Darshan V. Trivedi

Switch II Mutants Reveal Coupling between the Nucleotide- and Actin-Binding Regions in Myosin V

Conserved active-site elements in myosins and other P-loop NTPases play critical roles in nucleotide binding and hydrolysis; however, the mechanisms of allosteric communication among these mechanoenzymes remain unresolved. In this work we introduced the E442A mutation, which abrogates a salt-bridge between switch I and switch II, and the G440A mutation, which abolishes a main-chain hydrogen bond associated with the interaction of switch II with the γ phosphate of ATP, into myosin V. We used fluorescence resonance energy transfer between mant-labeled nucleotides or IAEDANS-labeled actin and FlAsH-labeled myosin V to examine the conformation of the nucleotide- and actin-binding regions, respectively. We demonstrate that in the absence of actin, both the G440A and E442A mutants bind ATP with similar affinity and result in only minor alterations in the conformation of the nucleotide-binding pocket (NBP). In the presence of ADP and actin, both switch II mutants disrupt the formation of a closed NBP actomyosin.ADP state. The G440A mutant also prevents ATP-induced opening of the actin-binding cleft. Our results indicate that the switch II region is critical for stabilizing the closed NBP conformation in the presence of actin, and is essential for communication between the active site and actin-binding region.

Kinetics and thermodynamics of the rate-limiting conformational change in the actomyosin V mechanochemical cycle.

We used FRET to examine the kinetics and thermodynamics of structural changes associated with ADP release in myosin V, which is thought to be a strain-sensitive step in many muscle and non-muscle myosins. We also explored essential dynamics using FIRST/FRODA starting with three different myosin V X-ray crystal structures to examine intrinsic flexibility and correlated motions. Our steady-state and time-resolved FRET analysis demonstrates a temperature-dependent reversible conformational change in the nucleotide-binding pocket (NBP). Our kinetic results demonstrate that the NBP goes from a closed to an open conformation prior to the release of ADP, while the actin-binding cleft remains closed. Interestingly, we find that the temperature dependence of the maximum actin-activated myosin V ATPase rate is similar to the pocket opening step, demonstrating that this is the rate-limiting structural transition in the ATPase cycle. Thermodynamic analysis demonstrates that the transition from the open to closed NBP conformation is unfavorable because of a decrease in entropy. The intrinsic flexibility analysis is consistent with conformational entropy playing a role in this transition as the MV.ADP structure is highly flexible compared to the MV.APO structure. Our experimental and modeling studies support the conclusion of a novel post-power-stroke actomyosin.ADP state in which the NBP and actin-binding cleft are closed. The novel state may be important for strain sensitivity as the transition from the closed to open NBP conformation may be altered by lever arm position.

Direct measurements of the coordination of lever arm swing and the catalytic cycle in myosin V.

Significance Myosins interact with actin filaments and convert the chemical energy from ATP hydrolysis into mechanical work. The swinging lever arm hypothesis describes the molecular mechanism of actomyosin-based force generation that is essential for cell motility, muscle contraction, and cell division. In this model the light chain-binding region of myosin undergoes a major conformational change, which drives force generation. However, the temporal kinetics of structural changes in the lever arm in relation to the product release steps of the catalytic cycle are not well established. By using a FRET-based strategy, we demonstrate the lever arm swing occurs in two steps, a rapid step prior to phosphate release and a slower step prior to ADP release. Myosins use a conserved structural mechanism to convert the energy from ATP hydrolysis into a large swing of the force-generating lever arm. The precise timing of the lever arm movement with respect to the steps in the actomyosin ATPase cycle has not been determined. We have developed a FRET system in myosin V that uses three donor–acceptor pairs to examine the kinetics of lever arm swing during the recovery and power stroke phases of the ATPase cycle. During the recovery stroke the lever arm swing is tightly coupled to priming the active site for ATP hydrolysis. The lever arm swing during the power stroke occurs in two steps, a fast step that occurs before phosphate release and a slow step that occurs before ADP release. Time-resolved FRET demonstrates a 20-Å change in distance between the pre- and postpower stroke states and shows that the lever arm is more dynamic in the postpower stroke state. Our results suggest myosin binding to actin in the ADP.Pi complex triggers a rapid power stroke that gates the release of phosphate, whereas a second slower power stroke may be important for mediating strain sensitivity.

