Steady-state ATPase Research Articles

Dilated cardiomyopathy mutation in beta-cardiac myosin enhances actin activation of the power stroke and phosphate release.

Inherited mutations in human beta-cardiac myosin (M2β) can lead to severe forms of heart failure. The E525K mutation in M2β is associated with dilated cardiomyopathy (DCM) and was found to stabilize the interacting heads motif (IHM) and autoinhibited super-relaxed (SRX) state in dimeric heavy meromyosin. However, in monomeric M2β subfragment 1 (S1) we found that E525K enhances (threefold) the maximum steady-state actin-activated ATPase activity (k cat) and decreases (eightfold) the actin concentration at which ATPase is one-half maximal (K ATPase). We also found a twofold to fourfold increase in the actin-activated power stroke and phosphate release rate constants at 30 μM actin, which overall enhanced the duty ratio threefold. Loaded motility assays revealed that the enhanced intrinsic motor activity translates to increased ensemble force in M2β S1. Glutamate 525, located near the actin binding region in the so-called activation loop, is highly conserved and predicted to form a salt bridge with another conserved residue (lysine 484) in the relay helix. Enhanced sampling molecular dynamics simulations predict that the charge reversal mutation disrupts the E525-K484 salt bridge, inducing conformations with a more flexible relay helix and a wide phosphate release tunnel. Our results highlight a highly conserved allosteric pathway associated with actin activation of the power stroke and phosphate release and suggest an important feature of the autoinhibited IHM is to prevent this region of myosin from interacting with actin. The ability of the E525K mutation to stabilize the IHM likely overrides the enhanced intrinsic motor properties, which may be key to triggering DCM pathogenesis.

Abstract EC.03: Distinct Molecular Signatures Of HCM- And DCM-causing Mutations At The Same Residue In The Transducer Of Cardiac Myosin

Mutations in cardiac myosin cause both hypertrophic (HCM) and dilated cardiomyopathy (DCM), and the clinical phenotype ultimately depends on the precise effect of the mutation on the function of myosin. Using recombinant human cardiac myosin, we studied the molecular effects of a novel Q222H mutation in MYH7 found in a patient with severe, early-onset DCM, and compared the effects to the Q222K mutation that was previously reported to cause HCM. The Q222 residue is located within the transducer of the head domain near the ATP binding pocket. The Q222H (DCM) mutant myosin exhibited significantly reduced steady-state actin-activated ATPase activity and a decreased rate of ATP binding in transient kinetic studies, while Q222K (HCM) mutant protein showed an increase in ATPase activity and ATP binding kinetics. In vitro motility assays showed 43% reduction and 32% increase in actin gliding velocity in Q222H and Q222K mutations, respectively. Finally, single nucleotide turnover assays revealed that the fraction of myosin with a slow ATP hydrolysis rate (super relaxed state, or SRX) thought to represent myosin in an autoinhibited state was significantly reduced in Q222K and increased in Q222H. Taken together, the Q222H/K mutations exerted contrasting effects on the motor properties of the cardiac myosin catalytic domain. Cellular characterization and molecular dynamic simulations of cardiac myosin to assess the structural impact of mutant residues are underway. These results highlight how subtle structural differences between cardiomyopathy-causing mutations can lead to significant differences in cardiac myosin motor activity and thus phenotypic expression of cardiomyopathy.

Drosophila class-I myosins that can impact left-right asymmetry have distinct ATPase kinetics

Myosin-1D (myo1D) is important for Drosophila left-right asymmetry, and its effects are modulated by myosin-1C (myo1C). De novo expression of these myosins in nonchiral Drosophila tissues promotes cell and tissue chirality, with handedness depending on the paralog expressed. Remarkably, the identity of the motor domain determines the direction of organ chirality, rather than the regulatory or tail domains. Myo1D, but not myo1C, propels actin filaments in leftward circles in invitro experiments, but it is not known if this property contributes to establishing cell and organ chirality. To further explore if there are differences in the mechanochemistry of these motors, we determined the ATPase mechanisms of myo1C and myo1D. We found that myo1D has a 12.5-fold higher actin-activated steady-state ATPase rate, and transient kinetic experiments revealed myo1D has an 8-fold higher MgADP release rate compared to myo1C. Actin-activated phosphate release is rate limiting for myo1C, whereas MgADP release is the rate-limiting step for myo1D. Notably, both myosins have among the tightest MgADP affinities measured for any myosin. Consistent with ATPase kinetics, myo1D propels actin filaments at higher speeds compared to myo1C in invitro gliding assays. Finally, we tested the ability of both paralogs to transport 50 nm unilamellar vesicles along immobilized actin filaments and found robust transport by myo1D and actin binding but no transport by myo1C. Our findings support a model where myo1C is a slow transporter with long-lived actin attachments, whereas myo1D has kinetic properties associated with a transport motor.

