Single ATP Turnover Research Articles

Dilated cardiomyopathy mutation E525K in human beta-cardiac myosin stabilizes the interacting-heads motif and super-relaxed state of myosin.

The auto-inhibited, super-relaxed (SRX) state of cardiac myosin is thought to be crucial for regulating contraction, relaxation, and energy conservation in the heart. We used single ATP turnover experiments to demonstrate that a dilated cardiomyopathy (DCM) mutation (E525K) in human beta-cardiac myosin increases the fraction of myosin heads in the SRX state (with slow ATP turnover), especially in physiological ionic strength conditions. We also utilized FRET between a C-terminal GFP tag on the myosin tail and Cy3ATP bound to the active site of the motor domain to estimate the fraction of heads in the closed, interacting-heads motif (IHM); we found a strong correlation between the IHM and SRX state. Negative stain electron microscopy and 2D class averaging of the construct demonstrated that the E525K mutation increased the fraction of molecules adopting the IHM. Overall, our results demonstrate that the E525K DCM mutation may reduce muscle force and power by stabilizing the auto-inhibited SRX state. Our studies also provide direct evidence for a correlation between the SRX biochemical state and the IHM structural state in cardiac muscle myosin. Furthermore, the E525 residue may be implicated in crucial electrostatic interactions that modulate this conserved, auto-inhibited conformation of myosin.

Molecular Mechanisms and Cellular Models of Hypertrophic Cardiomyopathy: Insights from a Surprising Mutation

Hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, associated with over 1000 mutations, a third of which are in β-cardiac myosin (MYH7). Molecular studies of myosin with different HCM mutations have revealed a surprising diversity of effects on ATPase and load-sensitive rate of detachment from actin. It is difficult to predict how such diverse molecular effects combine to influence forces at the cellular level and how these forces further influence cellular phenotypes. We focused on the P710R mutation, which, in contrast to other MYH7 mutations, dramatically decreases in vitro motility and actin-activated ATPase. Using harmonic force spectroscopy, we further showed that this mutation reduces the step size of the myosin motor and also reduces the load-sensitivity of the actin detachment rate, which may cause dynamic, context dependent effects on force. Conversely, single ATP turnover studies of two-headed myosin constructs revealed that this mutation destabilizes the super-relaxed state, freeing more heads to generate force. Gene-edited micropatterned hiPSC-cardiomyocytes with the P710R mutation produced significantly increased force compared with isogenic control cells (measured by traction force microscopy). We used a computational model to integrate measured molecular changes and predict force traces matching the forces measured in cells. This model confirmed a key role for regulation of the super-relaxed state in driving hypercontractility. The P710R mutation also caused cardiomyocyte hypertrophy and cytoskeletal remodeling, measured by immunostaining and electron microscopy. Cellular hypertrophy was prevented in the P710R cells by inhibition of ERK or Akt, signaling proteins involved in mechanosensitive, hypertrophic signaling. Our multiscale approach reveals key mechanisms of disease in response to this unusual mutation and provides a framework which can be extended to new mutations.

Study of Hcm Causing β-Cardiac Myosin Mutations Located at Different Structurally Significant Regions of the Myosin-Head

β-cardiac myosin dwells in an equilibrium between an actin-interacting ‘on’ state and an inactive folded-back ‘off’ state. During actin-interaction, myosin hydrolyzes ATP and undergoes conformational changes that move the thin filament with respect to the thick filament, resulting in cardiac contraction. ∼35% of heritable hypertrophic cardiomyopathy has been linked to β-cardiac myosin mutations. Although the myosin motor is highly allosteric, different regions of the motor serve distinct functions. As myosin heads interact with the actin, an ATP molecule gets hydrolyzed at the ATP-binding site (p-loop), and the converter domain and relay helix bring about required cyclic-conformational changes. Throughout the whole process the 7-stranded β-sheet at the core of myosin-head structure provides required rigidity to transfer force. Residues in the mesa and converter regions in the myosin-head, and residues in the proximal part of myosin-tail control the equilibrium between the ‘on’ and ‘off’ states. We plan to investigate how pathological β-cardiac myosin mutations — Y115H (β-sheet region), R169G (mesa region/near p-loop), E497D (relay helix), E536D (actin interacting/mesa region), and I702N (near converter domain) — alter function by performing ensemble and single molecule measurements. We prepared two new two-headed human β-cardiac constructs having different length of heptad units in the tail region for our test. One of the constructs has fifteen heptad repeats (15-hep) and is designed to support head-tail interaction in the “off” state, while the other construct has just eight heptads and is predicted to lack the head-tail interaction sites. Preliminary experiments show that the 15-hep, but not the 8-hep, construct showed a significant population in the super relaxed state by a single ATP turnover assay, consistent with only the 15-hep forming the off-state. Both construct show similar single molecule force measurements, as expected.

