FUNCTIONAL CONSEQUENCES OF TRUNCATING AMINO-ACID SIDE-CHAINS LOCATED AT A CALMODULIN-PEPTIDE INTERFACE

To test the relevance of the calmodulin-peptide crystal structures to their respective calmodulin-enzyme interactions, amino acid side chain s in calmodulin were altered at positions that interact with the calmo dulin-binding peptide of smooth muscle myosin light chain kinase but n ot with the calmodulin kinase II alpha peptide, Since shortening the s ide chains of Trp-800, Arg-812, and Leu-813 in smooth muscle myosin li ght chain kinase abrogated calmodulin-dependent activation (Bagchi, I. C., Huang, Q., and Means, A, R. (1992) J. Biol. Chern. 267, 3024-3029 ), substitutions were introduced at positions in calmodulin which cont act residues corresponding to Arg-812 and Leu-813 in the smooth muscle myosin light chain kinase peptide. Assays of smooth muscle myosin lig ht chain kinase with the calmodulin mutants M51A, V55A, L32A,M51A,V55A , and L32A,M51A,V55A,F68L, M71A exhibited 60%, 25%, and less than 1% o f maximal activity respectively, whereas the mutants fully activated c almodulin kinase II alpha. Alanine substitutions at positions on the s mooth muscle myosin light chain kinase peptide, corresponding to Trp-8 00 and Arg-812 in the enzyme, produced an 8-fold increase in the enzym e inhibition constant in contrast with the abolition of calmodulin bin ding by similar mutations in the parent enzyme.