Structural and Functional Effects of Cardiomyopathy-Causing Mutations in the Troponin T‑Binding Region of Cardiac Tropomyosin Alexander M. Matyushenko, †,‡ Daniil V. Shchepkin, § Galina V. Kopylova, § Katerina E. Popruga, †,‡ Natalya V. Artemova, † Anastasia V. Pivovarova, † Sergey Y. Bershitsky, § and Dmitrii I. Levitsky* ,†,∥ † A. N. Bach Institute of Biochemistry, Research Center of Biotechnology, Russian Academy of Sciences, Leninsky Prospect 33, Moscow 119071, Russian Federation ‡ Department of Biochemistry, School of Biology, Moscow State University, Lenin Hills 1, bld 12, Moscow 119234, Russian Federation § Institute of Immunology and Physiology, Russian Academy of Sciences, Pervomayskaya Street 106, Yekaterinburg 620049, Russian Federation ∥ A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Lenin Hills 1, bld 40, Moscow 119234, Russian Federation ABSTRACT: Hypertrophic cardiomyopathy (HCM) is a severe heart disease caused by missense mutations in genes encoding sarcomeric proteins of cardiac muscle. Many of these mutations are identified in the gene encoding the cardiac isoform of tropomyosin (Tpm), an α-helical coiled-coil actin-binding protein that plays a key role in Ca 2+ -regulated contraction of cardiac muscle. We employed various methods to characterize structural and functional features of recombinant human Tpm species carrying HCM mutations that lie either within the troponin T-binding region in the C-terminal part of Tpm (E180G, E180V, and L185R) or near this region (I172T). The results of our structural studies show that all these mutations affect, although differently, the thermal stability of the C-terminal part of the Tpm molecule: mutations E180G and I172T destabilize this part of the molecule, whereas mutation E180V strongly stabilizes it. Moreover, various HCM-causing mutations have different and even opposite effects on the stability of the Tpm−actin complexes. Studies of reconstituted thin filaments in the in vitro motility assay have shown that those HCM-associated mutations that lie within the troponin T-binding region of Tpm similarly increase the Ca 2+ sensitivity of the sliding velocity of the filaments and impair their relaxation properties, causing a marked increase in the sliding velocity in the absence of Ca 2+ , while mutation I172T decreases the Ca 2+ sensitivity and has no influence on the sliding velocity under relaxing conditions. Finally, our data demonstrate that various HCM mutations can differently affect the structural and functional properties of Tpm and cause HCM by different molecular mechanisms. I nherited cardiomyopathies are severe heart diseases in all age groups. Clinical consequences of these diseases are very diverse and can vary from relatively mild hypertrophy and hypertension to severe hypertrophy leading to complete heart dysfunction and sudden death. Both hypertrophic cardiomy- opathy (HCM) and dilated cardiomyopathy (DCM) are mainly caused by autosomal dominant inheritance of missense mutations in genes encoding sarcomeric proteins of cardiac muscle. HCM is characterized by an abnormally thickened left ventricular wall and an abnormally thickened interventricular septum and diastolic dysfunction, and DCM is characterized by a dilated left ventricle and systolic dysfunction. 1 Cardiomyo- pathic mutations have been found in all genes encoding the proteins of the thin filament in the sarcomere (actin, tropomyosin, troponin I, troponin C, and troponin T). Among them, numerous mutations have been identified in the TPM1 gene encoding the cardiac isoform of tropomyosin (Tpm), Tpm1.1 or α-Tpm. From 11 to 17 HCM-causing mutations and from 4 to 11 mutations associated with DCM have been identified in Tpm1.1. 2,3 Tpm is an actin-binding protein that forms a ropelike structure along the entire length of the actin filament and plays, together with the troponin (Tn) complex, a key role in Ca 2+ - regulated contraction of striated muscles. 4−6 According to recent views, Tpm serves as a “gatekeeper” for actin−myosin interaction. 7 In the absence of Ca 2+ , it sterically blocks the myosin-binding sites on actin, and Ca 2+ binding to Tn during muscle activation leads Tpm to move away from the blocking (B) position and allows binding of myosin heads to actin. 5,6,8 In terms of structure, the Tpm molecule is a typical α-helical coiled-coil dimer whose amino acid sequence contains a heptad repeat (a-b-c-d-e-f-g) in which residues at positions a and d are Received: September 29, 2016 Revised: December 1, 2016 Published: December 8, 2016 Article pubs.acs.org/biochemistry © XXXX American Chemical Society A DOI: 10.1021/acs.biochem.6b00994 Biochemistry XXXX, XXX, XXX−XXX