Hypertrophic Cardiomyopathy Mutations Disrupt Human Beta Cardiac Myosin Intramolecular Interactions Leading to Increased Myosin Activity

Hypertrophic cardiomyopathy is caused by mutations in beta-cardiac myosin and is characterized by increased cardiac contractility. Myosin ensemble force depends upon its duty ratio, intrinsic force and the number of available heads (NA). We have hypothesized that NA may be regulated by altering the proportion of myosin heads in an “off-state”. Recent work from our lab suggests that HCM mutations located at the interface between the myosin head (S1) and tail (S2) disrupt the myosin folded back state, potentially switching the myosin heads from an “off-state” to an “on-state”. This may increase NA and thereby increase myosin contractility at the molecular level. To investigate how HCM mutations at the S1-S2 interface (R249Q, H251N) and the S1-S1 interface (R719W, D382Y) alter myosin activity, we introduced each mutant into two different two-headed myosin constructs containing either a 25-heptad myosin S2 tail (25-hep) or a minimal 2-heptad tail (2-hep). We hypothesized that 25-hep myosin will be able to fold back and form these intramolecular interactions, thereby sequestering heads and leading to lower myosin catalytic activity. Conversely, the 2-hep myosin, which lacks the necessary proximal tail, would be unable to form these interactions and would have higher catalytic activity. We tested this hypothesis using an actin-activated ATPase assay. The ATPase activity of WT 25-hep was 43% than that of WT 2-hep, suggesting that the WT 25-hep myosin is in an off-state. However, for each mutation tested, the 25-hep activity was only 5-15% lower, indicating that these mutations disrupt the myosin intramolecular interactions, thereby increasing NA and leading to increased contractility. Moreover, single ATP turnover experiments also suggest that a higher fraction (∼80%) of the HCM-mutant 25-hep myosins are in an open state as compared to WT 25-hep (∼40%).

Ablation of cardiac myosin binding protein-C disrupts the super-relaxed state of myosin in murine cardiomyocytes

Cardiac myosin binding protein-C (cMyBP-C) is a structural and regulatory component of cardiac thick filaments. It is observed in electron micrographs as seven to nine transverse stripes in the central portion of each half of the A band. Its C-terminus binds tightly to the myosin rod and contributes to thick filament structure, while the N-terminus can bind both myosin S2 and actin, influencing their structure and function. Mutations in the MYBPC3 gene (encoding cMyBP-C) are commonly associated with hypertrophic cardiomyopathy (HCM). In cardiac cells there exists a population of myosin heads in the super-relaxed (SRX) state, which are bound to the thick filament core with a highly inhibited ATPase activity. This report examines the role cMyBP-C plays in regulating the population of the SRX state of cardiac myosin by using an assay that measures single ATP turnover of myosin. We report a significant decrease in the proportion of myosin heads in the SRX state in homozygous cMyBP-C knockout mice, however heterozygous cMyBP-C knockout mice do not significantly differ from the wild type. A smaller, non-significant decrease is observed when thoracic aortic constriction is used to induce cardiac hypertrophy in mutation negative mice. These results support the proposal that cMyBP-C stabilises the thick filament and that the loss of cMyBP-C results in an untethering of myosin heads. This results in an increased myosin ATP turnover, further consolidating the relationship between thick filament structure and the myosin ATPase.

