STRUCTURE-ACTIVITY STUDIES OF THE REGULATORY INTERACTION OF THE 10 KILODALTON C-TERMINAL FRAGMENT OF CALDESMON WITH ACTIN AND THE EFFECT OFMUTATION OF CALDESMON RESIDUES 691-696

We have used isotope-edited nuclear magnetic resonance spectroscopy, b inding studies, and ATPase activity assays to investigate tile interac tion with F-actin of the 10 kDa C-terminal 658C fragment of chicken gi zzard caldesmon and two site-directed mutants of this fragment. Simult aneous dual-sited contacts with F-actin are observed for the segments of the 658C sequence flanking tryptophan residues 692 and 722. Competi tion experiments showed that both 658C contacts with actin are displac ed by substoichiometric concentrations of the shore inhibitory region of troponin-I indicative of different binding sites on actin for these regions of troponin-I and caldesmon, Substitution of caldesmon serine -702 by aspartic acid within the spacer region linking the two actin c ontacts of 658C led to weaker binding but with retention of equivalent affinity for each interaction site, Differential binding affinity of the two sites was achieved by replacement of the sequence Glu(691)-Trp -Leu-Thr-Lys-Thr(696) by Pro-Gly-His-Tyr-Asn-Asn. Consistent with thes e data, the concentration of this Cg1 mutant required to achieve 50% i nhibition of actin-tropomyosin-activated myosin ATPase was 4-fold grea ter than found for the 658C fragment. Although calmodulin binding to C g1 was observed, calmodulin proved ineffective in relieving the inhibi tion induced by the binding of this mutant to actin. These results are discussed in light of the actin contacts which are involved in the in hibitory activity possessed by different regions of the C-terminus of caldesmon.