The major myosin-binding site of caldesmon resides near its N-terminal extreme

Abstract

The primary myosin-binding site of caldesmon was thought to be in the N- terminal region of the molecule, but the exact nature of the caldesmon-myosin interaction has not been well characterized. A caldesmon fragment that encompasses residues 1-240 (N240) was found to bind full-length smooth muscle myosin on the basis of co-sedimentation experiments. The interaction between myosin and N240 was not affected by phosphorylation of myosin, but it was weakened by the presence of Ca2+/calmodulin. To locate the myosin-binding site, we have designed several synthetic peptides based on the N-terminal caldesmon sequence. We found that a peptide stretch corresponding to the first 27 residues (Met-1 to Tyr-27), but not that of the first 22 residues (Met-1 to Ala-22), exhibited a moderate affinity toward myosin. We also found that a peptide containing the segment from Ile/Leu-25 to Lys-53 bound both myosin and heavy meromyosin more strongly and was capable of displacing caldesmon from myosin. Our results demonstrate that the sequence near the N- terminal extreme of caldesmon harbors a major myosin-binding site of caldesmon, in which both the nonpolar residues and clusters of positively and negatively charged residues confer the specificity and affinity of the caldesmon-myosin interaction.