Structural Thermodynamics and Kinetics of the Cardiac Myosin/Omecamtiv Mecarbil Complex Revealed by Time-Resolved FRET

We are investigating the structural thermodynamics and kinetics of cardiac myosin in the presence of omecamtiv mecarbil (OM), a small molecule that is currently in clinical trials for treating heart failure. AM has been shown to increase thin-filament activated cardiac myosin ATPase activity, the apparent rate constant for actin-activated phosphate release from cardiac myosin, and contractility in the heart (Malik et al, Science 2011). How OM modulates theses activities remains poorly understood. Based on a published Biophysical Society Annual Meeting poster abstract (Liu et al. Biophys. J. 2013) and on published studies (Malik et al, Science 2011), we hypothesized that OM stabilizes the pre-powerstroke structural state in the presence of ATP analogs and increases the observed rate constant for the actin-activated powerstroke during actin-activated single ATP turnover. To test these predictions, we used a time-resolved FRET biosensor of β-cardiac myosin, which detects the mole faction of pre and post-powerstroke structural states of the myosin light-chain binding domain, to determine the apparent equilibrium constant for the recovery-stroke under saturating ADP, ADP.BeF, ADP.AlF and ADP.V nucleotide states in the presence of saturating OM or DMSO. We varied the temperatures of these experiments to determine whether OM changes the structural thermodynamics of the pre-powerstroke state. In a second set of experiments, we directly measure light-chain domain rotation during ATP binding to myosin and during actin binding to myosin in the presence of ATP. Taken together, these results address two of the key phenotypes that the drug exhibits in vitro, increased actin-activated phosphate release, and increased thin-filament activated steady-state ATPase cycling.

Modeling Smooth Muscle Myosin's Two Heads: Long-Lived Enzymatic Roles and Phosphorylation-Dependent Equilibria

Smooth muscle myosin has two heads, each capable of interacting with actin to generate force and/or motion as it hydrolyzes ATP. These heads are inhibited when their associated regulatory light chain is unphosphorylated (0P), becoming active and hydrolyzing ATP maximally when phosphorylated (2P). Interestingly, with only one of the two regulatory light chains phosphorylated (1P), smooth muscle myosin is active but its ATPase rate is <2P. To explain published 1P single ATP turnover and steady-state ATPase activities, we propose a kinetic model in which 1P myosin exists in an equilibrium between being fully active (2P) and inhibited (0P). Based on the single ATP turnover data, we also propose that each 2P head adopts a hydrolytic role distinct from its partner at any point in time, i.e., one head strongly binds actin and hydrolyzes ATP at its actin-activated rate while the other weakly binds actin. Surprisingly, the heads switch roles slowly (<0.1 s−1), suggesting that their activities are not independent. The phosphorylation-dependent equilibrium between active and inhibited states and the hydrolytic role that each head adopts during its interaction with actin may have implications for understanding regulation and mechanical performance of other members of the myosin family of molecular motors.

Modeling The Mechanics and Enzymatic Activities of Doubly, Singly and Unphosphorylated Smooth Muscle Myosin

