Characterization of the regulatory domain of gizzard calponin. Interactions of the 145-163 region with F-actin, calcium-binding proteins, and tropomyosin.
Earlier, we proposed that the interaction of gizzard calponin with F-actin, promoting the inhibition of the actomyosin ATPase activity, involves the NH2-terminal portion of the calponin segment Ala145-Tyr182 (Mezgueldi, M., Fattoum, A., Derancourt, J., and Kassab, R. (1992) J. Biol. Chem. 267, 15943-15951). In this work, we have directly probed this region for actin binding sites using five peptide analogs covering different stretches of the sequence Thr133-Ile163. Co-sedimentation with F-actin, actomyosin ATPase measurements, and zero-length cross-linking reactions demonstrated that the 19-residue sequence Ala145-Ile163 is essential for actin interaction and ATPase inhibition. Furthermore, each peptide was tested for binding to the Ca(2+)-dependent proteins, caltropin and calmodulin, in both an actomyosin ATPase assay and an affinity chromatographic assay. The results revealed the 11-residue segment Gln153-Ile163, representing the COOH-terminal moiety of the F-actin binding sequence, as a crucial region for the high affinity binding of these regulatory proteins with concomitant removal of the ATPase inhibition. The 153-163 stretch contained also interactive sites for tropomyosin as assessed by affinity chromatography and spectrofluorometry. Collectively, the data support our initial results and highlight the ability of the multifunctional 145-163 region to serve as a potent regulatory domain of the smooth muscle calponin.
Mezgueldi
M
M
Centre de Recherches de Biochimie Macromoléculaire du CNRS, INSERM U 249, Université de Montpellier I, France.
Mendre
C
C
Calas
B
B
Kassab
R
R
Fattoum
A
A
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D002645:Chickens; D002918:Chymotrypsin / Q000378:metabolism; D006868:Hydrolysis; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D009218:Myosins / Q000037:antagonists & inhibitors; D011485:Protein Binding; D011817:Rabbits; D014335:Tropomyosin / Q000378:metabolism*; D014422:Turkeys; D000096985:Calponins
D000199:Actins; D002135:Calcium-Binding Proteins; D002918:Chymotrypsin; D008840:Microfilament Proteins; D009218:Myosins; D014335:Tropomyosin; D000096985:Calponins