Skeletal Muscle Myopathy Mutations in Tropomyosin Gene TPM3 affect Thin Filament Transitions between the Inactive and Active States

Abstract

It has recently been discovered that nemaline myopathy and cap myopathy can be caused by mutations in β-tropomyosin (TPM2 gene) and that a third skeletal muscle myopathy, congenital fibre-type disproportion, is predominantly due to mutations in slow α-tropomyosin (TPM3 gene). We have expressed tropomyosin TPM3 mutations of residues R90, R167 and R244 in a baculovirus-Sf9 insect cells system and studied reconstituted actin-tropomyiosin mutant filaments biochemical properties. We analysed the effects of these TPM3 mutations on the biochemical properties of tropomyosin namely secondary structure, actin binding and the equilibrium constants of transitions between the ‘open’, ‘closed’ and ‘blocked’ states of thin filament and the cooperativity of these transitions. We also assessed their functional effects on the actomyosin ATPase. We found that TPM3 mutations did not affect secondary structure of tropomyosin and its interaction with actin. In contrast these mutations affected troponin-tropomyosin inhibition and activation of actomyosin ATPase. TPM3 mutations also affected the Blocked-closed transitions in the cooperative allosteric mechanism of regulation of the actomyosin complex. These findings are in agreement with recent structural studies of tropomyosin bound to actin in the closed and blocked conformations. We propose that destabilisation of the actin-tropomyosin interface by charge change in the blocked state may represent the underlying biochemical defect for the onset of skeletal muscle myopathies linked to these mutations.