Design, synthesis, molecular dynamics simulation, MM/GBSA studies and kinesin spindle protein inhibitory evaluation of some 4-aminoquinoline hybrids

The discovery of novel chemotherapeutic agents is always challenging for researchers in industry and academia. Among the recent promising anticancer therapeutic targets, an important modulatory factor in mitosis is the expression of the kinesin family motor protein (Eg5). In terms of chemotherapy treatment, mitosis has gained significant attention due to its role as one of the biological processes that can be intervened in it. This study was undertaken to design, synthesise and evaluation of 4-aminoquinoline hybrid compounds as potential Eg5 inhibitors. Based on data collected from Malachite green and steady state ATPase assays, it has been determined that compounds such as 6c, 6d, 6g, and 6h are sensitive to Eg5 inhibition. In special mention, compounds 4 and 6c showed promising inhibitory activity in Malachite green assay with IC50 values of 2.32 ± 0.23 µM and 1.97 ± 0.23 µM respectively. Compound 4 showed favourable inhibitory potential Steady state ATPase Assay with IC50 value of 5.39 ± 1.39 µM. We performed molecular docking, MM/GBSA calculations, and molecular dynamic simulations to evaluate the interactions between ligands and the binding site of the kinesin spindle protein to evaluate the functional consequences of these interactions. As a result of these findings, it can be concluded that these 4-amioquinoline Schiff's base hybrids may prove to be promising candidates for development as novel inhibitors of Eg5. Further in-vivo research in this area is required.

Distinct effects of two hearing loss–associated mutations in the sarcomeric myosin MYH7b

For decades, sarcomeric myosin heavy chain proteins were assumed to be restricted to striated muscle where they function as molecular motors that contract muscle. However, MYH7b, an evolutionarily ancient member of this myosin family, has been detected in mammalian nonmuscle tissues, and mutations in MYH7b are linked to hereditary hearing loss in compound heterozygous patients. These mutations are the first associated with hearing loss rather than a muscle pathology, and because there are no homologous mutations in other myosin isoforms, their functional effects were unknown. We generated recombinant human MYH7b harboring the D515N or R1651Q hearing loss-associated mutation and studied their effects on motor activity and structural and assembly properties, respectively. The D515N mutation had no effect on steady-state actin-activated ATPase rate or load-dependent detachment kinetics but increased actin sliding velocity because of an increased displacement during the myosin working stroke. Furthermore, we found that the D515N mutation caused an increase in the proportion of myosin heads that occupy the disordered-relaxed state, meaning more myosin heads are available to interact with actin. Although we found no impact of the R1651Q mutation on myosin rod secondary structure or solubility, we observed a striking aggregation phenotype when this mutation was introduced into nonmuscle cells. Our results suggest that each mutation independently affects MYH7b function and structure. Together, these results provide the foundation for further study of a role for MYH7b outside the sarcomere.

Dilated cardiomyopathy mutation (E525K) in human beta-cardiac myosin enhances actin-activated phosphate release but stabilizes the auto-inhibited super-relaxed state.