Magnesium modulates actin binding and ADP release in myosin motors

Background: Magnesium may be an important physiological regulator of myosin motor activity. Results: Mg2+ inhibits the ADP release rate constant in the subset of myosins examined and reduces actin affinity in the post-hydrolysis state in myosin V. Conclusion: Mg2+ alters contractile velocity without altering overall tension-generating capacity. Significance: Mg2+-dependent regulation of motor activity is conserved in myosin motors. We examined the magnesium dependence of five class II myosins, including fast skeletal muscle myosin, smooth muscle myosin, β-cardiac myosin (CMIIB), Dictyostelium myosin II (DdMII), and nonmuscle myosin IIA, as well as myosin V. We found that the myosins examined are inhibited in a Mg2+-dependent manner (0.3–9.0 mm free Mg2+) in both ATPase and motility assays, under conditions in which the ionic strength was held constant. We found that the ADP release rate constant is reduced by Mg2+ in myosin V, smooth muscle myosin, nonmuscle myosin IIA, CMIIB, and DdMII, although the ADP affinity is fairly insensitive to Mg2+ in fast skeletal muscle myosin, CMIIB, and DdMII. Single tryptophan probes in the switch I (Trp-239) and switch II (Trp-501) region of DdMII demonstrate these conserved regions of the active site are sensitive to Mg2+ coordination. Cardiac muscle fiber mechanic studies demonstrate cross-bridge attachment time is increased at higher Mg2+ concentrations, demonstrating that the ADP release rate constant is slowed by Mg2+ in the context of an activated muscle fiber. Direct measurements of phosphate release in myosin V demonstrate that Mg2+ reduces actin affinity in the M·ADP·Pi state, although it does not change the rate of phosphate release. Therefore, the Mg2+ inhibition of the actin-activated ATPase activity observed in class II myosins is likely the result of Mg2+-dependent alterations in actin binding. Overall, our results suggest that Mg2+ reduces the ADP release rate constant and rate of attachment to actin in both high and low duty ratio myosins.

Magnesium impacts myosin V motor activity by altering key conformational changes in the mechanochemical cycle.

We investigated how magnesium (Mg) impacts key conformational changes during the ADP binding/release steps in myosin V and how these alterations impact the actomyosin mechanochemical cycle. The conformation of the nucleotide binding pocket was examined with 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. We examined the maximum actin-activated ATPase activity of MV FlAsH at a range of free Mg concentrations (0.1-9 mM) and found that the highest activity occurs at low Mg (0.1-0.3 mM), while there is a 50-60% reduction in activity at high Mg (3-9 mM). The motor activity examined with the in vitro motility assay followed a similar Mg-dependence, and the trend was similar with dimeric myosin V. Transient kinetic FRET studies of mantdADP binding/release from actomyosin V FlAsH demonstrate that the transition between the weak and strong actomyosin.ADP states is coupled to movement of the upper 50 kDa domain and is dependent on Mg with the strong state stabilized by Mg. We find that the kinetics of the upper 50 kDa conformational change monitored by FRET correlates well with the ATPase and motility results over a wide range of Mg concentrations. Our results suggest the conformation of the upper 50 kDa domain is highly dynamic in the Mg free actomyosin.ADP state, which is in agreement with ADP binding being entropy driven in the absence of Mg. Overall, our results demonstrate that Mg is a key factor in coupling the nucleotide- and actin-binding regions. In addition, Mg concentrations in the physiological range can alter the structural transition that limits ADP dissociation from actomyosin V, which explains the impact of Mg on actin-activated ATPase activity and in vitro motility.

