Site-specific mutagenesis of the alpha-helices of calmodulin. Effects of altering a charge cluster in the helix that links the two halves of calmodulin.
Alteration of residues 82-84 in the alpha-helix that links the two halves of calmodulin results in a differential effect on activator activity. Previous studies (Lukas, T. J., Burgess, W. H., Prendergast, F. G., Lau, W., and Watterson, D. M. (1986) Biochemistry 25, 1458-1464) indicated the importance of positive charge clusters in the calmodulin-binding protein, myosin light chain kinase. This suggested the possible importance of complementary negative charge clusters in calmodulin. By using an efficient cassette mutagenesis approach and a synthetic calmodulin gene (Roberts, D. M., Crea, R., Malecha, M., Alvarado-Urbina, G., Chiarello, R. H., and Watterson, D. M. (1985) Biochemistry 24, 5090-5098), this possibility was directly addressed by engineering a new calmodulin, VU-8 calmodulin, in which the glutamate cluster at residues 82-84 in the synthetic gene product (VU-1 calmodulin) was replaced by three lysines. VU-8 calmodulin activated phosphodiesterase to the same maximal extent as VU-1 calmodulin, although there was an alteration in the concentration of calmodulin required for half-maximal stimulation. In contrast, myosin light chain kinase was activated to only 30% of maximal activity and NAD kinase was not activated. These results provide insight into the functional role of the unusual central helix structure found in the calmodulin family of proteins and indicate that different, although possibly overlapping, chemical complementarities are employed in the interaction between calmodulin and its various physiological targets.
D000595:Amino Acid Sequence; D001483:Base Sequence; D002147:Calmodulin / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D002942:Circular Dichroism; D003488:Cyanogen Bromide; D004247:DNA / Q000235:genetics; D004274:DNA, Recombinant; D004789:Enzyme Activation; D009154:Mutation; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D010446:Peptide Fragments; D010770:Phosphotransferases / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor); D010957:Plasmids; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D002147:Calmodulin; D004274:DNA, Recombinant; D010446:Peptide Fragments; D004247:DNA; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C021844:NAD kinase; D009219:Myosin-Light-Chain Kinase; D003488:Cyanogen Bromide