Abstract
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.
Highlights
The precise location of the regulatory F-actin- and The regulatory function of the caldesmon molecule is known calmodulin-binding sites in the COOH-terminal se- to reside within its well conserved carboxyl-terminal part of quence Trpsas-Pr076o6f gizzard caldesmon was investi- about 150 amino acids, designated domain 4,which harbors gated by subjecting the corresponding 10-kDaCNBr theactin-and calmodulin-binding sites as well as theside fragment, characterized earlier (Bartegi, A., Fattoum, chains phosphorylated by the proline-directed protein kinases A., Derancourt, J., and Kassab, R. (1990)J
2651, 5231-15238), to limited chymotryptic reactions the sequences involved in the twoformercriticalactivities conducted in the absence and presence of F-actin-tro- and for a better understandingof the molecular mechanism of pomyosin.As a result, the F-actin-bindingand actomyo- this protein, we have previouslyisolated for the first time, sin ATPase inhibitory activity was separated from the from a total CNBr digest of gizzard caldesmon, a 10-kDa fragregulatory Ca2+-calmodulin-bindinsgite
Tchhearacterization of the complete amino acid sequences were established by functional properties of the purified material indicated that combiningNH,terminal sequencing, mass spectrom- this stretch contains major regulatory determinants for F-acetry, and gel electrophoresis
Summary
As the three large peptides share a common sequence mobilities of the 3- and 6-kDa bands first observed upon gel corresponding to P4 ( L e ~ - ~ ~ ~ T rtphe' ~o~ve)r,all data strongly electrophoresis of the total digest (Fig. lA).Most importantly, suggest that F-actin has afforded protectioangainst the combination of the measured molecular mass values and the scission of the peptide bonds at Trp6" and Trp722 flanking the NH,-terminal sequencing of the peptides has permitted an the sequenceof P4.the 60-min digest of the CBlO unambiguous assignment of their COOH-terminal amino acid fragment alone was supplemented with F-actin-tropomyosin as recently describedby le Maire et al (24).The CBlO fragment and, aftecrentrifugation, the resulting pellet was analyzed digest produced in the presence of F-actin-tropomyosin dis- by HPLC in comparison with the pellet isolated directly from of Caldesmon
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