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The impact of two switch II mutations, G440A and E442A, on the conformation of the nucleotide binding pocket and actin binding cleft were examined with temperature dependent FRET analysis of FlAsH labeled myosin V (MV FlAsH). E442A MV FlAsH, which abrogates the salt bridge between switch I and switch II, remains in a closed nucleotide binding pocket state at all temperatures between 4 and 35°C in the presence of ATP indicating a highly stable closed pocket similar to wild-type MV FlAsH. The G440A MV mutant prevents the formation of a highly conserved hydrogen bond to the gamma-phosphate of ATP, and is able to form a closed pocket conformation at 25°C similar to E442A and WT MV. In the presence of ATP,G440A MV FlAsH populates a closed cleft conformation while E442A MV FlAsH forms an open cleft conformation. Our results suggest the switch II region is not critical for formation of the closed nucleotide binding pocket conformation while it is critical for communicating the conformational changes from the nucleotide binding region to the actin binding cleft. These results are supported by essential dynamic analyses using FIRST/FRODA applied to the myosin V crystal structures. To compare our FRET analysis to the thermal unfolding profile of myosin V we examined alpha-helical content by circular dichroism (CD) spectrometry as a function of temperature. We observed a broad transition at lower temperatures and a steep transition at higher temperatures in WT MV FlAsH. Comparing our FRET results with CD will allow us to determine if the conformational changes are associated with changes in secondary structure. Our results are interpreted in the context of identifying communication pathways essential to the energy transduction pathway of myosin motors.

Previously we have demonstrated in fluorescence resonance energy transfer (FRET) studies that mant labeled nucleotides and IAEDANS actin can act as good donor probes for a FlAsH labeled acceptor site in the upper 50 kDa domain of myosin V. We examined the temperature dependence of the FRET signal between mantADP and MV WT FlAsH in the presence and absence of actin. We found that at low temperature (4-15°C) a high FRET state dominates (closed pocket) while at high temperature (30-35°C) a low FRET state dominates (open pocket). This transition is reversible suggesting a temperature-dependent conformational change. However, the mutant E442A, which is incapable of hydrolyzing ATP, remains in a high FRET state (closed pocket) with mantATP bound in the presence or absence of actin. Our results suggest a more flexible conformation of myosin in the presence of ADP compared to ATP which allows myosin to populate two actomyosin.ADP state conformations. These results are supported by the lifetime FRET analysis, and by computational FIRST/FRODA analysis of the intrinsic flexibility found in different x-ray crystal structures. We also plan to explore the temperature dependent conformational dynamics of the actin binding cleft using the IAEDANS actin (donor) and MV FlAsH (acceptor) pair in the presence of ATP, ADP, and absence of nucleotide using steady state and lifetime based FRET measurements. Our results will provide critical insights into the mechanocoupling that may occur between the nucleotide-binding pocket and actin binding cleft in myosin motors.

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.

Kinetic and structural studies of both muscle and non-muscle myosins have revealed that the enzymatic cycle of these motors frequently contains more than one actomyosin ADP state. Interestingly, the rate of ADP release in myosin motors is thought to be the main determinant of sliding velocity in muscle, suggesting strain dependent ADP release may be a critical mechanism of mechanochemical coupling. Our previous work has demonstrated that labeling myosin V in the upper 50 kDa domain with the biarsenical dye FlAsH (MV FlAsH) can serve as an acceptor for fluorescence resonance energy transfer studies with mant labeled nucleotides. We also determined that this donor-acceptor pair likely monitors opening/closing of the nucleotide binding pocket. Currently, we utilized the FRET signal to examine the kinetics of nucleotide binding pocket opening during the process of mantADP release from acto-MV FlAsH. We obtained evidence that the nucleotide binding pocket goes from a closed to an open conformation prior to the release of ADP. We also explored the temperature dependence of the closed to open transition and nucleotide release steps. We find that at lower temperatures the closed conformation is favored while at higher temperature the open conformation is favored. The more rapid ADP release step which follows nucleotide binding pocket opening is also temperature dependent. Therefore, since both steps are temperature-dependent they likely require significant conformational changes. We also compared our FRET results to the rate of ATP-induced dissociation from actin in the presence of ADP monitored by light scatter. Understanding how strain alters either of these two steps may be critical for elucidating the structural mechanism of strain-dependent ADP release in myosins.