We have measured the kinetics of inorganic phosphate (Pi) release during a single turnover of actomyosin nucleoside triphosphate (NTP) hydrolysis using a double-mixing stopped-flow spectrofluorometer, at very low ionic strength to increase the affinity of myosin-ATP and myosin-ADP-Pi to actin. Myosin subfragment 1 and a series of nucleoside triphosphates were mixed and incubated for approximately 1-10 s to allow NTP to bind to myosin and generate a steady state mixture of myosin-NTP and myosin-NDP-Pi. The steady state intermediates were then mixed with actin. The kinetics of Pi release were measured using a fluorescent probe for Pi, based on a phosphate binding protein [Brune et al. (1994) Biochemistry 33, 8262-8271]. These data are correlated with quenched-flow data, where the extent of the rapid burst of hydrolysis during the first turnover of ATP hydrolysis was followed by chemical quenching of the reaction mix at various times after rapidly mixing ATP and myosin subfragment 1. From the double-mixing actomyosin measurements, the kinetics of Pi release are biphasic. The fast phase corresponds to Pi release from the associated actomyosin-ADP-Pi complex. The slow phase measures the rate of the cleavage step on associated actomyosin. At saturating actin, there is a correlation between the amplitude of the fast phase and the size of the Pi burst observed by quenched flow in the absence of actin: the size of this phase corresponds to the amount of myosin-ADP-Pi formed during the first mix. For ATP at 20 degrees C the rate of the Pi release step is 75 (+/-5) s-1, 25-fold larger than the cleavage step, which is the rate-limiting step of actomyosin ATP hydrolysis at saturating actin. The rate constant of Pi release varies only slightly with nucleoside structure. The rate constant of the slow phase of the Pi release (measuring cleavage) is highly dependent upon the structure of the NTP substrate.

We determined the effect of Omecamtiv Mecarbil, a novel allosteric effector of cardiac muscle myosin, on the kinetic and "in vitro" motility properties of the porcine ventricular heavy meromyosin (PV-HMM). Omecamtiv Mecarbil increases the equilibrium constant of the hydrolysis step (M-ATP ⇄ M-ADP-Pi) from 2.4 to 6 as determined by quench flow, but the maximal rates of both the hydrolysis step and tryptophan fluorescence increase are unchanged by the drug. OM also increases the amplitude of the fast phase of phosphate dissociation (AM-ADP-Pi → AM-ADP + Pi) that is associated with force production in muscle by 4-fold. These results suggest a mechanism in which hydrolysis of M-ATP to M-ADP-Pi occurs both before and after the recovery stroke, but rapid acceleration of phosphate dissociation by actin occurs only on post-recovery stroke A-M-ADP-Pi. One of the more dramatic effects of OM on PV-HMM is a 14-fold decrease in the unloaded shortening velocity measured by the in vitro motility assay. The increase in flux through phosphate dissociation and the unchanged rate of ADP dissociation (AM-ADP → AM + ADP) by the drug produce a higher duty ratio motor in which a larger fraction of myosin heads are strongly bound to actin filaments. The increased internal load produced by a larger fraction of strongly attached crossbridges explains the reduced rate of in vitro motility velocity in the presence of OM and predicts that the drug will produce slower and stronger contraction of cardiac muscle.

Muscle contraction relies on the interaction of myosin motors with F-actin, which is regulated through a translocation of tropomyosin by the troponin complex in response to Ca 2+ . The current model of muscle regulation holds that at relaxing (low-Ca 2+ ) conditions tropomyosin blocks myosin binding sites on F-actin, whereas at activating (high-Ca 2+ ) conditions tropomyosin translocation only partially exposes myosin binding sites on F-actin so that binding of rigor myosin is required to fully activate the thin filament (TF). Here we used a single-particle approach to helical reconstruction of frozen hydrated native cardiac TFs under relaxing and activating conditions to reveal the azimuthal movement of the tropomyosin on the surface of the native cardiac TF upon Ca 2+ activation. We demonstrate that at either relaxing or activating conditions tropomyosin is not constrained in one structural state, but rather is distributed between three structural positions on the surface of the TF. We show that two of these tropomyosin positions restrain actomyosin interactions, whereas in the third position, which is significantly enhanced at high Ca 2+ , tropomyosin does not block myosin binding sites on F-actin. Our data provide a structural framework for the enhanced activation of the cardiac TF over the skeletal TF by Ca 2+ and lead to a mechanistic model for the regulation of the cardiac TF.thin filament | cardiac muscle regulation | cryoelectron microscopy

