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 Ca²⁺-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 Ca²⁺-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.