Precise identification of the regulatory F-actin- and calmodulin-binding sequences in the 10-kDa carboxyl-terminal domain of caldesmon

• Published in: The Journal of biological chemistry Vol. 269; no. 17; pp. 12824 - 12832
• Main Authors: Mezgueldi, M, Derancourt, J, Calas, B, Kassab, R, Fattoum, A
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 29-04-1994
• Subjects: Actins - metabolism; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Binding and carrier proteins; Binding Sites; Biological and medical sciences; Calmodulin - metabolism; Calmodulin-Binding Proteins - chemistry; Calmodulin-Binding Proteins - metabolism; Chymotrypsin; Cyanogen Bromide; Fundamental and applied biological sciences. Psychology; Hydrolysis; Molecular Sequence Data; Myosins - antagonists & inhibitors; Peptide Mapping; Proteins; Rabbits; Turkeys; Calcium; C terminal peptide; Enzyme; Adenosinetriphosphatase; Contractile protein; Actins; Binding site; Binding protein; Enzymatic activity; Hydrolases; Regulatory sequence; Calmodulin; Aminoacid sequence
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)99950-3

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.