C.P.17 A repeating structural motif in tropomyosin that is responsible for multiple gain-of-function skeletal myopathy mutations

Abstract

It was recently shown that the beta-tropomyosin ΔK7 mutation caused a gain of function, detected by increased Ca2+-sensitivity in single filament assays and increased Ca2+-sensitivity and maximium force in skinned fibres. Lysine 7 is adjacent to lysine 6 that has been shown to interact with actin Asp25 in the structure of actin-tropomyosin when tropomyosin is in the closed (inactive) state. We hypothesised that the mutation one amino acid from the amino acid that interacts with actin destabilises the closed state and therefore increase the probability of the muscle being in the open (active) state. Tropomyosin has a 7-fold repeated motif of about 40 amino acids that corresponds to the 7 actins that one tropomyosin molecule is in contact with. The contact with actin Asp 25 is repeated at K6, K48, R90, K128, R167, K205 and R244. We therefore asked whether further gain of function mutations might be associated with the other interaction sites. ΔK49 in beta-tropomyosin (second repeat) is reported to be a cap myopathy-causing mutation. We found that in vitro, this mutation increased myofilament Ca2+-sensitivity like ΔK7. R91G in beta-tropomyosin (third period) is a well document mutation associated with distal arthrogryposis that also increases Ca2+-sensitivity. K168E (fifth period) and R245G (sixth period) in gamma-tropomyosin have been reported associated with congenital fibre-type disproportion, but have not been studied in vitro. In summary, disease-related mutations resulting in the loss of positive charge in the amino acid immediately downstream of the basic amino acid that interacts with Asp25 have been noted in 5 of the 7 repeating motifs in tropomyosin. Three of these have been investigated and show an increased Ca2+-sensitivity and the remainder have not yet been tested. We propose that the repeating motif demonstrates one molecular mechanism for gain of function mutations.