Investigation of the Structural Requirements of the Troponin C Central Helix for Function

Abstract

The two globular Ca(2+)-binding domains of troponin C are connected by a three-turn, exposed central helix. The requirements of this helical linker for regulatory function are not fully understood. In the present work we investigated the structural requirement of the linker using a series of insertion mutations that differ in predicted flexibility. TnCinrc has a nine-residue flexible random coil insert, TnCinpp has a nine-residue rigid polyproline insert (three turns), and TnCin alpha h has a seven-residue insert with high potential of forming alpha-helix. TnCinrc and TnCinpp were defective in the activation of the regulated actomyosin ATPase activity in the presence of Ca2+ when compared to wild type or TnCin alpha h, suggesting that altering the flexibility of the central helix impairs the regulatory function of troponin C. TnCin alpha h, TnCinrc, and TnCinpp had 87% +/- 3, 62% +/- 3, and 58% +/- 2 of the wild type activity, respectively (n = 6). All insertions in the central helix resulted in elongation of molecule compared to wild type TnC as determined by Stokes' radius. The Ca(2+)-affinity, the Ca(2+)-dependence of the actomyosin ATPase, and the stability of the insertion mutants were similar to wild type. Deletions of up to two turns of the central helix have little effect on troponin C function [Dobrowolski, Z., Xu, G-Q., & Hitchcock-DeGregori, S. E. (1991) J. Biol. Chem. 266, 5703-5710]. In another mutant (TnCd11) the entire central helix, 87KEDAKGKSEEE97, was deleted. With TnCd11, activation of the actomyosin ATPase activity in the presence of Ca2+ was normal, but inhibition in the absence of Ca2+ was less effective. Interaction of TnCd11 with TnI was altered. There was a 2-fold excess of TnCd11 in reconstituted Tn complex, consistent with another report [Babu, A., Rao, V. G, Su, H., & Gulati, J. (1993) J. Biol. Chem. 268, 19232-19238]. Our results suggest that the native length and structure of the central helix are optimal for normal regulatory function and that connectivity alone is insufficient for TnC function.

Conformational Changes of Troponin C Within the Thin Filaments Detected by Neutron Scattering

“…The role of the D/E linker in TnC appears to be in its flexibility that allows the molecule to adopt multiple conformations. This is in agreement with the studies of TnC mutants in which the D/E linker were modified, 43,44 showing that the mutants with the D/E linker deletions do not seriously impair the regulatory activity, while those that reduce the flexibility of the D/E linker impair the ability to activate the thin filament.…”

Solution Structure of the Chicken Skeletal Muscle Troponin Complex Via Small-angle Neutron and X-ray Scattering

“…This mechanism could explain the necessary flexibility of the link between the N and C-terminal domains of TnC. 54,55 Finally, we note a report of a crystal structure of chicken skeletal muscle troponin TnC-TnI-TnT2 ternary complex in the CCa 2C state (M. V. Vinogradova et al, unpublished results). Our structure for the CCa 2C state is in agreement with this crystal structure, in which TnC adopts a dumbbell conformation.…”

Ca 2+ -regulated structural changes in troponin

“…Consistent with this suggestion, an intact central helix linker was shown to be important for orienting the structural and regulatory domains of TnC and maintaining its function in skeletal troponin (41)(42)(43). According to neutron-scattering data, skeletal TnC is extended in the Ca 2ϩ -activated ternary complex and in the TnC-TnI binary complex (30,31,44); these data agree with the intact central helix conformation seen here.…”