The 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 (cTnI34−71) was reduced (K D = 3.0 ± 0.6 μM) compared to that of the wild type (K D < 1 μM). Overall, the structure and dynamics of the G159D·cTnI34−71 complex were very similar to those of the cCTnC·cTnI34−71 complex. There were, however, significant changes in the 1H, 13C, and 15N NMR chemical shifts, especially for the residues in direct contact with cTnI34−71, and the changes in NOE connectivity patterns between the G159D·cTnI34−71 and cCTnC·cTnI34−71 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 (cTnI128−147 and cTnI147−163) and/or the cardiac specific N-terminus of cTnI (cTnI1−29) to the N-domain of cTnC. These important interactions are all spatially close in the X-ray structure of the cardiac TnC core.