Mutations in beta-cardiac myosin (M2β) are a common cause of inherited cardiomyopathies including dilated (DCM) and hypertrophic cardiomyopathy (HCM). We investigated the impact of a DCM mutation (E525K) in the human M2β motor domain, which has been shown to stabilize both the interacting heads motif (IHM) and auto-inhibited super-relaxed (SRX) state via head-tail electrostatic interactions in 15 heptad heavy meromyosin (M2β HMM). These findings suggest E525K may reduce muscle force and power by triggering an increase in the IHM/SRX state. However, it is unclear how E525K impacts the intrinsic motor properties of M2β. Thus, we introduced the mutation into single-headed human cardiac myosin subfragment 1 (M2β-S1), unable to form the IHM, and examined its impact on the ATPase mechanism. We revealed that E525K induces a 2.8-fold increase in maximum steady-state actin-activated ATPase activity (kcat), a 6-fold decrease in the actin concentration at which ATPase is half maximal (KATPase), and a 3.4-fold increase in the fast phase of actin-activated phosphate release. Glutamate 525 is located in the conserved activation loop that is proposed to trigger phosphate release by interacting with the negatively charged N-terminus of actin. We propose the mutation may enhance the rate of rotation of the lower 50-kDa domain, movement of the relay helix/switch II, and lever arm rotation associated with the power stroke. Thus, we will directly examine the impact on the actin-activated power stroke utilizing a FRET approach that measures lever arm rotation. Our results suggest the E525K mutation triggers an increase in intrinsic motor properties, while its ability to stabilize the SRX/IHM state in the myosin dimer likely dominates the molecular mechanism that leads to decreased muscle force and power and DCM pathogenesis.

Molecular characterization of a novel MYH7 mutation Q222H in a patient with severe dilated cardiomyopathy.

Mutations in the cardiac myosin heavy chain gene (MYH7) are known to cause both hypertrophic and dilated cardiomyopathy (HCM and DCM), and the use of recombinant human cardiac myosin has enabled the study of the precise biochemical and biophysical effects of these mutations on myosin function at the molecular level. Here we report a case of a patient with severe, early-onset DCM who carries a previously unreported Q222H mutation in MYH7. This mutation affects a residue at the distal end of the ATP binding pocket of cardiac myosin. The Q222H mutant myosin exhibited significantly reduced steady-state actin-activated ATPase activity and a decreased rate of ATP binding in transient kinetic studies. It also showed a ∼50% reduction in actin gliding velocity in an in vitro motility assay. Single-nucleotide turnover assays were performed comparing Q222H mutant myosin 2-headed constructs with either a long S2 tail capable of forming a folded-back auto-inhibited state or a short S2 tail that cannot stably fold back and sequester heads. These studies revealed that the fraction of myosin with a slow ATP hydrolysis rate (super relaxed state, or SRX) thought to represent myosin in an autoinhibited state did not differ significantly between WT and the Q222H mutant myosins. Taken together, the Q222H mutation exerts substantial effects on the motor properties of the cardiac myosin catalytic domain, rather than affecting the availability of myosin heads in the sarcomere. Further transient kinetic assays are underway to delineate precise steps in the actin-myosin ATPase cycle affected by the mutation. Future work also includes comparing these effects with the Q222K mutation that has been reported to cause HCM, to gain further insights into the molecular basis for phenotypic determination of cardiomyopathy.

Modeling the stimulation by glutathione of the steady state kinetics of an adenosine triphosphate binding cassette transporter.

We report the steady state ATPase activities of the ATP Binding Cassette (ABC) exporter NaAtm1 in the absence and presence of a transported substrate, oxidized glutathione (GSSG), in detergent, nanodiscs, and proteoliposomes. The steady state kinetic data were fit to the “nonessential activator model” where the basal ATPase rate of the transporter is stimulated by GSSG. The detailed kinetic parameters varied between the different reconstitution conditions, highlighting the importance of the lipid environment for NaAtm1 function. The increased ATPase rates in the presence of GSSG more than compensate for the modest negative cooperativity observed between MgATP and GSSG in lipid environments. These studies highlight the central role of the elusive ternary complex in accelerating the ATPase rate that is at the heart of coupling mechanism between substrate transport and ATP hydrolysis.

