Structural kinetics of cardiac troponin C mutants linked to familial hypertrophic and dilated cardiomyopathy in troponin complexes

Abstract

The key events in regulating cardiac muscle contraction involve Ca 2+ binding to and release from cTnC (troponin C) and structural changes in cTnC and other thin filament proteins triggered by Ca2+ movement. Single mutations L29Q and G159D in human cTnC have been reported to associate with familial hypertrophic and dilated cardiomyopathy, respectively. We have examined the effects of these individual mutations on structural transitions in the regulatory N-domain of cTnC triggered by Ca2+ binding and dissociation. This study was carried out with a double mutant or triple mutants of cTnC, reconstituted into troponin with tryptophanless cTnI and cTnT. The double mutant, cTnC(L12W/N51C) labeled with 1,5-IAEDANS at Cys-51, served as a control to monitor Ca2+-induced opening and closing of the N-domain by Förster resonance energy transfer (FRET). The triple mutants contained both L12W and N51C labeled with 1,5-IAEDANS, and either L29Q or G159D. Both mutations had minimal effects on the equilibrium distance between Trp-12 and Cys-51-AEDANS in the absence or presence of bound Ca2+. L29Q had no effect on the closing rate of the N-domain triggered by release of Ca2+, but reduced the Ca2+-induced opening rate. G159D reduced both the closing and opening rates. Previous results showed that the closing rate of cTnC N-domain triggered by Ca2+ dissociation was substantially enhanced by PKA phosphorylation of cTnI. This rate enhancement was abolished by L29Q or G159D. These mutations alter the kinetics of structural transitions in the regulatory N-domain of cTnC that are involved in either activation (L29Q) or deactivation (G159D). Both mutations appear to be antagonistic toward phosphorylation signaling between cTnI and cTnC. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.