Congenital myopathy‐related mutations in tropomyosin disrupt regulatory function through altered actin affinity and tropomodulin binding

Tropomyosin (Tpm) binds along actin filaments and regulates myosin binding to control muscle contraction. Tropomodulin binds to the pointed end of a filament and regulates actin dynamics, which maintains the length of a thin filament. To define the structural determinants of these Tpm functions, we examined the effects of two congenital myopathy mutations, A4V and R91C, in the Tpm gene, TPM3, which encodes the Tpm3.12 isoform, specific for slow‐twitch muscle fibers. Mutation A4V is located in the tropomodulin‐binding, N‐terminal region of Tpm3.12. R91C is located in the actin‐binding period 3 and directly interacts with actin. The A4V and R91C mutations resulted in a 2.5‐fold reduced affinity of Tpm3.12 homodimers for F‐actin in the absence and presence of troponin, and a two‐fold decrease in actomyosin ATPase activation in the presence of Ca2+. Actomyosin ATPase inhibition in the absence of Ca2+ was not affected. The Ca2+ sensitivity of ATPase activity was decreased by R91C, but not by A4V. In vitro, R91C altered the ability of tropomodulin 1 (Tmod1) to inhibit actin polymerization at the pointed end of the filaments, which correlated with the reduced affinity of Tpm3.12‐R91C for Tmod1. Molecular dynamics simulations of Tpm3.12 in complex with F‐actin suggested that both mutations reduce the affinity of Tpm3.12 for F‐actin binding by perturbing the van der Waals energy, which may be attributable to two different molecular mechanisms—a reduced flexibility of Tpm3.12‐R91C and an increased flexibility of Tpm3.12‐A4V. In muscle, tropomyosin (Tpm) binds to thin filaments to control actin interactions with myosin. At the pointed end, Tpm increases tropomodulin‐dependent inhibition of actin elongation. Myopathy‐causing substitutions A4V and R91C alter Tpm3.12 flexibility. Both mutations reduce activation of actin–myosin interactions. R91C decreases inhibition of the pointed end elongation by tropomodulin 1.