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

Abstract

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.

Thin filament dysfunctions caused by mutations in tropomyosin Tpm3.12 and Tpm1.1

“…Modeling of the full length Tpm3.12 by molecular dynamics did not detect significant differences in flexibility of the N-terminal segment between wild type and the Ala4Val mutant. Hence interactions with Tmod1 seem not to be disturbed (Moraczewska et al 2019).…”

“…Met8 is located in the core of the coiled coil (a position of the heptapeptide repeat); therefore substitution with Arg greatly destabilizes the structure of the N-terminus , which distorts the interactions with Tmod. In contrast, the substitution Ala4Val in Tpm3.12, which is associated with CFTD (Marttila et al 2014b), does not interfere with binding of full length Tmod1 and only mildly reduces Tmod1's ability to inhibit the thin filament elongation at the pointed end (Moraczewska et al 2019). Ala4 is at the c position of the heptapeptide repeat, and therefore it is located outside the core of the coiled coil.…”

“…In contrast, decreased acto-myosin interactions underlie hypocontractile phenotypes associated with substitutions in Tpm1.1 and Tpm3.12, causing DCM and congenital myopathies. Various in vitro assays as well as fiber experiments show that this phenotype manifests itself through reduced activation of actin-myosin ATPase with preserved inhibitory function, decreased Ca-sensitivity, and reduced motility of the thin filaments (Farman et al 2018;Gupte et al 2015;Ly et al 2019;Marston et al 2013;Moraczewska et al 2019;Robaszkiewicz et al 2012;Yuen et al 2015). However, hypocontraction cannot be directly linked to stabilization of the inhibitory state, because the mutations do not show a regular pattern in terms of changes in actin affinity.…”

“…However, Arg91 is a conserved residue which interacts electrostatically with actin (Li et al 2011). Molecular dynamics simulations predicted that charge reduction in this site can increase the distance between Tpm3.12 and the axis of the filament (Moraczewska et al 2019). Such a shift may distort the interface between Tpm3.12 and Tmod1, leading to reduced binding.…”

Looking for Targets to Restore the Contractile Function in Congenital Myopathy Caused by Gln147Pro Tropomyosin

“…Even local changes in Tpm structure are able to change the pattern of the protein-protein interaction in the complex of Actin‒Myosin‒Tpm‒Troponin and to disrupt the precise mechanisms of the actin-myosin motor functioning [ 32 , 37 , 38 , 39 , 40 ]. In addition to the disturbance of the actin‒Tpm‒troponin relationship, a change in the functioning of some other proteins that interplays with Tpm and actin, in particular, tropomodulin, nebulin, leiomodin, cofilin may also occur [ 41 , 42 , 43 , 44 , 45 ]. A serious consequence of this disruption may be muscle weakness and the development of myopathy in human and animals.…”