Dilated cardiomyopathy G159D mutation in cardiac troponin C weakens the anchoring interaction with troponin I

NMR spectroscopy has been employed to elucidate the molecular consequences of the DCM G159D mutation on the structure and dynamics of troponin C, and its interaction with troponin I (TnI). Since the molecular effects of human mutations are often subtle, all NMR experiments were conducted as direct side-by-side comparisons of the wild-type C-domain of troponin C (cCTnC) and the mutant protein, G159D. With the mutation, the affinity toward the anchoring region of cTnI (cTnI(34-71)) was reduced (K(D) = 3.0 +/- 0.6 mu M) compared to that of the wild type (K(D) < 1 mu M). Overall, the structure and dynamics of the G159D center dot CTnI(34-71) complex were very similar to those of the cCTnC center dot cTnI(34-71) complex. There were, however, significant changes in the (1)H, (13)C, and (15)N NMR chemical shifts, especially for the residues in direct contact with cTnI(34-71), and the changes in NOE connectivity patterns between the G159D center dot cTnI(34-71) and cCTnC center dot cTnI(34-71) complexes. Thus, the most parsimonious hypothesis is that the development of disease results from the poor anchoring of cTnI to cCTnC, with the resulting increase in the level of acto-myosin inhibition in agreement with physiological data. Another possibility is that long-range electrostatic interactions affect the binding of the inhibitory and switch regions of cTnI (cTnI(128-147) and cTnI(147-163)) and/or the cardiac specific N-terminus of cTnI (cTnI(1-29)) to the N-domain of cTnC. These important interactions are all spatially close in the X-ray structure of the cardiac TnC core.