Troponin Structural Dynamics in the Native Cardiac Thin Filament Revealed by Cryo Electron Microscopy.

Cardiac muscle contraction occurs due to repetitive interactions between myosin thick and actin thin filaments (TF) regulated by Ca2+ levels, active cross-bridges, and cardiac myosin-binding protein C (cMyBP-C). The cardiac TF (cTF) has two nonequivalent strands, each comprised of actin, tropomyosin (Tm), and troponin (Tn). Tn shifts Tm away from myosin-binding sites on actin at elevated Ca2+ levels to allow formation of force-producing actomyosin cross-bridges. The Tn complex is comprised of three distinct polypeptides - Ca2+-binding TnC, inhibitory TnI, and Tm-binding TnT. The molecular mechanism of their collective action is unresolved due to lack of comprehensive structural information on Tn region of cTF. C1 domain of cMyBP-C activates cTF in the absence of Ca2+ to the same extent as rigor myosin. Here we used cryo-EM of native cTFs to show that cTF Tn core adopts multiple structural conformations at high and low Ca2+ levels and that the two strands are structurally distinct. At high Ca2+ levels, cTF is not entirely activated by Ca2+ but exists in either partially or fully activated state. Complete dissociation of TnI C-terminus is required for full activation. In presence of cMyBP-C C1 domain, Tn core adopts a fully activated conformation, even in absence of Ca2+. Our data provide a structural description for the requirement of myosin to fully activate cTFs and explain increased affinity of TnC to Ca2+ in presence of active cross-bridges. We suggest that allosteric coupling between Tn subunits and Tm is required to control actomyosin interactions.

Effect of the N-terminal extension in myosin essential light chain A1 on the mechanism of actomyosin ATP hydrolysis.

Myosin essential light chains A1 and A2 are identical isoforms except for an extension of ∼40 amino acids at the N terminus of A1 that binds F-actin. The extension has no bearing on the burst hydrolysis rate (M-ATP → M-ADP-Pi) as determined by chemical quench flow (100 µM isoenzyme). Whereas actomyosin-S1A2 steady state MgATPase (low ionic strength, 20 °C) is hyperbolically dependent on concentration: Vmax 7.6 s-1, Kapp 6.4 µM (F-actin) and Vmax 10.1 s-1, Kapp 5.5 µM (native thin filaments, pCa 4), the relationship for myosin-S1A1 is bimodal; an initial rise at low concentration followed by a decline to one-third the Vmax of S1A2, indicative of more than one rate-limiting step and A1-enforced flux through the slower actomyosin-limited hydrolysis pathway. In double-mixing stopped-flow with an indicator, Ca(II)-mediated activation of Pi dissociation (regulatedAM-ADP-Pi → regulatedAM-ADP + Pi) is attenuated by A1 attachment to thin filaments (pCa 4). The maximum accelerated rates of Pi dissociation are: 81 s-1 (S1A1, Kapp 8.9 µM) versus 129 s-1 (S1A2, Kapp 58 µM). To investigate apomyosin-S1-mediated activation, thin filaments (EGTA) are premixed with a given isomyosin-S1 and double-mixing is repeated with myosin-S1A1 in the first mix. Similar maximum rates of Pi dissociation are observed, 44.5 s-1 (S1A1) and 47.1 s-1 (S1A2), which are lower than for Ca(II) activation. Overall, these results biochemically demonstrate how the longer light chain A1 can contribute to slower contraction and higher force and the shorter version A2 to faster contraction and lower force, consistent with their distribution in different types of striated muscle.

High-resolution cryo-EM structure of the junction region of the native cardiac thin filament in relaxed state.

Cardiac contraction depends on molecular interactions among sarcomeric proteins coordinated by the rising and falling intracellular Ca2+ levels. Cardiac thin filament (cTF) consists of two strands composed of actin, tropomyosin (Tm), and equally spaced troponin (Tn) complexes forming regulatory units. Tn binds Ca2+ to move Tm strand away from myosin-binding sites on actin to enable actomyosin cross-bridges required for force generation. The Tn complex has three subunits-Ca2+-binding TnC, inhibitory TnI, and Tm-binding TnT. Tm strand is comprised of adjacent Tm molecules that overlap "head-to-tail" along the actin filament. The N-terminus of TnT (e.g., TnT1) binds to the Tm overlap region to form the cTF junction region-the region that connects adjacent regulatory units and confers to cTF internal cooperativity. Numerous studies have predicted interactions among actin, Tm, and TnT1 within the junction region, although a direct structural description of the cTF junction region awaited completion. Here, we report a 3.8 Å resolution cryo-EM structure of the native cTF junction region at relaxing (pCa 8) Ca2+ conditions. We provide novel insights into the "head-to-tail" interactions between adjacent Tm molecules and interactions between the Tm junction with F-actin. We demonstrate how TnT1 stabilizes the Tm overlap region via its interactions with the Tm C- and N-termini and actin. Our data show that TnT1 works as a joint that anchors the Tm overlap region to actin, which stabilizes the relaxed state of the cTF. Our structure provides insight into the molecular basis of cardiac diseases caused by missense mutations in TnT1.

