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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.

Myosins are proposed to utilize a conserved structural mechanism to generate force in which small conformational changes in the active site result in a large swing of the lever arm or light chain binding region. The converter domain is a flexible region that provides a link between the catalytic domain and lever arm and is proposed to play a critical role in the allosteric communication between these two domains. We introduced the R712G mutation in the converter domain and examined the impact of this mutation on the structural and functional properties of myosin V. The mutation resulted in a 16% reduction in the maximum actin-activated ATPase rate and no change in the actin concentration at which the ATPase activity is one-half maximal (KATPase). The sliding velocities examined in the in vitro motility assay were very similar between WT and R712G MV. We have developed a novel FRET system in myosin V (MV) that allows examination of the dynamics of lever arm motion. We labeled MV 11IQ containing an N-terminal (NT) tetracysteine motif with the bisarsenical dye FlAsH (MV.NT.FlAsH). The first IQ motif of MV.NT.FlAsH was exchanged with QSY-9 labeled CaM, a non-fluorescent acceptor. We followed the motion of the lever-arm during the ATP binding (recovery stroke) and actin-activated product release (power stroke) steps using stopped-flow FRET. The R712G mutation reduced the rate of the recovery stroke by 23% while having little impact on the fast power stroke that occurs prior to phosphate release. Thus, a mutation in the converter domain can specifically impact the recovery stroke without altering the power stroke demonstrating different allosteric mechanisms are responsible for these two key structural transitions.

Cardiac myosin interacts cyclically with actin in the presence of ATP in the sarcomere to drive heart contraction. Previous fiber studies suggested that the structure and function of myosin in the thick filaments are regulated by its own regulatory light chain (RLC) phosphorylation and through its interaction with myosin binding protein-C (MyBP-C). Interestingly, ∼80% of mutations that cause hypertrophic cardiomyopathy (HCM), a relatively common genetic cardiac disorder characterized by hypercontractility, have been found in the genes encoding human β-cardiac myosin and human cardiac MyBP-C. It has been hypothesized that the heads of the two-headed motor can adopt a sequestered structure where the heads interact asymmetrically with their own tail as Ser-15 residue of RLC remains dephosphorylated, and the N-terminal domains of MyBP-C binds to myosin to stabilize the structure. We designed two different two-headed human β-cardiac myosin motor constructs differing in the length of their S2 tail domains and co-expressed them with both of the human light chains to test the hypothesis that the number of myosin heads available for interaction with actin is regulated by phosphorylation of the RLC and MyBP-C. The shorter construct (2-hep HMM) is devoid of the tail region needed for the interaction of S2 with its S1 heads whereas the longer one (25-hep HMM) has the required length of S2 tail region to allow such interactions. We found that the maximal actin-activated ATPase rate for 2-hep HMM and the single-headed myosin construct S1 are essentially the same, and are only slightly changed by RLC phosphorylation. On the other hand, de-phosphorylated 25-hep HMM has a significantly lower maximal actin-activated ATPase than the 2-hep HMM, consistent with the former having fewer actin-accessible S1 heads than the latter. Additionally, binding experiments of 2-hep and 25-hep HMM with the N-terminal C0-C2 domain of human cardiac MyBP-C showed that the affinity of complex formation is weakened by phosphorylation of either RLC or MyBP-C.