Design, synthesis, and molecular docking study of some 2-((7-chloroquinolin-4-yl) amino) benzohydrazide Schiff bases as potential Eg5 inhibitory agents

In Search of new microtubule-targeting compounds and to identify a promising Eg5 inhibitory agents, a series of 2-((7-chloroquinolin-4-yl) amino) benzohydrazide Schiff bases molecules (6 a-r) were synthesized using appropriate synthetic method. The synthesized compounds were characterized by using FTIR, Proton NMR, Carbon NMR and mass spectral analysis. All eighteen compounds were evaluated for their Eg5 inhibitory activity. Among the evaluated compounds, only seven compounds are shown inhibitory activity. The results of Steady state ATPase reveled that compounds 6b, 6l and 6p exhibited promising inhibitory activity with IC50 Values of 2.720 ± 0.69, 2.676 ± 0.53 and 2.408 ± 0.46 respectively. Malachite Green Assay results reveled that 6q compound showed better inhibitory activity with IC50 Value of 0.095 ± 0.27. In vitro antioxidant capacity of the synthesized compounds was investigated. A molecular docking studies were performed to evaluate interaction in to binding site of kinesin spindle protein, these interaction influencing may support Eg5 inhibitory activity. The drug like parameters of the eighteen synthesized compounds were also computed using Qikprop software. In conclusion, some of 2-((7-chloroquinolin-4-yl) amino) benzohydrazide Schiff base compounds represent promising drug like agents for discovery of effective anticancer molecules.

Mechanistic analysis of actin-binding compounds that affect the kinetics of cardiac myosin–actin interaction

Actin–myosin mediated contractile forces are crucial for many cellular functions, including cell motility, cytokinesis, and muscle contraction. We determined the effects of ten actin-binding compounds on the interaction of cardiac myosin subfragment 1 (S1) with pyrene-labeled F-actin (PFA). These compounds, previously identified from a small-molecule high-throughput screen (HTS), perturb the structural dynamics of actin and the steady-state actin-activated myosin ATPase activity. However, the mechanisms underpinning these perturbations remain unclear. Here we further characterize them by measuring their effects on PFA fluorescence, which is decreased specifically by the strong binding of myosin to actin. We measured these effects under equilibrium and steady-state conditions, and under transient conditions, in stopped-flow experiments following addition of ATP to S1-bound PFA. We observed that these compounds affect early steps of the myosin ATPase cycle to different extents. They increased the association equilibrium constant K1 for the formation of the strongly bound collision complex, indicating increased ATP affinity for actin-bound myosin, and decreased the rate constant k+2 for subsequent isomerization to the weakly bound ternary complex, thus slowing the strong-to-weak transition that actin–myosin interaction undergoes early in the ATPase cycle. The compounds' effects on actin structure allosterically inhibit the kinetics of the actin–myosin interaction in ways that may be desirable for treatment of hypercontractile forms of cardiomyopathy. This work helps to elucidate the mechanisms of action for these compounds, several of which are currently used therapeutically, and sets the stage for future HTS campaigns that aim to discover new drugs for treatment of heart failure.

Evaluating Phenyl Propanoids Isolated from Citrus medica as Potential Inhibitors for Mitotic kinesin Eg5

Background:Human mitotic kinesins play an essential role in mitotic cell division. Targeting the spindle separation phase of mitosis has gained much attention in cancer chemotherapy. Spindle segregation is carried out mainly by the kinesin, Eg5. Many Eg5 inhibitors are in different phases of clinical trials as cancer drugs. This enzyme has two allosteric binding sites to which the inhibitors can bind. The first site is formed by loop L5, helix α2 and helix α3 and all the current drug candidates bind un-competitively to this site with ATP/ADP. The second site, formed by helix α4 and helix α6, which has gained attention recently, has not been explored well. Some inhibitors that bind to this site are competitive, while others are uncompetitive to ATP/ADP. Phenylpropanoids are pharmacologically active secondary metabolites.Methods:In this study, we have evaluated fourteen phenyl propanoids extracted from Citrus medica for inhibitory activity against human mitotic kinesin Eg5 in vitro steady-state ATPase assay. Ther interactions and stability using molecular docking and molecular dynamics simulations.Results and Discussions:Of the fourteen compounds tested, naringin and quercetin showed good activity with IC50 values in the micromolar range. Molecular docking studies of these complexes showed that both the molecules interact with the key residues of the active site predominantly thorough hydrophobic & aromatic π–π interactions consistent with the known inhibitors. Besides, these molecules also form hydrogen bonding interactions stabilizing the complexes. Molecular dynamics simulations of these complexes confirm the stability of these interactions.Conclusion:These results can be used as a strong basis for further modification of these compounds to design new inhibitors with higher potency using structure-based drug design.