Smooth muscle myosin II has two heads, each capable of ATP hydrolysis and actin binding. However, both heads are required for maximal force and motion generation (Tyska et al., 1999), where one head, by weakly interacting with actin, may serve as a “tether and guide,” to optimize the second head's mechanical performance (Kad et al., 2003). In addition to these non-equivalent mechanical roles, an asymmetric head-head interaction inhibits the mechanical and ATPase activities of unphosphorylated (0P) smooth muscle myosin where one head's (blocked head) actin-binding domain associates with the converter region of the second head (free head). How does the equilibrium between active and inhibited states depend on myosin's phosphorylation state and specifically when only one of its two heads is phosphorylated? Once fully active, how quickly do the heads exchange their mechanical roles? We have addressed these questions in a multistate kinetic and mechanical model.Based on single ATP turnover and steady-state ATPase data for fully active doubly phosphorylated (2P) smooth muscle myosin (Ellison et al. 2000, Rovner et al. 2006), we predict that, surprisingly, the two heads exchange their mechanical roles (i.e. weak vs. strong-binding head) slowly - at a rate of 0.1-0.3s^-1. By comparing ATPase and single molecule mechanical data (step size, attachment time, force-velocity) from fully active (2P), inhibited (0P), and smooth muscle myosin with only one head phosphorylated (1P), we predict that an equilibrium between active and inhibited conformations, which is phosphorylation-state dependent, can explain the enzymatic and mechanical data for all three phosphorylation states (2P, 1P and 0P). However, an alternative model can be proposed where the active and inhibited state equilibrium in the 1P state depends on the mechanical role of the phosphorylated head.

Step-size Analyses of the Mitochondrial Hsp70 Import Motor Reveal the Brownian Ratchet in Operation

Newly synthesized mitochondrial precursor proteins have to become unfolded by the mitochondrial Hsp70 (mtHsp70) import motor to cross the mitochondrial membranes. To assess the mechanism of unfolding of precursor proteins by mtHsp70, we designed a system to measure step sizes of the mtHsp70 import motor, which are distances at which the motor system moves along polypeptide chains during a single turnover of ATP. We made a series of fusion proteins consisting of a mitochondrial presequence containing the first mtHsp70 binding site, a spacer sequence containing an Hsp70 avoidance segment followed by the second mtHsp70 binding site, and different folded mature domains. Analyses of the dependence of the import rates of those fusion proteins on the lengths of Hsp70 avoidance segments allowed us to estimate the step sizes, which differ for different mature domains and different lengths of the spacers. These results suggest that the mtHsp70 import motor functions at least as a molecular Brownian ratchet to unfold mitochondrial precursor proteins.

Orientational Changes of Crossbridges During Single Turnover of ATP

Muscle contraction results from rotation of actin-bound myosin crossbridges. Crossbridges consist of the globular N-terminal catalytic domain and the α-helical C-terminal regulatory domain containing the essential and regulatory light chains. The essential light chain exists in two isoforms, of which the larger one has a 41-amino acid extension piece added at the N-terminus. The catalytic domain is responsible for binding to actin and for setting the stage for the main force-generating event, which is a “swing” of the regulatory domain. We measured the kinetics of the swing associated with the turnover of a single molecule of ATP. Muscle was labeled at the regulatory domain by replacing native essential or regulatory light chain with fluorescent adducts. The rotations were measured by the anisotropy of fluorescence originating from ∼400 crossbridges residing in a small volume defined by a confocal aperture of a microscope. The crossbridges were synchronized by rapid photogeneration of a stoichiometric amount of ATP. The rotations reflected dissociation from thin filaments followed by a slow reattachment. The dissociation was the same for each light chain (halftime ∼120 ms) but the rate of reattachment depended on the type of light chain. The halftimes were 920 ± 50 ms and 660 ± 100 ms for isoforms 1 and 3 of the essential light chain, respectively. The reason that the lifetimes were so long was creation of a small amount of ATP, enough only for a single turnover of crossbridges. A model was constructed that quantitized this effect. After accounting for the slowdown, the halftimes of dissociation and attachment were 34 and 200 ms, respectively.

101生体分子モータの運動と化学反応を1分子で観察

The motilities of life are produced by motor proteins, for example, myosin molecule in muscle contraction, kinesin molecule in vesicle transportation. We reviewed the mechanics reaction and single ATP turnover of individual kinesin and myosin molecules. The displacement of kinesin during force generation bound to bead was determined by measuring the position of the beads with nanometer accuracy. The analysis of movement indicates that each kinesin head mainly steps by 16 urn between the binding sites while kinesin molecules exist at 8 nm intervals. The single ATP turnover was observed using fluorescence labeled ATP. The results suggest that myosin has a memory state which stores chemical energy from ATP hydrolysis.

