Identification of the Regions Conferring Calmodulin-like Properties to Troponin

The structural and functional correlations between troponin C (TnC) and calmodulin (CaM) were investigated by mutagenizing a synthetic cDNA coding rabbit skeletal muscle TnC. Compared with TnC, calmodulin lacks the N-terminal a-helical arm (N-helix), and its central helix is shorter due to the absence of SaKGKso residues. Deleting both regions concomitantly (ANtAKGK) elicited CaM-like regulation as tested (i) by smooth muscle contractility (maximal tension = 80 .C 6% Po of control) and (ii) by the activation of phosphodiesterase (Vm= 75 f 2% of control). The Ca2+binding capacity of the mutant and the effect of the mutant on maximally Ca2+-activated tension of skinned rabbit psoas muscle fibers were both conserved. Furthermore, in the linker region of the central helix, replacing the TnC-characteristic a6EDAKGKso successive residues with CaM-specific DTD residues generated a highly effective CaM mimic (Vm= 96 2 2%) whether or not the N-helix was also retained. Apparent KO values (i.e. concentrations for half-maximal response) for the successful mutants were similar to each other but about 200-fold higher than that for CaM. A part of the a-helical linker region in CaM may unfold and bend to promote multiplicity of target interaction using all four hands (Ikura, M., Clore, G. M., Gronenborn, A. M., Zhu, G., Klee, C. B., and Bax, A. (1992) Science 256,632-638; Meador, W. E., Means, A. R., and Quiocho, F. A. (1992) Science 257, 12511256). In contrast, our results suggest that the TnC central helix evolved to be less aliable by the combined influences of s6EDAKGKeo residues and the a-helical extension in N terminus, thereby keeping the N-terminal hands well separated from their C-terminal counterparts.