Lead Generation for Human Mitotic Kinesin Eg5 Using Structure-based Virtual Screening and Validation by In-vitro and Cell-based Assays.

Human mitotic kinesins play a crucial role in mitotic cell division. Targeting the spindle separation phase of mitosis has gained much attention pharmaceutically in cancer chemotherapy. Spindle segregation is carried out mainly by Eg5 kinesin, and currently, it has many inhibitors in different phases of clinical trials. All the current drug candidates bind un-competitively with ATP/ADP at allosteric site 1 (formed by loop L5, helix α2 and helix α3). Recent experiments show that inhibitors that bind to the site 2 (formed by helix α4 and helix α6) are either competitive or uncompetitive to ATP/ADP. To identify suitable lead compounds that target the mitotic kinesin Eg5, using in silico screening and their validation using in vitro and cell-based assays. Potential inhibitors were screened for human Eg5 (kinesin-5) through structurebased virtual screening and the top-scoring compounds were validated using steady-state ATPase assay, differential scanning fluorimetry, and microscale thermophoresis. The anti-cancer activity of the compounds was evaluated in the epithelial (A549) and chronic myelogenous leukemia (K562) cancer cell lines. A known strong binding inhibitor, S-trityl-L-cystine, is used as a reference compound. Out of the many compounds tested, MM01 and MM03 showed good cell-based activity against the cancer cell lines A549 and K562 and can be further studied in animal models. In this study, a structure-based approach was used to identify the potential inhibitors and validate them using different in-vitro and cell-based assays.

A Mathematical Model for the Kinetics of the MalFGK[Formula: see text] Maltose Transporter.

The MalFGK[Formula: see text] transporter regulates the movement of maltose across the inner membrane of E. coli and serves as a model system for bacterial ATP binding cassette (ABC) importers. Despite the wealth of biochemical and structural data available, a general model describing the various translocation pathways is still lacking. In this study, we formulate a mathematical model with the goal of determining the transporter reaction pathway, specifically looking at the order of binding events and conformation changes by which transport proceeds. Fitting our mathematical model to equilibrium binding data, we estimate the unknown equilibrium parameters of the system, several of which are key determinants of the transport process. Using these estimates along with steady-state ATPase rate data, we determine which of several possible reaction pathways is dominant, as a function of five underdetermined kinetic parameter values. Because neither experimental measurements nor estimates of certain kinetic rate constants are available, the problem of deciding which of the reaction pathways is responsible for transport remains unsolved. However, using the mathematical framework developed here, a firmer conclusion regarding the dominant reaction pathway as a function of MalE and maltose concentration could be drawn once these unknown kinetic parameters are determined.

Functional Differences in MYH7b That Contribute to Distinct Biological Roles across Species and in Health and Disease

Myosin heavy chain 7b (MYH7b) is a recently identified sarcomeric myosin gene family member that has a unique expression pattern in mammals. MYH7b protein is absent from the heart and skeletal muscle of mammals and is only expressed in specialized muscles and, surprisingly, in the inner ear and brain. Additionally, compound heterozygous mutations in MYH7b (one in the motor domain and one in the rod domain) cause hearing loss in humans by an unknown mechanism (Haraksingh et al., BMC Genomics, 2014) indicating that MYH7b has an important yet undefined role in mammalian non-muscle tissue. Intriguingly, in snakes, MYH7b protein is abundant in cardiac and skeletal muscles. Despite these divergent roles in mammals and reptiles, MYH7b shares high sequence identity between these vertebrate classes, so it is unclear how mammalian MYH7b function may deviate in non-muscle tissues from its expected sarcomeric role. To address this question, we have generated recombinant protein for human MYH7b, human MYH7b with the motor domain hearing loss mutation (D515N), and python MYH7b, and have measured their biochemical and biophysical properties. We have observed differences in the steady-state actin-activated ATPase activities and actin translocation as assessed by in vitro motility assays between these three recombinant proteins. Comparing the motor activity of human MYH7b and python MYH7b provides both an evolutionary perspective and functional analysis of highly conserved myosins that operate in different cell types. Further, comparing the function of human wild type MYH7b and D515N MYH7b provides greater insight into the etiology of human hearing loss.