Cryo-Electron Microscopy Reveals Cardiac Myosin Binding Protein-C M-Domain Interactions with the Thin Filament.

Cardiac myosin binding protein C (cMyBP-C) modulates cardiac contraction via direct interactions with cardiac thick (myosin) and thin (actin) filaments (cTFs). While its C-terminal domains (e.g. C8-C10) anchor cMyBP-C to the backbone of the thick filament, its N-terminal domains (NTDs) (e.g. C0, C1, M, and C2) bind to both myosin and actin to accomplish its dual roles of inhibiting thick filaments and activating cTFs. While the positions of C0, C1 and C2 on cTF have been reported, the binding site of the M-domain on the surface of the cTF is unknown. Here, we used cryo-EM to reveal that the M-domain interacts with actin via helix 3 of its ordered tri-helix bundle region, while the unstructured part of the M-domain does not maintain extensive interactions with actin. We combined the recently obtained structure of the cTF with the positions of all the four NTDs on its surface to propose a complete model of the NTD binding to the cTF. The model predicts that the interactions of the NTDs with the cTF depend on the activation state of the cTF. At the peak of systole, when bound to the extensively activated cTF, NTDs would inhibit actomyosin interactions. In contrast, at falling Ca2+ levels, NTDs would not compete with the myosin heads for binding to the cTF, but would rather promote formation of active cross-bridges at the adjacent regulatory units located at the opposite cTF strand. Our structural data provides a testable model of the cTF regulation by the cMyBP-C.

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy.

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures and information on more challenging systems. Sample preparation for cryo-EM is undergoing a similar revolution with new approaches being developed to supersede the traditional blotting systems. These include the use of piezo-electric dispensers, pin printing and direct spraying. As a result of these developments, the speed of grid preparation is going from seconds to milliseconds, providing new opportunities, especially in the field of time-resolved cryo-EM where proteins and substrates can be rapidly mixed before plunge freezing, trapping short lived intermediate states. Here we describe, in detail, a standard protocol for making grids on our in-house time-resolved EM device both for standard fast grid preparation and also for time-resolved experiments. The protocol requires a minimum of about 50 µL sample at concentrations of ≥ 2 mg/mL for the preparation of 4 grids. The delay between sample application and freezing can be as low as 10 ms. One limitation is increased ice thickness at faster speeds and compared to the blotting method. We hope this protocol will aid others in designing their own grid making devices and those interested in designing time-resolved experiments.

On-grid and in-flow mixing for time-resolved cryo-EM.

Time-resolved cryo-electron microscopy (TrEM) allows the study of proteins under non-equilibrium conditions on the millisecond timescale, permitting the analysis of large-scale conformational changes or assembly and disassembly processes. However, the technique is developing and there have been few comparisons with other biochemical kinetic studies. Using current methods, the shortest time delay is on the millisecond timescale (∼5-10 ms), given by the delay between sample application and vitrification, and generating longer time points requires additional approaches such as using a longer delay line between the mixing element and nozzle, or an incubation step on the grid. To compare approaches, the reaction of ATP with the skeletal actomyosin S1 complex was followed on grids prepared with a 7-700 ms delay between mixing and vitrification. Classification of the cryo-EM data allows kinetic information to be derived which agrees with previous biochemical measurements, showing fast dissociation, low occupancy during steady-state hydrolysis and rebinding once ATP has been hydrolysed. However, this rebinding effect is much less pronounced when on-grid mixing is used and may be influenced by interactions with the air-water interface. Moreover, in-flow mixing results in a broader distribution of reaction times due to the range of velocities in a laminar flow profile (temporal spread), especially for longer time delays. This work shows the potential of TrEM, but also highlights challenges and opportunities for further development.

