Magnesium Regulates Myosin V Motor Activity by Altering Key Conformational Changes in the Nucleotide Binding Pocket

Abstract

We investigated how magnesium impacts key conformational changes in the nucleotide binding pocket of myosin V and how these alterations impact the mechanochemical cycle. The conformation of the nucleotide binding pocket was examined using our established FRET system in which myosin V labeled with FlAsH in the upper 50 kDa domain participates in energy transfer with mant labeled nucleotides. Our previous work demonstrated the rate limiting conformation change in the actomyosin V ATPase cycle is opening of the nucleotide binding pocket which precedes ADP release from the open state. We examined the maximum actin-activated ATPase activity of MV FlAsH at a range of free magnesium concentrations (0-10 mM) and find that the highest activity occurs at 0.5 mM magnesium, while there is a 50-60% reduction in activity above 4 mM magnesium. We also demonstrate that the motor activity assayed by in vitro motility is similarly dependent on magnesium concentrations. Transient kinetic studies of mantADP binding/release with actomyosin V FlAsH demonstrate the equilibrium between the open and closed nucleotide binding pocket conformations is dependent on magnesium with the closed state stabilized by magnesium. We find that the kinetics of the nucleotide binding pocket opening step correlates well with the ATPase and motility results over a wide range of magnesium concentrations. In the absence of magnesium (presence of 4 mM EDTA) the nucleotide binding pocket populates a single conformation that is dramatically open at higher temperatures. In addition, magnesium significantly slows the rate of ADP release from the open state. Our results shed light on the structural mechanism of ADP release in myosin V and allow us to speculate about the conserved conformational pathways involved in strain sensitive ADP release in myosins.