FRET and Optical Trapping Measurements Reveal Relationship between Phosphate Release and the Power Stroke in Myosin V

Myosin V is able to processively walk along actin filaments by coordinating its mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. Our previous FRET measurements of the power stroke in myosin V containing a single IQ domain (MV 1IQ) revealed a fast rotation of the lever arm prior to the phosphate release step and a slower rotation of the lever that occurs during the transition between actomyosin.ADP states. Here we introduce a mutation in the active site (S217A) that is proposed to slow entry of phosphate into the exit tunnel. The mutation causes both fast lever arm rotation and phosphate release to occur with the same rate constant (slowing the maximum rate 10-fold), suggesting entry of phosphate into the exit tunnel gates the lever arm swing. In single molecule laser trapping studies we found the step size of S217A was similar to WT (4nm). Furthermore, raising the Pi concentration in the laser trap assay, which should increase the population of the actomyosin.ADP.Pi state, also did not affect the step size of S217A MV 1IQ. Interestingly, the attached time increased dramatically in the presence of 15 mM Pi. We also found that Pi can inhibit actin gliding in the vitro motility assay in both S217A and WT MV 1IQ in a dose-dependent manner, which is consistent with the optical trapping studies, while Pi did not impact steady-state ATPase activity. Our results suggest a model in which attachment to actin and the power stroke (lever arm swing) occur rapidly before phosphate is released in myosin V. In addition, Pi rebinding to the actomyosin.ADP state may be capable of reversing the power stroke without altering the actin binding interface.

Identification of benzo[d]pyrrolo[2,1-b]thiazole derivatives as CENP-E inhibitors

Kinesin centromere-associated protein E (CENP-E) has emerged as a potential target for the development of anticancer drugs due to its involvement in the mitotic progression of the cell cycle. Although several CENP-E inhibitors have been reported, more knowledge of chemical structures and inhibitory mechanisms is necessary for developing CENP-E inhibitors. Here, we describe the identification of new CENP-E inhibitors. Screening of a small-molecule chemical library identified benzo[d]pyrrolo[2,1-b]thiazole derivatives, including 1, as compounds with inhibitory activity against the microtubule-stimulated ATPase of the CENP-E motor domain. Among the mitotic kinesins examined, 1 selectively inhibited the kinesin ATPase activity of CENP-E. In a steady-state ATPase assay, 1 exhibited ATP-competitive behavior, which was different from the CENP-E inhibitor GSK923295. Compound 1 inhibited the proliferation of tumor-derived HeLa and HCT116 cells more efficiently than that of non-cancerous WI-38 cells. The inhibition of cell proliferation was attributed to the ability of 1 to induce apoptotic cell death. The compound showed antimitotic activity, which caused cell cycle arrest at mitosis via interference with proper chromosome alignment. We identified 1 and its derivatives as the lead compounds that target CENP-E, thus providing a new opportunity for the development of anticancer agents targeting kinesins.

Functional Dissection of a Viral DNA Packaging Machine's Walker B Motif

Many viruses employ ATP-powered motors for genome packaging. We combined genetic, biochemical, and single-molecule techniques to confirm the predicted Walker-B ATP-binding motif in the phage λ motor and to investigate the roles of the conserved residues. Most changes of the conserved hydrophobic residues resulted in >107-fold decrease in phage yield, but we identified nine mutants with partial activity. Several were cold-sensitive, suggesting that mobility of the residues is important. Single-molecule measurements showed that the partially active A175L exhibits a small reduction in motor velocity and increase in slipping, consistent with a slowed ATP binding transition, whereas G176S exhibits decreased slipping, consistent with an accelerated transition. All changes to the conserved D178, predicted to coordinate Mg2+•ATP, were lethal except conservative change D178E. Biochemical interrogation of the inactive D178N protein found no folding or assembly defects and near-normal endonuclease activity, but a ∼200-fold reduction in steady-state ATPase activity, a lag in the single-turnover ATPase time course, and no DNA packaging, consistent with a critical role in ATP-coupled DNA translocation. Molecular dynamics simulations of related enzymes suggest that the aspartate plays an important role in enhancing the catalytic activity of the motor by bridging the Walker motifs and precisely contributing its charged group to help polarize the bound nucleotide. Supporting this prediction, single-molecule measurements revealed that change D178E reduces motor velocity without increasing slipping, consistent with a slowed hydrolysis step. Our studies thus illuminate the mechanistic roles of Walker-B residues in ATP binding, hydrolysis, and DNA translocation by this powerful motor.

