CHARACTERIZATION OF THE FUNCTIONAL-PROPERTIES OF SMOOTH-MUSCLE CALDESMON DOMAIN 4A - EVIDENCE FOR AN INDEPENDENT INHIBITORY ACTIN-TROPOMYOSIN BINDING DOMAIN

Recent analysis has shown the presence of three sequences in the C-ter minal 170 amino acids of human caldesmon (domain 4) which are involved in actin binding and tropomyosin-dependent inhibition of actomyosin A TPase. Two are in domain 4b (amino acids 715-793) and one is in domain 4a (amino acids 636-714). In the present work we have compared recomb inant peptides containing either domain 4a or domain 4b to address the question as to whether domain 4a alone has any inhibitory activity. W e have produced three new recombinant fragments containing domain 4a: H10 [622-708], H12 [506-708] and H13 [622-726] and we have characteriz ed their functional properties. All three fragments bound to actin and tropomyosin. Caldesmon, but not domain 4b, was able to displace the f ragments H10, H12 and H13 from actin. Thus the isolated caldesmon doma in 4a peptides bind to the same region on actin as in the whole molecu le while domains 4a and 4b occupy different sites on the actin molecul e. Unlike domain 4b, none of the domain 4a fragments inhibited the act omyosin ATPase in the absence of tropomyosin. However both domain 4a a nd 4b fragments displayed an inhibitory activity in the presence of tr opomyosin. H13 and H12 were more potent inhibitors than H10. Ca2+-calm odulin bound to H13 and reversed the inhibitory activity of this fragm ent but did not bind to H10 and H12. We conclude that domain 4a can ac t as an independent inhibitory actin-tropomyosin binding domain, but i ts properties are very different from the extreme C-terminal domain 4b .