PRECISE IDENTIFICATION OF THE REGULATORY F-ACTIN-BINDING AND CALMODULIN-BINDING SEQUENCES IN THE 10-KDA CARBOXYL-TERMINAL DOMAIN OF CALDESMON

The precise location of the regulatory F-actin- and calmodulin-binding sites in the COOH-terminal sequence Trp(659)-Pro(756) of gizzard cald esmon was investigated by subjecting the corresponding 10-kDa CNBr fra gment, characterized earlier (Bartegi, A., Fattoum, A., Derancourt, J. , and Kassab, R. (1990) J. Biol. Chem. 265, 15231-15238), to limited c hymotryptic reactions conducted in the absence and presence of F-actin -tropomyosin. As a result, the F-actin-binding and actomyosin ATPase i nhibitory activity was separated from the regulatory Ca2+-calmodulin-b inding site. Seven chymotryptic peptides accounting for the entire pri mary structure of the CB10 fragment were isolated, and their complete amino acid sequences were established by combining NH2-terminal sequen cing, mass spectrometry, and gel electrophoresis. Reversed-phase high performance liquid chromatography analyses of the binding of F-actin t o these peptides revealed the 30-residue sequence Leu(693)-Trp(722) as the unique crucial stretch for actin interaction and ATPase inhibitio n. This segment was also specifically protected by F-actin against pro teolytic degradation. We further determined the functional properties of three synthetic peptides which successively cover the sequences Asn (675)-Lys(695), Leu(693)-Trp(722), and Arg(711)-Lys(729). The first pe ptide segment specifically bound Ca2+-calmodulin as assessed by affini ty chromatography and spectrofluorometry and should contain a potent n ovel calmodulin-binding subsite. The second immediately adjacent pepti de inhibited the actomyosin ATPase in a tropomyosin-sensitive manner, as expected. In contrast, the third peptide displayed no detectable fu nction, The results indicate that the overall sequence Asn(675)-Trp(72 2) represents the essential regulatory unit of the COOH terminal 10-kD a domain of caldesmon.