Converter domain mutations in myosin alter structural kinetics and motor function

Myosins are molecular motors that use a conserved ATPase cycle to generate force. We investigated two mutations in the converter domain of myosin V (R712G and F750L) to examine how altering specific structural transitions in the motor ATPase cycle can impair myosin mechanochemistry. The corresponding mutations in the human β-cardiac myosin gene are associated with hypertrophic and dilated cardiomyopathy, respectively. Despite similar steady-state actin-activated ATPase and unloaded in vitro motility-sliding velocities, both R712G and F750L were less able to overcome frictional loads measured in the loaded motility assay. Transient kinetic analysis and stopped-flow FRET demonstrated that the R712G mutation slowed the maximum ATP hydrolysis and recovery-stroke rate constants, whereas the F750L mutation enhanced these steps. In both mutants, the fast and slow power-stroke as well as actin-activated phosphate release rate constants were not significantly different from WT. Time-resolved FRET experiments revealed that R712G and F750L populate the pre- and post-power-stroke states with similar FRET distance and distance distribution profiles. The R712G mutant increased the mole fraction in the post-power-stroke conformation in the strong actin-binding states, whereas the F750L decreased this population in the actomyosin ADP state. We conclude that mutations in key allosteric pathways can shift the equilibrium and/or alter the activation energy associated with key structural transitions without altering the overall conformation of the pre- and post-power-stroke states. Thus, therapies designed to alter the transition between structural states may be able to rescue the impaired motor function induced by disease mutations.

Using the SpyTag SpyCatcher system to label smooth muscle myosin II filaments with a quantum dot on the regulatory light chain

The regulatory light chain (RLC) of myosin is commonly tagged to monitor myosin behavior in vitro, in muscle fibers, and in cells. The goal of this study was to prepare smooth muscle myosin (SMM) filaments containing a single head labeled with a quantum dot (QD) on the RLC. We show that when the RLC is coupled to a QD at Cys-108 and exchanged into SMM, subsequent filament assembly is severely disrupted. To address this, we used a novel approach for myosin by implementing the SpyTag002 SpyCatcher002 system to prepare SMM incorporated with RLC constructs fused to SpyTag or SpyCatcher. We show that filament assembly, actin-activated steady-state ATPase activities, ability to be phosphorylated, and selected enzymatic and mechanical properties were essentially unaffected if either SpyTag or SpyCatcher were fused to the C-terminus of the RLC. Crucially for our application, we also show that a QD coupled to SpyCatcher can be covalently attached to a RLC-Spy incorporated into a SMM filament without disrupting the filament, and that the filaments can move along actin in vitro.

Structure-activity relationship of pyrazolo pyrimidine derivatives as inhibitors of mitotic kinesin Eg5 and anticancer agents

Human kinesin Eg5 is a potential inhibiting site for cancer chemotherapy. Blocking metaphase by binding foreign inhibitors with Eg5 eventually leads to apoptotic cell death. Here, we report the pyrazolopyrimidine derivates as potent inhibitors of Eg5 that prevents mitotic kinesin progression. IC50 values were evaluated against the motor domain of Eg5 using steady-state ATPase assay. To better understanding, we have performed molecular docking simulation. It reveals that the interactions of the proposed inhibitors with both the allosteric sites (helices α2, α3 and loopL5, and helices α4 & α6). Out of fifteen pyrazolopyrimidine derivates, three compounds (12, 25, and 27) have shown significant inhibition of Eg5. The synthesized compounds (12, 25, and 27) were tested for their in-vitro anticancer activity against cervical cancer cell line (HeLa).