Molecular Mechanism of Novel Deletions in TPM3 that cause a Hypercontractile Phenotype with Congenital Muscle Stiffness

Abstract

Patients with ‘stiff child’ syndrome usually have mutations at the interface of actin and tropomyosin that could affect the equilibrium of the Ca2+-dependent switch of muscle. ACTA1 K326N was previously reported and we now report two de novo TPM3 (Tpm3.12 protein) mutations, ΔE218 and ΔE224, resulting in a significant hypercontractile phenotype with marked congenital muscle stiffness associated with ventilatory failure in one case.The atomic resolution structure of tropomyosin bound to actin in the ‘switched off’ state shows that tropomyosin makes contact with actin at only two points: one of which is a cluster of basic amino acids: actin K326, K328 and R147. We have demonstrated that two Tpm2.2 mutations, ΔE139 and E181K, and the actin K326N mutation destabilize this actin-tropomyosin interface. We predicted that equivalent charge loss mutations at Tpm3.12 EE 218-219, EE 224-224, or ED 257-258 would also destabilise the interaction with actin leading to a partial switch-on of the muscle. The two newly discovered stiff patient mutation are located at two of the three predicted gain of function sites.We used the quantitative in vitro motility assay and skeletal muscle thin filaments containing recombinant mutant Tpm3.12 expressed in a Baculovirus/sf9 system. ΔE218 led to a 2.5-fold increase in Ca2+-sensitivity (EC50 ratio ΔE218/WT = 0.40 ± 0.07, ). ΔE224 also showed an increase in Ca2+-sensitivity by 2.2-fold (EC50 ratio ΔE224/WT for = 0.46 ± 0.09). It has been previously shown that there was a 2.5 fold increase in Ca2+ sensitivity for ACTC K326N mutation (EC50 ratio K326N/WT = 0.4 ± 0.05, p=0.07).The increased Ca2+-sensitivity indicates that both mutations cause a gain of function that was predicted from the structural analysis and that can account for the stiff patient syndrome.