Interaction of the C2 Ig-like Domain of Cardiac Myosin Binding Protein-C with F-actin.

Cardiac muscle contraction depends on interactions between thick (myosin) and thin (actin) filaments (TFs). TFs are regulated by intracellular Ca2+ levels. Under activating conditions Ca2+ binds to the troponin complex and displaces tropomyosin from myosin binding sites on the TF surface to allow actomyosin interactions. Recent studies have shown that in addition to Ca2+, the first four N-terminal domains (NTDs) of cardiac myosin binding protein C (cMyBP-C) (e.g. C0, C1, M and C2), are potent modulators of the TF activity, but the mechanism of their collective action is poorly understood. Previously, we showed that C1 activates the TF at low Ca2+ and C0 stabilizes binding of C1 to the TF, but the ability of C2 to bind and/or affect the TF remains unknown. Here we obtained 7.5 Å resolution cryo-EM reconstruction of C2-decorated actin filaments to demonstrate that C2 binds to actin in a single structural mode that does not activate the TF unlike the polymorphic binding of C0 and C1 to actin. Comparison of amino acid sequences of C2 with either C0 or C1 shows low levels of identity between the residues involved in interactions with the TF but high levels of conservation for residues involved in Ig fold stabilization. This provides a structural basis for strikingly different interactions of structurally homologous C0, C1 and C2 with the TF. Our detailed analysis of the interaction of C2 with the actin filament provides crucial information required to model the collective action of cMyBP-C NTDs on the cardiac TF.

The structure of the native cardiac thin filament at systolic Ca2+ levels.

Every heartbeat relies on cyclical interactions between myosin thick and actin thin filaments orchestrated by rising and falling Ca2+ levels. Thin filaments are comprised of two actin strands, each harboring equally separated troponin complexes, which bind Ca2+ to move tropomyosin cables away from the myosin binding sites and, thus, activate systolic contraction. Recently, structures of thin filaments obtained at low (pCa ∼9) or high (pCa ∼3) Ca2+ levels revealed the transition between the Ca2+-free and Ca2+-bound states. However, in working cardiac muscle, Ca2+ levels fluctuate at intermediate values between pCa ∼6 and pCa ∼7. The structure of the thin filament at physiological Ca2+ levels is unknown. We used cryoelectron microscopy and statistical analysis to reveal the structure of the cardiac thin filament at systolic pCa = 5.8. We show that the two strands of the thin filament consist of a mixture of regulatory units, which are composed of Ca2+-free, Ca2+-bound, or mixed (e.g., Ca2+ free on one side and Ca2+ bound on the other side) troponin complexes. We traced troponin complex conformations along and across individual thin filaments to directly determine the structural composition of the cardiac native thin filament at systolic Ca2+ levels. We demonstrate that the two thin filament strands are activated stochastically with short-range cooperativity evident only on one of the two strands. Our findings suggest a mechanism by which cardiac muscle is regulated by narrow range Ca2+ fluctuations.

High-Resolution Cryo-EM Structure of the Cardiac Actomyosin Complex.

Heart contraction depends on a complicated array of interactions between sarcomeric proteins required to convert chemical energy into mechanical force. Cyclic interactions between actin and myosin molecules, controlled by troponin and tropomyosin, generate the sliding force between the actin-based thin and myosin-based thick filaments. Alterations in this sophisticated system due to missense mutations can lead to cardiovascular diseases. Numerous structural studies proposed pathological mechanisms of missense mutations at the myosin-myosin, actin-tropomyosin, and tropomyosin-troponin interfaces. However, despite the central role of actomyosin interactions a detailed structural description of the cardiac actomyosin interface remained unknown. Here, we report a cryo-EM structure of a cardiac actomyosin complex at 3.8 Å resolution. The structure reveals the molecular basis of cardiac diseases caused by missense mutations in myosin and actin proteins.

Need for Speed: Examining Protein Behavior during CryoEM Grid Preparation at Different Timescales.

A host of new technologies are under development to improve the quality and reproducibility of cryoelectron microscopy (cryoEM) grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot, chameleon, and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms (the fastest reported to date), to interrogate particle behavior at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin-film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimize. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic "silver bullet" for improving the quality of every cryoEM sample preparation.

Sample deposition onto cryo-EM grids: from sprays to jets and back.

