Identification of amino acids essential for calmodulin binding and activation of smooth muscle myosin light chain kinase.
Smooth muscle myosin light chain kinase (smMLCK) is a Ca(2+)-calmodulin (CaM)-dependent enzyme that phosphorylates the 20-kDa light chains of myosin. In a previous study (Bagchi, I.C., Kemp, B.E., and Means, A.R. (1989) J. Biol. Chem. 264, 15843-15849), we expressed in bacteria a 40-kDa fragment of smMLCK that displayed Ca(2+)-CaM-regulated catalytic activity. Initial mutagenesis experiments indicated that Gly811 and Arg812 were important for CaM-dependent activation of this 40-kDa enzyme. We have now carried out site-directed mutagenesis within the CaM-binding domain (Ser787 to Leu813) of this enzyme to identify amino acids that are critical for CaM binding and activation. Our studies reveal that the individual mutation of several hydrophobic amino acid residues such as Leu813, Ile810, and Trp800 and the glycine residue Gly804 also resulted in a severe decrease in or complete loss of CaM binding and activation of smMLCK. The hydrophobic residue (Trp800) and the basic residue (Arg812), both of which are mandatory for CaM binding to smMLCK, occur in analogous positions within the CaM-binding domain of a number of CaM-regulated enzymes. We conclude from these results that CaM binding by smMLCK is determined by an interplay of specific hydrophobic and electrostatic interactions which appear to be conserved among various target enzymes of CaM.
Bagchi
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Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Huang
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Means
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism* / Q000494:pharmacology; D002645:Chickens; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D009219:Myosin-Light-Chain Kinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D002147:Calmodulin; D011994:Recombinant Proteins; D009219:Myosin-Light-Chain Kinase