Abstract
Hypertrophic cardiomyopathy (HCM) is one of the most common cardiomyopathies and a major cause of sudden death in young athletes. The Ca2+ sensor of the sarcomere, cardiac troponin C (cTnC), plays an important role in regulating muscle contraction. Although several cardiomyopathy-causing mutations have been identified in cTnC, the limited information about their structural defects has been mapped to the HCM phenotype. Here, we used high-resolution electron-spray ionization mass spectrometry (ESI-MS), Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG-RD), and affinity measurements of cTnC for the thin filament in reconstituted papillary muscles to provide evidence of an allosteric mechanism in mutant cTnC that may play a role to the HCM phenotype. We showed that the D145E mutation leads to altered dynamics on a μs-ms time scale and deactivates both of the divalent cation-binding sites of the cTnC C-domain. CPMG-RD captured a low populated protein-folding conformation triggered by the Glu-145 replacement of Asp. Paradoxically, although D145E C-domain was unable to bind Ca2+, these changes along its backbone allowed it to attach more firmly to thin filaments than the wild-type isoform, providing evidence for an allosteric response of the Ca2+-binding site II in the N-domain. Our findings explain how the effects of an HCM mutation in the C-domain reflect up into the N-domain to cause an increase of Ca2+ affinity in site II, thus opening up new insights into the HCM phenotype.
Highlights
Hypertrophic cardiomyopathy (HCM)6 is the most common familial heart disorder
Based on electron-spray ionization mass spectrometry (ESI-MS) and nuclear spin relaxation data, the results suggest that D145E impairs both of the Ca2ϩ-binding sites of the cardiac troponin C (cTnC) C-domain: site IV, due to a direct effect of the mutation at the coordination sphere, and site
TNNC1 is included in routine genetic screening for HCM mutations [15], but up to this point there has appeared very little structural information showing how these mutations affect the binding affinity of cTnC for Ca2ϩ and for its protein partners on the thin filament
Summary
Hypertrophic cardiomyopathy (HCM) is the most common familial heart disorder. Macroscopic features include myocardial disarray and left ventricle thickening accompanied by significant calcium imbalance at a molecular level [1, 2]. New mutations in TNNC1 (A8V, A31S, and D145E), the gene that codifies cardiac troponin C (cTnC), have been observed in patients with HCM [4, 5]. Cardiac troponin C, a subunit of the troponin complex (Tn), is a key player in regulating muscle contraction at the level of the thin filament [6, 7] and belongs to the EF-hand family of Ca2ϩ-binding proteins. The binding of Ca2ϩ to this site leads to the exposure of hydrophobic patches and a slight opening that favors interaction with troponin I [7]. HCM-causing mutations can lead to changes in the cTnC three-dimensional structure and tend to impair its ability to regulate the Ca2ϩ binding process and interact with biological partners on the thin filament [10].
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