Identification of the regions conferring calmodulin-like properties to troponin C.
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 alpha-helical arm (N-helix), and its central helix is shorter due to the absence of 88KGK90 residues. Deleting both regions concomitantly (delta Nt delta KGK) elicited CaM-like regulation as tested (i) by smooth muscle contractility (maximal tension = 80 +/- 5% Po of control) and (ii) by the activation of phosphodiesterase (Vmax = 75 +/- 2% of control). The Ca(2+)-binding capacity of the mutant and the effect of the mutant on maximally Ca(2+)-activated tension of skinned rabbit psoas muscle fibers were both conserved. Furthermore, in the linker region of the central helix, replacing the TnC-characteristic 85EDAKGK90 successive residues with CaM-specific DTD residues generated a highly effective CaM mimic (Vmax = 96 +/- 2%) whether or not the N-helix was also retained. Apparent KD 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 alpha-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, 1251-1255). In contrast, our results suggest that the TnC central helix evolved to be less pliable by the combined influences of 85EDAKGK90 residues and the alpha-helical extension in N terminus, thereby keeping the N-terminal hands well separated from their C-terminal counterparts.
Gulati
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Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461.
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D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D003001:Cloning, Molecular; D003106:Colon / Q000187:drug effects / Q000502:physiology; D005818:Genetic Engineering; D006168:Guinea Pigs; D066298:In Vitro Techniques; D008961:Models, Structural; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects / Q000502:physiology; D009132:Muscles / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011817:Rabbits; D015183:Restriction Mapping; D017384:Sequence Deletion; D014336:Troponin / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D019209:Troponin C
D002147:Calmodulin; D014336:Troponin; D019209:Troponin C; D010727:Phosphoric Diester Hydrolases; D002118:Calcium