The N-terminus is a highly variable region among the myosin superfamily and has been associated with alterations in rotation of the lever arm and step size. We examined the structural dynamics of the N-terminal region of myosin V using steady-state and stopped-flow FRET. We introduced a tetra-cysteine site at the extreme N-terminus of a Myosin V 1IQ construct and labeled it with a bisarsenical fluorescin derivative (FlAsH) (MV NT-FLAsH). Energy transfer between FlAsH and mant or deac labeled nucleotides (dmantATP, dmantADP and deacATP) was monitored. Steady-state FRET experiments with the mant-FlAsH pair demonstrated a high FRET state in the presence of dmantATP and a low FRET state in the presence of dmantADP. Sequential-mix, in which MV NT-FLAsH is first mixed with dmantATP, aged to allow formation of the M.ADP.Pi state, and then mixed with saturating actin allowed us to explore the actin-acitvated product release steps. Biphasic transients with rates corresponding to the fast and slow rate of dmantADP release were observed. The sequential mix experiments with deacATP yielded three phases; a fast, actin dependent, phosphate release phase followed by the two phases of ADP release. Our results suggest the FRET signal monitors a structural change in the N-terminus associated with the sequential release of products. A high FRET state upon binding of ATP is associated with formation of the pre-powerstroke state of the lever arm and a low or no FRET state in the presence of ADP suggests formation of the post-powerstroke state. Our results allow us to hypothesize that movement of the N-terminal domain follows the movement of the converter/lever arm. Further experiments will explore the dynamics of the N-terminal domain and examine how its motion correlates with the conformation of the nucleotide binding pocket and lever arm.

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.

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.

Our previous work has demonstrated that labeling myosin V in the upper 50 kDa domain with the biarsenical dye FlAsH can serve as an acceptor for fluorescence resonance energy transfer studies with mant labeled nucleotides and IAEDANS actin. These FRET studies suggest that myosin V can adopt a conformation in which the nucleotide binding pocket and the actin binding cleft are in a closed conformation. Our studies suggest the upper 50 kDa domain may be highly flexible in certain nucleotide-states which allows tight binding to nucleotide and actin. Molecular geometric simulations demonstrate the upper 50 kDa domain is most flexible in the myosin V.ADP state, consistent with this state having a high affinity for ADP and actin. Currently, we examined the temperature dependence of the FRET signal between mantADP and MV FlAsH. We found that at low temperature (4-15°C) a high FRET state dominates (closed pocket) while at high temperature (30-37°C) a low FRET state dominates (open pocket). This transition is reversible suggesting a temperature-dependent conformational change. We also found that FlAsH labeled G440A MV, a non-hydrolyzable mutant, has a similar temperature-dependent transition in the presence of mantATP. In contrast, the transition does not occur in the presence of mantADP.BeFx or with the non-hydrolyzable E44A MV mutant in the presence of mantATP. Our results suggest coordination of the gamma-phosphate of ATP rigidifies the upper 50 kDa domain which results in a weak actin affinity state (open actin binding cleft and closed nucleotide binding pocket). However, upon phosphate release the upper 50kDa domain becomes more flexible which allows myosin to adopt a conformation in which it has a high affinity for both nucleotide and actin (closed nucleotide binding pocket and actin binding cleft).

We examined the impact of temperature and free magnesium concentration on monomeric FlAsH labeled myosin V (MV FlAsH), dimeric myosin V (MV HMM), and dimeric fast skeletal muscle myosin II (SK HMM) using ATPase and motility assays. Our results indicate that MV HMM and SK HMM both have a linear dependence on temperature that is similar in both ATPase and motility assays. However, MV FlAsH contains a different temperature dependence in ATPase and motility assays suggesting its short lever arm may impart a high strain dependence in the motility assay. MV HMM and MV FlAsH are inhibited by high concentrations of magnesium in both ATPase and motility assays. The rate-limiting step in myosin V is known to be ADP release, which we demonstrate correlates well with the magnesium and temperature dependence of ATPase and motility assays. Interestingly, SK HMM exhibits magnesium inhibition in ATPase assays, but only a slight decrease is observed in the motility assay. In SK HMM the rate-limiting step in ATPase assays is thought to be attachment to actin or phosphate release, while in motility assays it is controversial. Our results indicate that SK HMM is better described by an attachment limited model in the motility assay. Magnesium may reduce the duty ratio of SK HMM which alters ATPase activity but not velocity in the motility assay. Future experiments will determine if magnesium alters the actin binding and/or the product release steps in myosin V and skeletal muscle myosin. Myosin V contains a tyrosine (residue 439) in the switch II region, which is an alanine at the corresponding position in myosin II, suggesting this residue may play a key role in differentially altering magnesium coordination in the active site of myosins.

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.