The thick filaments of mammalian and avian skeletal muscle fibers are disordered at low temperature, but become increasingly ordered into an helical structure as the temperature is raised. Wray and colleagues (Schlichting, I., and J. Wray. 1986. J. Muscle Res. Cell Motil. 7:79; Wray, J., R. S. Goody, and K. Holmes. 1986. Adv. Exp. Med. Biol. 226:49-59) interpreted the transition as reflecting a coupling between nucleotide state and global conformation with M.ATP (disordered) being favored at 0 degrees C and M.ADP.P(i) (ordered) at 20 degrees C. However, hitherto this has been limited to a qualitative correlation and the biochemical state of the myosin heads required to obtain the helical array has not been unequivocally identified. In the present study we have critically tested whether the helical arrangement of the myosin heads requires the M.ADP.P(i) state. X-ray diffraction patterns were recorded from skinned rabbit psoas muscle fiber bundles stretched to non-overlap to avoid complications due to interaction with actin. The effect of temperature on the intensities of the myosin-based layer lines and on the phosphate burst of myosin hydrolyzing ATP in solution were examined under closely matched conditions. The results showed that the fraction of myosin mass in the helix closely followed that of the fraction of myosin in the M.ADP.P(i) state. Similar results were found by using a series of nucleoside triphosphates, including CTP and GTP. In addition, fibers treated by N-phenylmaleimide (Barnett, V. A., A. Ehrlich, and M. Schoenberg. 1992. Biophys. J. 61:358-367) so that the myosin was exclusively in the M.ATP state revealed no helical order. Diffraction patterns from muscle fibers in nucleotide-free and in ADP-containing solutions did not show helical structure. All these confirmed that in the presence of nucleotides, the M.NDP.P(i) state is required for helical order. We also found that the spacing of the third meridional reflection of the thick filament is linked to the helical order. The spacing in the ordered M.NDP.P(i) state is 143.4 A, but in the disordered state, it is 144. 2 A. This may be explained by the different interference functions for the myosin heads and the thick filament backbone.

SUMMARY Muscle contraction relies on interaction between myosin-based thick filaments and actin-based thin filaments. Myosin binding protein-C (MyBP-C) is a key regulator of actomyosin interactions. Recent studies established that the N’-terminal domains (NTDs) of MyBP-C can either activate or inhibit thin filaments, but the mechanism of their collective action is poorly understood. Cardiac MyBP-C (cMyBP-C) harbors an extra NTD which is absent in skeletal isoforms of MyBP-C and its role in regulation of cardiac contraction is unknown. Here we show that the first two domains of human cMyPB-C (i.e., C0 and C1) cooperate to activate the thin filament. We demonstrate that C1 interacts with tropomyosin via a positively charged loop and that this interaction, stabilized by the C0 domain, is required for thin filament activation by cMyBP-C. Our data reveal a mechanism by which cMyBP-C can modulate cardiac contraction and demonstrate a function of the C0 domain.

Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure-function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the "open" structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca 2+ have been achieved. We suggest that Ca 2+ modulates the interaction of cMyBP-C with the TF in the sarcomere.

