Precise identification of the regulatory F-actin- 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 Trp659-Pro756 of gizzard caldesmon was investigated by subjecting the corresponding 10-kDa CNBr fragment, characterized earlier (Bartegi, A., Fattoum, A., Derancourt, J., and Kassab, R. (1990) J. Biol. Chem. 265, 15231-15238), to limited chymotryptic reactions conducted in the absence and presence of F-actin-tropomyosin. As a result, the F-actin-binding and actomyosin ATPase inhibitory activity was separated from the regulatory Ca(2+)-calmodulin-binding site. Seven chymotryptic peptides accounting for the entire primary structure of the CB10 fragment were isolated, and their complete amino acid sequences were established by combining NH2-terminal sequencing, mass spectrometry, and gel electrophoresis. Reversed-phase high performance liquid chromatography analyses of the binding of F-actin to these peptides revealed the 30-residue sequence Leu693-Trp722 as the unique crucial stretch for actin interaction and ATPase inhibition. This segment was also specifically protected by F-actin against proteolytic degradation. We further determined the functional properties of three synthetic peptides which successively cover the sequences Asn675-Lys695, Leu693-Trp722, and Arg711-Lys729. The first peptide segment specifically bound Ca(2+)-calmodulin as assessed by affinity chromatography and spectrofluorometry and should contain a potent novel calmodulin-binding subsite. The second immediately adjacent peptide inhibited the actomyosin ATPase in a tropomyosin-sensitive manner, as expected. In contrast, the third peptide displayed no detectable function. The results indicate that the overall sequence Asn675-Trp722 represents the essential regulatory unit of the COOH-terminal 10-kDa domain of caldesmon.
Mezgueldi
M
M
Centre de Recherches de Biochimie Macromoléculaire du CNRS, INSERM U 249, Université de Montpellier I, France.
Derancourt
J
J
Calas
B
B
Kassab
R
R
Fattoum
A
A
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002918:Chymotrypsin; D003488:Cyanogen Bromide; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009218:Myosins / Q000037:antagonists & inhibitors; D010449:Peptide Mapping; D011817:Rabbits; D014422:Turkeys
D000199:Actins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D002918:Chymotrypsin; D009218:Myosins; D003488:Cyanogen Bromide