Despite the great strides made in the field of single-particle cryogenic electron microscopy (cryo-EM) in microscope design, direct electron detectors and new processing suites, the area of sample preparation is still far from ideal. Traditionally, sample preparation involves blotting, which has been used to achieve high resolution, particularly for well behaved samples such as apoferritin. However, this approach is flawed since the blotting process can have adverse effects on some proteins and protein complexes, and the long blot time increases exposure to the damaging air-water interface. To overcome these problems, new blotless approaches have been designed for the direct deposition of the sample on the grid. Here, different methods of producing droplets for sample deposition are compared. Using gas dynamic virtual nozzles, small and high-velocity droplets were deposited on cryo-EM grids, which spread sufficiently for high-resolution cryo-EM imaging. For those wishing to pursue a similar approach, an overview is given of the current use of spray technology for cryo-EM grid preparation and areas for enhancement are pointed out. It is further shown how the broad aspects of sprayer design and operation conditions can be utilized to improve grid quality reproducibly.

A cryo-EM grid preparation device for time-resolved structural studies.

Structural biology generally provides static snapshots of protein conformations that can provide information on the functional mechanisms of biological systems. Time-resolved structural biology provides a means to visualize, at near-atomic resolution, the dynamic conformational changes that macromolecules undergo as they function. X-ray free-electron-laser technology has provided a powerful tool to study enzyme mechanisms at atomic resolution, typically in the femtosecond to picosecond timeframe. Complementary to this, recent advances in the resolution obtainable by electron microscopy and the broad range of samples that can be studied make it ideally suited to time-resolved approaches in the microsecond to millisecond timeframe to study large loop and domain motions in biomolecules. Here we describe a cryo-EM grid preparation device that permits rapid mixing, voltage-assisted spraying and vitrification of samples. It is shown that the device produces grids of sufficient ice quality to enable data collection from single grids that results in a sub-4 Šreconstruction. Rapid mixing can be achieved by blot-and-spray or mix-and-spray approaches with a delay of ∼10 ms, providing greater temporal resolution than previously reported mix-and-spray approaches.

Investigation into the mechanism of thin filament regulation by transient kinetics and equilibrium binding: Is there a conflict?

Striated muscle contraction occurs when myosin undergoes a lever-type structural change. This process (the power stroke) requires ATP and is governed by the thin filament, a complex of actin, tropomyosin, and troponin. The authors have used a fast-mixing instrument to investigate the mechanism of regulation. Such (pre-steady-state kinetic) experiments allow biochemical intermediates in a working actomyosin cycle to be monitored. The regulatory focal point is demonstrated to be the step that involves the departure of inorganic phosphate (i.e., AM-ADP-Pi → AM-ADP). This part of the cycle, which lies on the main kinetic pathway and coincides with the drive stroke, is maximally accelerated ∼100-fold by the combined association of ligands (Ca[II] and rigor myosin heads) with the thin filament. However, the observed ligand dependencies of the rates of Pi dissociation that are reported herein are at variance with predictions of models derived from experiments where ATP hydrolysis is not taking place (and myosin exists in a nonphysiological form). It is concluded that the principal influence of the thin filament is in setting the rate of Pi dissociation and that physiological levels of regulation are dependent upon the liganded state of the thin filament as well as the conformation of myosin.

N-Terminal Domains of Cardiac Myosin Binding Protein C Cooperatively Activate the Thin Filament.

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.

Distinct sites in tropomyosin specify shared and isoform-specific regulation of myosins II and V.

Muscle contraction, cytokinesis, cellular movement, and intracellular transport depend on regulated actin-myosin interaction. Most actin filaments bind one or more isoform of tropomyosin, a coiled-coil protein that stabilizes the filaments and regulates interactions with other actin-binding proteins, including myosin. Isoform-specific allosteric regulation of muscle myosin II by actin-tropomyosin is well-established while that of processive myosins, such as myosin V, which transport organelles and macromolecules in the cell periphery, is less certain. Is the regulation by tropomyosin a universal mechanism, the consequence of the conserved periodic structures of tropomyosin, or is it the result of specialized interactions between particular isoforms of myosin and tropomyosin? Here, we show that striated muscle tropomyosin, Tpm1.1, inhibits fast skeletal muscle myosin II but not myosin Va. The non-muscle tropomyosin, Tpm3.1, in contrast, activates both myosins. To decipher the molecular basis of these opposing regulatory effects, we introduced mutations at conserved surface residues within the six periodic repeats (periods) of Tpm3.1, in positions homologous or analogous to those important for regulation of skeletal muscle myosin by Tpm1.1. We identified conserved residues in the internal periods of both tropomyosin isoforms that are important for the function of myosin Va and striated myosin II. Conserved residues in the internal and C-terminal periods that correspond to Tpm3.1-specific exons inhibit myosin Va but not myosin II function. These results suggest that tropomyosins may directly impact myosin function through both general and isoform-specific mechanisms that identify actin tracks for the recruitment and function of particular myosins.

