Mutations in repeating structural motifs of tropomyosin cause gain of function in skeletal muscle myopathy patients

Abstract

The congenital myopathies include a wide spectrum of clinically, histologically and genetically variable neuromuscular disorders many of which are caused by mutations in genes for sarcomeric proteins. Some congenital myopathy patients have a hypercontractile phenotype. Recent functional studies demonstrated that ACTA1 K326N and TPM2 ΔK7 mutations were associated with hypercontractility that could be explained by increased myofibrillar Ca(2+) sensitivity. A recent structure of the complex of actin and tropomyosin in the relaxed state showed that both these mutations are located in the actin-tropomyosin interface. Tropomyosin is an elongated molecule with a 7-fold repeated motif of around 40 amino acids corresponding to the 7 actin monomers it interacts with. Actin binds to tropomyosin electrostatically at two points, through Asp25 and through a cluster of amino acids that includes Lys326, mutated in the gain-of-function mutation. Asp25 interacts with tropomyosin K6, next to K7 that was mutated in the other gain-of-function mutation. We identified four tropomyosin motifs interacting with Asp25 (K6-K7, K48-K49, R90-R91 and R167-K168) and three E-E/D-K/R motifs interacting with Lys326 (E139, E181 and E218), and we predicted that the known skeletal myopathy mutations ΔK7, ΔK49, R91G, ΔE139, K168E and E181K would cause a gain of function. Tests by an in vitro motility assay confirmed that these mutations increased Ca(2+) sensitivity, while mutations not in these motifs (R167H, R244G) decreased Ca(2+) sensitivity. The work reported here explains the molecular mechanism for 6 out of 49 known disease-causing mutations in the TPM2 and TPM3 genes, derived from structural data of the actin-tropomyosin interface.

Aberrant movement of β-tropomyosin associated with congenital myopathy causes defective response of myosin heads and actin during the ATPase cycle

“…2b). As in our experiments the flexibility of TM was estimated in the presence of F-actin, this discrepancy can result from the conformational changes in the mutant TMs induced by F-actin binding which through electrostatic bonds between these proteins [73,79] can alter the flexibility of TM strands [23]. The increase in the flexibility of F-actin-E41K TM complex can be explained by a change in the nature of electrostatic bonds between F-actin and TM.…”

“…Thus, thin filaments reconstituted with R91G and E139del mutant TM species show an increased Ca 2+ sensitivity and a slightly higher maximum sliding speed in an in vitro motility assay [37,79]. Conversely, the E41K mutation is associated with a hypocontractile phenotype that is manifested both clinically (hypotonia and muscle weakness in patients [34]) and biochemically (a reduced Ca 2+ sensitivity and cross-bridge cycling rate in vitro [35]).…”

“…(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) [79] may result from the movement of TM strands towards the inner and outer actin domain, respectively.…”

Investigating the effects of tropomyosin mutations on its flexibility and interactions with filamentous actin using molecular dynamics simulation

“…Extensive structure-function analyses have been conducted for Tpm mutations that are linked to hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) (Marston 2011), and skeletal muscle myopathy (Marston et al 2013;Marttila et al 2012;Yuen et al 2015). As revealed by electron microscopy (EM) studies of Tpmdecorated F-actin, Tpm adopts three average azimuthal positions (i.e.…”

“…Previous MD simulation studies have provided rich structural and dynamic information for the shape and flexibility of Tpm Li et al 2010aLi et al , b, c, 2011Li et al , 2014Zheng et al 2013), for the effects of Tpm mutations Moore et al 2011), and for actin dynamics (Zheng et al 2007). In complement with the MD approach, an energy landscape method was recently used to probe how disease mutations perturb the interactions between Tpm and F-actin as rigid bodies (Marston et al 2013;Orzechowski et al 2014). …”