Two domains of interaction with calcium binding proteins can be mapped using fragments of calponin

Abstract

Native calponin is able to bind 2 mol of calcium binding protein (CaBP) per mole calponin. This study extends this observation to define the 2 domains of interaction, one of which is near the actin binding site, and the other in the amino-terminal region of calponin. Also, the first evidence for a differentiation in the response of calponin to interaction with caltropin versus calmodulin is demonstrated. The binding of caltropin to cleavage and recombinant fragments of calponin was determined by 3 techniques: tryptophan fluorescence of the fragments, CD measurements to determine secondary structure changes, and analytical ultracentrifugation. In order to delineate the sites of interaction, 3 fragments of calponin have been studied. From a cyanogen bromide cleavage of calponin, residues 2-51 were isolated. This fragment is shown to bind to CaBPs and the affinity for caltropin is slightly higher than that for calmodulin. A carboxyl-terminal truncated mutant of calponin comprising residues 1-228 (CP 1-228) has been produced by recombinant techniques. Analytical ultracentrifugation has shown that C P 1-228, like the parent calponin, is able to bind 2 mol of caltropin per mol of 1-228 in a Ca'+-dependent fashion, indicating that there is a second site of interaction between residues 52-228. Temperature denaturation of the carboxylterminal truncated fragment compared with whole calponin show that the carboxyl-terminal region does not change the temperature at which calponin melts; however, there is greater residual secondary structure with whole calponin versus the fragment. A second mutant produced through recombinant techniques comprises residues 45-228 and is also able to bind caltropin, thus mapping the location of the second site of interaction to near the actin binding site.

Localization of Protein Regions Involved in the Interaction between Calponin and Myosin

“…These data indicate that the myosin binding function of CaP resides within residues 7-182 and not residues 183-292. It is interesting to note that actin (4,5), tropomyosin (4,5,8,9), calmodulin (5, 6, 11), and caltropin (27) binding sites are located in the same NH 2 -terminal portion of CaP.…”

Calponin reduces shortening velocity in skinned taenia coli smooth muscle fibres

“…An extinction coefficient, El%276nm, of 7.7 and a molecular weight of 25 kDa were used. The fragment was dialysed prior to experiments as described above for calponin [15].…”

“…The inhibition of the actomyosin/Mg-ATPase activity can be reversed by Ca2*-calmodulin [13] or calcium-caltropin [15] and by phosphorylation of calponin [12]. The role of these regulatory mechanisms in vivo is, however, unknown and in part also controversial [8,16,17,18,19].…”

“…Itoh et al [20] recently reported that exogenously added calponin reduced active tension in smooth muscle. In this report we describe the application of native calponin as well as a recently engineered fragment of the molecule [15] to chemically skinned taenia coli muscles activated by calcium or thiophosphorylation. The activity of the protein preparations was also assayed in parallel in actin filament sliding assays, which have formerly shown the inhibitory effects of calponin on filament velocity [21,22].…”

The Cytoskeleton and Its Regulation by Calcium and Protons

“…Among the class 14 kinesins, there are several that possess calponin homology domains (for review, see Richardson et al, 2006;Collings, 2008;Reddy and Day, 2011;Schneider and Persson, 2015). These kinesins are interesting for two reasons: first, they are often noted for binding actin and thus could link MTs and MFs (Wills et al, 1994;Schneider and Persson, 2015); and second, the calponin homology domain can be involved in binding to calmodulin (Wills et al, 1994). Should the latter be shown to be present, then there is a strong likelihood that calcium modulation will be found.…”