Regulation by calcium and myosin-S1 of the acceleration of the rate of phosphate release from myosin-ADP-inorganic phosphate (M-ADP-Pi) by the thin filament actin-tropomyosin (Tm)-troponin (Tn), was measured directly by using double mixing stopped-flow experiments with fluorescent phosphate binding protein. At low calcium and without rigor myosin-S1, saturating concentrations of thin filaments accelerate the rate of phosphate dissociation from M-ADP-Pi 8-fold, from 0.08 to 0.64 s ؊1 . If either myosin-S1 or calcium is bound to the thin filaments, phosphate release is a biphasic process in which the fast phase is the dissociation of Pi from actoTmTnM-ADP-Pi and the slow phase is limited by the hydrolysis of actoTmTnM-ATP to actoTmTnM-ADP-Pi. The maximum accelerations of the fast components by saturating thin filaments (relative to M-ADP-Pi alone) are: Ϸ200-fold, 16 s ؊1 (calcium only); Ϸ400-fold, 30 s ؊1 (EGTA and rigor S1); and Ϸ1,000-fold, 75 s ؊1 (calcium and rigor S1). The maximum rate of phosphate dissociation attained with S1 and calcium bound to the thin filament is the same as for unregulated actin. Regulation of the rate of phosphate dissociation by calcium and myosin-S1 is partially explained by the model of Geeves In striated muscle the constitutive activation of myosin by actin (1) is prevented by the actin binding proteins, tropomyosin (Tm) and troponin (Tn). These proteins assemble into the repeating units of the thin filament, which contains seven actin monomers, one Tn, and one Tm. Although the atomic-level structure of the thin filament is not yet available, the broad structural features are well established (2, 3). Tm lies end to end in the grove created by the helical strands of actin monomers. There is no direct interaction between adjacent Tn molecules that are bound to the underlying filament at regular intervals via two of three subunits, Tn-T and Tn-I. The third subunit, Tn-C, is the calcium sensor. Although there is only one Tn per regulatory unit, protein-protein contacts within the thin filament expand the range of any signal resulting from the binding of ligand (4-7).How the structure of the thin filament is modified by ligand interaction has provided considerable insight into the mechanism of regulation. Despite the lack of a crystal structure, different conformations of the thin filament can be distinguished at low resolution (8). The three-state model (9), consistent with the interpretation of 3D reconstructions of electron micrographs (8), requires the formation of three different conformers produced by the binding of calcium and myosin-S1. In a resting muscle, Tn straddles actin and Tm, tethering the latter to the outside of the actin filament on subdomains 1 and 2. In this state the productive interaction of actin and myosin is said to be blocked. Calcium, which instigates a structural change in Tn (10-12), induces a new state in which Tm has moved toward subdomains 3 and 4 of actin, exposing a portion of the myosin binding site of the weak binding intermediates, myosin-ATP (M-A...

We have determined the kinetic mechanism and motile properties of the switch 1 mutant S217A of myosin Va. Phosphate dissociation from myosin V-ADP-P i (inorganic phosphate) and actomyosin V-ADP-P i and the rate of the hydrolysis step (myosin V-ATP 3 myosin V-ADP-P i ) were all ϳ10-fold slower in the S217A mutant than in wild type (WT) myosin V, resulting in a slower steady-state rate of basal and filamentous actin (actin)-activated ATP hydrolysis. Substrate binding and ADP dissociation kinetics were all similar to or slightly faster in S217A than in WT myosin V and mechanochemical gating of the rates of dissociation of ADP between trail and lead heads is maintained. The reduction in the rate constants of the hydrolysis and phosphate dissociation steps reduces the duty ratio from ϳ0.85 in WT myosin V to ϳ0.25 in S217A and produces a motor in which the average run length on actin at physiological concentrations of ATP is reduced 10-fold. Thus we demonstrate that, by mutational perturbation of the switch 1 structure, myosin V can be converted into a low duty ratio motor that is processive only at low substrate concentrations.During the past 2 decades a considerable number of different myosins have been discovered (1). Myosin V is the best characterized among the so-called unconventional myosins (i.e. those not belonging to class II), and it serves as an important model molecule for studying actomyosin interactions and single molecule processive motility (2). Myosin V is a highly processive motor whose role is to transport cargo along actin filaments or bundles inside the cell (3-5). The kinetic mechanism of myosin V is significantly different from that of conventional myosins such as muscle myosin II, as it remains bound to actin (filamentous actin) through a number of ATPase cycles (6 -8). Myosin V has a high duty ratio: a single-headed myosin V-S1 (myosin V, subfragment 1) is in the strongly bound AM-ADP state 80 -90% of the time during ATP hydrolysis. An additional mechanism for promoting highly processive runs is the preferential release of ADP from the trail head because of mechanochemical gating, which causes a drastic reduction of the rate constant of ADP release from the lead head (9 -11). Although there are significant differences between the ATPase mechanisms of the different myosins, the structure of the nucleotide binding pocket (composed of the switch 1 and 2 regions and the P-loop) is highly conserved. The position of the Ser 217 (Ser 236 in Dictyostelium myosin II) residue of the switch 1 loop (the first serine in the NDNSSRFG sequence) is shown in Fig. 1. It had been shown previously by mutagenesis in Dictyostelium (12) and in smooth muscle myosin II (13) that the substitution of serine 236 to alanine retains at least partial enzymatic and motile function in these mutant myosins. Therefore, the OH group is not an essential part of the catalytic mechanism, but the rate of steady-state ATP hydrolysis is reduced several fold. However, neither of these studies includes a detailed kinetic analysis to det...