Regulation of scallop myosin by calcium. Cooperativity and the "off" state.

Scallop subfragment 1 (S1) is an unregulated molecule; it differs from heavy meromyosin (HMM) and myosin in that it has no "off" state, although it contains the full complement of light chains and the triggering calcium binding site. S1 differs from myosin by lacking the head-rod junction and being single-headed. The contribution of the head-rod junction was evaluated by studying single-headed myosin. Isolated single-headed myosins show some regulation; their actin activated ATPase is stimulated about 3-fold by calcium. However, in contrast to HMM and myosin, the calcium dependence of ATPase activation of single-headed myosin is non-cooperative. The single ATP turnover rate of single-headed myosin in the absence of calcium is less than 30 seconds (our experimental resolution) compared to the approximately 5 minute turnover rate of myosin. HMM and myosin exhibit several cooperative features not shown by S1. Calcium binding becomes cooperative in the presence of nucleotide analogues in HMM and myosin, but not in S1. Nucleotide analogues are bound cooperatively by myosin and HMM in the absence of calcium; the introduction of calcium to the system reduces the affinity and abolishes the cooperative binding of nucleotide in the double headed molecules. Conversely, S1 shows normal binding curves for nucleotide analogues both in the presence and absence of calcium. Therefore, there is direct communication between the calcium binding sites and nucleotide binding sites in regulated molecules that is mediated by interaction between the two heads. .

A two-site mechanism for ATP hydrolysis by the asymmetric Rep dimer P2S as revealed by site-specific inhibition with ADP-A1F4.

The Escherichia coli Rep helicase is a dimeric motor protein that catalyzes the transient unwinding of duplex DNA to form single-stranded (ss) DNA using energy derived from the binding and hydrolysis of ATP. In an effort to understand this mechanism of energy transduction, we have used pre-steady-state methods to study the kinetics of ATP binding and hydrolysis by an important intermediate in the DNA unwinding reaction--the asymmetric Rep dimer state, P2S, where ss DNA [dT(pT)15] is bound to only one subunit of the Rep dimer. To differentiate between the two potential ATPase active sites inherent in the dimer, we constructed dimers with one subunit covalently cross-linked to ss DNA and where one or the other of the ATPase sites was selectively complexed to the tightly bound transition state analog ADP-A1F4. We found that when ADP-A1F4 is bound to the Rep subunit in trans from the subunit bound to ss DNA, steady-state ATPase activity of 18 s(-1) per dimer (equivalent to wild-type P2S) was recovered. However, when the ADP-A1F4 and ss DNA are both bound to the same subunit (cis), then a titratable burst of ATP hydrolysis is observed corresponding to a single turnover of ATP. Rapid chemical quenched-flow techniques were used to resolve the following minimal mechanism for ATP hydrolysis by the unligated Rep subunit of the cis dimer: E + ATP <==> E-ATP <==> E'-ATP <==> E'-ADP-Pi <==> E-ADP-Pi <==> E-ADP + Pi <==> E + ADP + Pi, with K1 = (2.0 +/- 0.85) x 10(5) M(-1), k2 = 22 +/- 3.5 s(-1), k(-2) < 0.12 s(-1), K3 = 4.0 +/- 0.4 (k3 > 200 s(-1)), k4 = 1.2 +/- 0.14 s(-1), k(-4) << 1.2 s(-1), K5 = 1.0 +/- 0.2 mM, and K6 = 80 +/- 8 microM. A salient feature of this mechanism is the presence of a kinetically trapped long-lived tight nucleotide binding state, E'-ADP-Pi. In the context of our "subunit switching" model for Rep dimer translocation during processive DNA unwinding [Bjornson, K. B., Wong, I., & Lohman, T. M. (1996) J. Mol. Biol. 263, 411-422], this state may serve an energy storage function, allowing the energy from the binding and hydrolysis of ATP to be harnessed and held in reserve for DNA unwinding.

A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems.

