TY - JOUR T1 - Molecular mechanisms and structural features of cardiomyopathy-causing troponin T mutants in the tropomyosin overlap region. JF - Proc Natl Acad Sci U S A Y1 - 2017 A1 - Gangadharan, Binnu A1 - Sunitha, Margaret S A1 - Mukherjee, Souhrid A1 - Chowdhury, Ritu Roy A1 - Haque, Farah A1 - Sekar, Narendrakumar A1 - Sowdhamini, Ramanathan A1 - Spudich, James A A1 - Mercer, John A AB -

Point mutations in genes encoding sarcomeric proteins are the leading cause of inherited primary cardiomyopathies. Among them are mutations in the gene that encodes cardiac troponin T (TnT). These mutations are clustered in the tropomyosin (Tm) binding region of TnT, TNT1 (residues 80-180). To understand the mechanistic changes caused by pathogenic mutations in the TNT1 region, six hypertrophic cardiomyopathy (HCM) and two dilated cardiomyopathy (DCM) mutants were studied by biochemical approaches. Binding assays in the absence and presence of actin revealed changes in the affinity of some, but not all, TnT mutants for Tm relative to WT TnT. HCM mutants were hypersensitive and DCM mutants were hyposensitive to Ca in regulated actomyosin ATPase activities. To gain better insight into the disease mechanism, we modeled the structure of TNT1 and its interactions with Tm. The stability predictions made by the model correlated well with the affinity changes observed in vitro of TnT mutants for Tm. The changes in Ca sensitivity showed a strong correlation with the changes in binding affinity. We suggest the primary reason by which these mutations between residues 92 and 144 cause cardiomyopathy is by changing the affinity of TnT for Tm within the TNT1 region.

VL - 114 IS - 42 ER - TY - JOUR T1 - Mechanistic heterogeneity in contractile properties of α-tropomyosin (TPM1) mutants associated with inherited cardiomyopathies. JF - J Biol Chem Y1 - 2015 A1 - Gupte, Tejas M A1 - Haque, Farah A1 - Gangadharan, Binnu A1 - Sunitha, Margaret S A1 - Mukherjee, Souhrid A1 - Anandhan, Swetha A1 - Rani, Deepa Selvi A1 - Mukundan, Namita A1 - Jambekar, Amruta A1 - Thangaraj, Kumarasamy A1 - Sowdhamini, Ramanathan A1 - Sommese, Ruth F A1 - Nag, Suman A1 - Spudich, James A A1 - Mercer, John A KW - Actins KW - Adenosine Triphosphatases KW - Calcium KW - Cardiomyopathies KW - Humans KW - Models, Molecular KW - Myosins KW - Point Mutation KW - Protein Stability KW - Tropomyosin AB -

The most frequent known causes of primary cardiomyopathies are mutations in the genes encoding sarcomeric proteins. Among those are 30 single-residue mutations in TPM1, the gene encoding α-tropomyosin. We examined seven mutant tropomyosins, E62Q, D84N, I172T, L185R, S215L, D230N, and M281T, that were chosen based on their clinical severity and locations along the molecule. The goal of our study was to determine how the biochemical characteristics of each of these mutant proteins are altered, which in turn could provide a structural rationale for treatment of the cardiomyopathies they produce. Measurements of Ca(2+) sensitivity of human β-cardiac myosin ATPase activity are consistent with the hypothesis that hypertrophic cardiomyopathies are hypersensitive to Ca(2+) activation, and dilated cardiomyopathies are hyposensitive. We also report correlations between ATPase activity at maximum Ca(2+) concentrations and conformational changes in TnC measured using a fluorescent probe, which provide evidence that different substitutions perturb the structure of the regulatory complex in different ways. Moreover, we observed changes in protein stability and protein-protein interactions in these mutants. Our results suggest multiple mechanistic pathways to hypertrophic and dilated cardiomyopathies. Finally, we examined a computationally designed mutant, E181K, that is hypersensitive, confirming predictions derived from in silico structural analysis.

VL - 290 IS - 11 ER -