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.
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Related Subject Headings
- Tryptophan
- Structure-Activity Relationship
- Protein Conformation
- Myosin-Light-Chain Kinase
- Muscle, Smooth
- Muscle, Skeletal
- Leucine
- Kinetics
- Enzyme Activation
- Crystallography, X-Ray
Citation
Published In
DOI
ISSN
Publication Date
Volume
Issue
Start / End Page
Location
Related Subject Headings
- Tryptophan
- Structure-Activity Relationship
- Protein Conformation
- Myosin-Light-Chain Kinase
- Muscle, Smooth
- Muscle, Skeletal
- Leucine
- Kinetics
- Enzyme Activation
- Crystallography, X-Ray