A spectrophotometric method for the measurement of inorganic phosphate (P(i)) has been developed by using 2-amino-6-mercapto-7-methylpurine ribonucleoside and purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1). This substrate gives an absorbance increase at 360 nm on phosphorolysis at pH 6.5-8.5, and at pH 7.6 the change in extinction coefficient is 11,000 M-1.cm-1. The Michaelis-Menten constants of the two substrates with the enzyme are 70 microM for the nucleoside and 26 microM for P(i); the kcat is 40 s-1 (25 degrees C). The assay was shown to quantitate P(i) in solution at concentrations at least down to 2 microM. It can be used to measure the kinetics of P(i) release from phosphatases, such as GTPases and ATPases, by coupling the two enzymic reactions. The utility of this assay was shown by three test systems: glycerol kinase plus D-glyceraldehyde acting as an ATPase and actin-activated myosin ATPase, and myosin subfragment 1, hydrolyzing a single turnover of ATP, releasing P(i) with a rate constant the same as the steady-state ATPase activity.

Presteady state kinetic analysis of vanadate-induced inhibition of the dynein ATPase.

The effects of vanadate on the kinetics of ATP binding and hydrolysis by Tetrahymena 30 S dynein were examined by presteady state kinetic analysis. Up to a concentration of 400 microM, vanadate did not inhibit the rate or amplitude of the ATP binding-induced dissociation of the microtubule-dynein complex measured by stopped flow light-scattering methods. Chemical quench flow experiments showed that vanadate (80 microM) did not alter the rate or amplitude of the presteady state ATP binding or ATP hydrolysis transients, but the steady state hydrolysis of ATP was blocked immediately after a single turnover of ATP. Preincubation of the enzyme with ADP and vanadate inhibited both presteady state and steady state hydrolysis. These data suggest that vanadate acts as a phosphate analog to form an enzyme-ADP-vanadate complex, analogous to the transition state during catalysis, by the following pathway: (formula; see text) where V represents vanadate and D represents a dynein active site. ADP and vanadate, added together, induced dissociation of the microtubule-dynein complex at a maximum rate of 0.6 S-1. These observations imply that a microtubule-dynein-ADP-vanadate complex was formed which subsequently dissociated as shown below: (formula; see text) where M denotes a microtubule. The ADP plus vanadate-induced dissociation may represent the reverse of the normal forward pathway involving the binding of a dynein-ADP-phosphate complex to a microtubule.

Kinetic comparison of normal and thyrotoxic bovine cardiac myosin subfragment-1.

A comparison of the transient kinetics of cardiac ventricular normal and hyperthyroid modified myosin subfragment-1 reveals substantial similarities between the two proteins. The nucleotide-binding kinetics are nonexponential for both proteins, but the large tryptophan fluorescence changes, 34% for ATP binding and 12% for ADP binding which are comparable to those of rabbit skeletal myosin subfragment-1, permit the kinetic data to be resolved into a sum of two exponentials. Both the fast and slow forms of the proteins reach limiting rate constants at high nucleotide concentration. The fast forms of normal and thyrotoxic cardiac subfragment-1 are kinetically identical for nucleotide binding at 20 degrees C and pH 7 and the slow forms differ by less than a factor of 2. The kinetic data for ADP release and the single turnover of ATP could neither be fit by a single exponential nor resolved into two components, which indicates a difference in the rate constants by a factor of 2 or less. The largest difference found was in the steady state turnover of ATP for which thyrotoxic subfragment-1 had a 2.5 times faster turnover as compared to normal subfragment-1. The fractions of fast and slow forms of the two proteins are dependent on the nucleotide concentration and the fractions as well as the rate constants are a function of the protein concentration. This is consistent with the kinetic heterogeneity of cardiac myosin subfragment-1 resulting from aggregation. The differences in the rate constant for the steady state turnover of ATP and in aggregation properties between normal and hyperthyroid cardiac subfragment-1 are consistent with the induction of a myosin isozyme by thyroxine treatment. Moreover, the increase in the steady state turnover of ATP is consistent with the increase in contractility of the muscle in the hyperthyroid state.