis; D010446:Peptide Fragments / Q000302:isolation & purification; D010447:Peptide Hydrolases
D002147:Calmodulin; D009842:Oligopeptides; D010446:Peptide Fragments; D001224:Aspartic Acid; D010447:Peptide Hydrolases; D003488:Cyanogen Bromide