On the Molecular Basis of Monogenic Human Hypertrophic and Dilated Cardiomyopathies

After 40 years of developing and utilizing assays to understand the molecular basis of energy transduction by the myosin family of molecular motors, all members of my laboratory are now focused on understanding the underlying biochemical and biophysical bases of human hypertrophic (HCM) and dilated (DCM) cardiomyopathies. HCM and DCM are most often a result of single missense mutations in one of several sarcomeric proteins, the sarcomere being the fundamental contractile unit of the cardiomyocyte. Associated with HCM and DCM worldwide are heart failure, arrhythmias, and sudden cardiac death at any age. We are using in vitro molecular studies of biochemically reconstituted human sarcomeric protein complexes to lay the foundation for understanding the effects of HCM- and DCM-causing mutations on power generation by the contractile apparatus of the sarcomere. With such a detailed understanding at the molecular level, one should be able to exquisitely design and screen for appropriate small molecule therapies that are desperately needed for treatment of these diseases.

Kinetics and Thermodynamics of the Rate Limiting Conformational Change in the Myosin V Mechanochemical Cycle

We have used FRET to examine the kinetics and thermodynamics of the structural changes associated with ADP release in myosin V, which is thought to be a strain sensitive step in many muscle and non-muscle myosins. We also use essential dynamics using FIRST/FRODA starting with three different myosin V X-ray crystal structures to examine the intrinsic flexibility and correlated motions. Our kinetic and steady-state FRET results demonstrate that the nucleotide binding pocket goes from a closed to an open conformation prior to the release of ADP while the actin binding cleft remains closed. Thermodynamic analysis of ADP binding to actomyosin V suggests the collision complex formation is driven by a large enthalpy change and a small change in entropy. The transition from the open to closed pocket actomyosin.ADP state is associated with a large unfavorable decrease in entropy, which suggests the closed pocket conformation is more rigid than the open pocket conformation. Although no crystal structure is available of the closed pocket myosin V.ADP state, our FRET analysis reveals that this conformation may be similar to the myosin V.ATP state. FIRST/FRODA analysis is consistent with these conclusions as the myosin V.ADP structure is more flexible than the Apo structure, while the myosin V.ATP structure is more rigid than myosin V.ADP. Principal component analysis demonstrates that opening and closing of the nucleotide binding pocket correlates with the motions of loop 1 and the transducer region in all three crystal structures. Interestingly, we find that the temperature dependence of the maximum actin-activated myosin V ATPase rate correlates with the pocket opening step, suggesting this is the rate limiting step in the ATPase cycle. Our results provide insight into the structural mechanism of strain-dependent ADP release in myosins.

The HCM Loop Plays a Role in Actin-Activated Product Release in Myosin V

We examined the functional role of the upper 50 kDa hypertrophic cardiomyopathy (HCM) loop in myosin V. Hypertrophic cardiomyopathy is caused by missense mutations in highly conserved regions of myo2β and one deadly mutation occurs in the HCM loop (R403Q). Since the R403Q mutation has been shown to enhance or decrease the ATPase activity and in vitro motility of myosin II, it may be expected that the HCM loop plays a role in actin-activated product release. In our previous work we correlated the conformational change associated with ADP release and maximum ATPase rate in myosin V using FRET analysis. We engineered the R403Q mutation at an analogous site in myosin V 1IQ (R378Q) so that we would be able to investigate the impact of the mutation on actin-activated product release, maximum ATPase rate, in vitro motility, and FRET in myosin V. The R378Q mutation reduces the maximum ATPase rate two-fold while it slightly enhances sliding velocity compared to wild-type MV 1IQ. Our results suggest the duty ratio may be reduced as a result of the R378Q mutation. We will directly examine both ADP-release and phosphate-release to evaluate this possibility. To examine the impact of the point mutation on structural dynamics we will determine if conformational changes in the nucleotide-binding pocket and actin-binding cleft are disrupted using our established FRET probes. Our studies further establish a strategy for examining the mechanism of product release in myosin using the three assays: FRET, ATPase, and motility.

The Switch II Region is Critical for the Formation of the Open Cleft Weak Binding Conformation in Myosin V

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.