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 chains in calmodulin were altered at positions that interact with the calmodulin-binding peptide of smooth muscle myosin light chain kinase but not with the calmodulin kinase IIalpha peptide. Since shortening the side chains of Trp-800, Arg-812, and Leu-813 in smooth muscle myosin light chain kinase abrogated calmodulin-dependent activation (Bagchi, I. C., Huang, Q., and Means, A. R. (1992) J. Biol. Chem. 267, 3024-3029), substitutions were introduced at positions in calmodulin which contact residues corresponding to Arg-812 and Leu-813 in the smooth muscle myosin light chain kinase peptide. Assays of smooth muscle myosin light chain kinase with the calmodulin mutants M51A,V55A, L32A,M51A,V55A, and L32A,M51A,V55A,F68L, M71A exhibited 60%, 25%, and less than 1% of maximal activity respectively, whereas the mutants fully activated calmodulin kinase IIalpha. Alanine substitutions at positions on the smooth muscle myosin light chain kinase peptide, corresponding to Trp-800 and Arg-812 in the enzyme, produced an 8-fold increase in the enzyme inhibition constant in contrast with the abolition of calmodulin binding by similar mutations in the parent enzyme.
Chin
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Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Sloan
D J
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Quiocho
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Means
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AR
D000596:Amino Acids / Q000032:analysis*; D001120:Arginine; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D007700:Kinetics; D007930:Leucine; D018482:Muscle, Skeletal / Q000201:enzymology; D009130:Muscle, Smooth / Q000201:enzymology; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014364:Tryptophan
D000596:Amino Acids; D002147:Calmodulin; D014364:Tryptophan; D001120:Arginine; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase; D007930:Leucine