Magnesium-Dependence and Active Site Dynamics in Myosin V and Myosin II

Abstract

We examined the magnesium-dependence of heavy meromyosin V (MV HMM) and two class II muscle myosins, skeletal muscle myosin (SK HMM) and smooth muscle myosin (SM HMM), in the in vitro motility and solution actin-activated ATPase assays. We find that all three myosins are inhibited in a Mg2+-dependent manner (0.5-10 mM MgCl2) in both ATPase and motility assays, under conditions in which the ionic strength was held constant. Interestingly, MV was more steeply dependent on magnesium than both SK and SM. In addition, actin filament breaking in the motility assay was two-fold faster with MV HMM at high (10 mM) compared to low (1 mM) MgCl2 conditions. To begin to understand how the active site of myosin V is different from myosin II we examined the time resolved fluorescence from dmantADP bound to myosin V 1IQ (MV 1IQ) or Dicty myosin II S1 (Dicty S1) in the presence of 1 mM MgCl2. We found that dmantADP bound to acto-MV 1IQ contained two lifetime components (8 and 3 ns). Time-resolved anisotropy studies of the two lifetime components in myosin V reveal that the long lifetime component was more immobilized (correlation time = 12 ns) while the short lifetime component was highly dynamic (correlation time = 0.4 ns). Interestingly, Dicty S1 contained a single lifetime component (8 ns). The two conformations of the myosin V active site may allow Mg2+ to more efficiently bind and reduce key steps in the ATPase cycle such as ADP release. Overall, our results suggest that differences in the structural dynamics of the active site of myosins may play a role in their dependence on free Mg2+, which could explain why Mg2+ differentially alters the motile and force generating properties of myosins.