Myosin filaments from many muscles are activated by phosphorylation of their regulatory light chains (RLCs). To elucidate the structural mechanism of activation, we have studied RLC phosphorylation in tarantula thick filaments, whose high resolution structure is known. In the relaxed state, tarantula RLCs are ~50% non- and 50% mono-phosphorylated, while on activation mono-phosphorylation increases and some RLCs become bi-phosphorylated. Mass spectrometry shows that relaxed-state mono-phosphorylation occurs on Ser35 while Ca2+-activated phosphorylation is on Ser45, both located near the RLC N-terminus. The sequences around these serines suggest they are the targets for protein kinase C (PKC) and myosin light chain kinase (MLCK) respectively. The atomic model of the tarantula filament shows that the two myosin heads (“free” and “blocked”) are in different environments, with only the free head serines readily accessible to kinases. Thus PKC Ser35 mono-phosphorylation in relaxed filaments would occur only on the free heads. Structural considerations suggest these heads are less strongly bound to the filament backbone, and may oscillate occasionally between attached and detached states (“swaying” heads). These heads would be available for immediate actin interaction upon Ca2+-activation of the thin filaments. Once MLCK becomes activated, it phosphorylates free heads on Ser45. These heads become fully mobile, exposing blocked-head Ser45 to MLCK. This would release the blocked-heads, allowing their interaction with actin. On this model, twitch force would be produced by rapid interaction of swaying free heads with activated thin filaments, while prolonged exposure to Ca2+ on tetanus would recruit new, MLCK-activated heads, resulting in force potentiation.
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