The shaker-1 mouse myosin VIIa deafness mutation results in a severely reduced rate of the ATP hydrolysis step.

Mutations in the MYO7A gene, encoding the motor protein myosin VIIa, can cause Usher 1B, a deafness/blindness syndrome in humans, and the shaker-1 phenotype, characterized by deafness, head tossing, and circling behavior, in mice. Myosin VIIa is responsible for tension bearing and the transduction mechanism in the stereocilia and for melanosome transport in the retina, in line with the phenotypic outcomes observed in mice. However, the effect of the shaker-1 mutation, a R502P amino acid substitution, on the motor function is unclear. To explore this question, we determined the kinetic properties and the effect on the filopodial tip localization of the recombinant mouse myosin VIIa-5IQ-SAH R502P (myoVIIa-sh1) construct. Interestingly, although residue 502 is localized to a region thought to be involved in interacting with actin, the kinetic parameters for actin binding changed only slightly for the mutant construct. However, the rate constant for ATP hydrolysis (k+H + k-H) was reduced by ∼200-fold from 12 s-1 to 0.05 s-1, making the hydrolysis step the rate-limiting step of the ATPase cycle in the presence and absence of actin. Given that wild-type mouse myosin VIIa is a slow, high-duty ratio, monomeric motor, this altered hydrolysis rate would reduce activity to extremely low levels. Indeed, the translocation to the filopodial tips was hampered by the diminished motor function of a dimeric construct of the shaker-1 mutant. We conclude that the diminished motor activity of this mutant is most likely responsible for impaired hearing in the shaker-1 mice.

Ca2+-induced movement of tropomyosin on native cardiac thin filaments revealed by cryoelectron microscopy.

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 Ca2+ The current model of muscle regulation holds that at relaxing (low-Ca2+) conditions tropomyosin blocks myosin binding sites on F-actin, whereas at activating (high-Ca2+) 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 Ca2+ 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 Ca2+, 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 Ca2+ and lead to a mechanistic model for the regulation of the cardiac TF.

C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation.

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.

Omecamtiv Mecarbil modulates the kinetic and motile properties of porcine ß-cardiac myosin.

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.

Modulation of thin filament activation of myosin ATP hydrolysis by N-terminal domains of cardiac myosin binding protein-C.

We have used enzyme kinetics to investigate the molecular mechanism by which the N-terminal domains of human and mouse cardiac MyBP-C (C0C1, C1C2, and C0C2) affect the activation of myosin ATP hydrolysis by F-actin and by native porcine thin filaments. N-Terminal domains of cMyBP-C inhibit the activation of myosin-S1 ATPase by F-actin. However, mouse and human C1C2 and C0C2 produce biphasic activating and inhibitory effects on the activation of myosin ATP hydrolysis by native cardiac thin filaments. Low ratios of MyBP-C N-terminal domains to thin filaments activate myosin-S1 ATP hydrolysis, but higher ratios inhibit ATP hydrolysis, as is observed with F-actin alone. These data suggest that low concentrations of C1C2 and C0C2 activate thin filaments by a mechanism similar to that of rigor myosin-S1, whereas higher concentrations inhibit the ATPase rate by competing with myosin-S1-ADP-Pi for binding to actin and thin filaments. In contrast to C0C2 and C1C2, the activating effects of the C0C1 domain are species-dependent: human C0C1 activates actomyosin-S1 ATPase rates, but mouse C0C1 does not produce significant activation or inhibition. Phosphorylation of serine residues in the m-linker between the C1 and C2 domains by protein kinase-A decreases the activation of thin filaments by huC0C2 at pCa > 8 but has little effect on the activation mechanism at pCa = 4. In sarcomeres, the low ratio of cMyBP-C to actin is expected to favor the activating effects of cMyBP-C while minimizing inhibition produced by competition with myosin heads.