Phosphorylated Smooth Muscle Heavy Meromyosin Shows an Open Conformation: Implications for the Structure of Myosin with One Head Phosphorylated

Abstract

Smooth muscle myosin (smM) and heavy meromyosin (smHMM) are activated by regulatory light chain (RLC) phosphorylation but the mechanism remains unclear. Dephosphorylated, inactive smHMM assumes a closed conformation with asymmetric intramolecular head-head interactions involving motor domains and the essential light chain (ELC) [Wendt et al., PNAS 98: 4361 (2001)]. The “free head” can bind to actin, but the actin-binding interface of the “blocked head” is involved in interactions with the free head. We report here a 3-D structure for phosphorylated, active smHMM obtained using electron crystallography of 2-D arrays, and an atomic model obtained by fitting using normal mode flexible fitting. Head-head interactions of phosphorylated smHMM resemble those found in the dephosphorylated state, but occur between separate molecules. The interface between heads of phosphorylated smHMM is less extensive and somewhat altered in orientation compared with that of dephosphorylated smHMM. The light chain binding domain of phosphorylated and several dephosphorylated myosin structures show systematic differences. However, the major difference appears to be the relationship between the motor domain and the ELC in a phosphorylated head compared to that of the “blocked head” of dephosphorylated smHMM. We hypothesize that RLC phosphorylation disrupts the inhibited conformation primarily by its effect on the “blocked head” rather than the “free head”. Singly phosphorylated smHMM is not compatible with the closed conformation if the “blocked head” is phosphorylated. The implications of this observation for myosin activation at low levels of phosphorylation in smooth muscle will be discussed. Supported by grants from the NIAMS, NHLBI and NSF-MCB.