Troponin C Mutations Partially Stabilize the Active State of Regulated Actin and Fully Stabilize the Active State When Paired with Δ14 TnT.

Striated muscle contraction is regulated by the actin-associated proteins tropomyosin and troponin. The extent of activation of myosin ATPase activity is lowest in the absence of both Ca2+ and activating cross-bridges (i.e., S1-ADP or rigor S1). Binding of activating species of myosin to actin at a saturating Ca2+ concentration stabilizes the most active state (M state) of the actin-tropomyosin-troponin complex (regulated actin). Ca2+ binding alone produces partial stabilization of the active state. The extent of stabilization at a saturating Ca2+ concentration depends on the isoform of the troponin subunits, the phosphorylation state of troponin, and, in the case of cardiac muscle, the presence of hypertrophic cardiomyopathy-producing mutants of troponin T and troponin I. Cardiac dysfunction is also associated with mutations of troponin C (TnC). Troponin C mutants A8V, C84Y, and D145E increase the Ca2+ sensitivity of ATPase activity. We show that these mutants change the distribution of regulated actin states. The A8V and C84Y TnC mutants decreased the inactive B state distribution slightly at low Ca2+ concentrations, but the D145E mutants had no effect on that state. All TnC mutants increased the level of the active M state compared to that of the wild type, at a saturating Ca2+ concentration. Troponin complexes that contained two mutations that stabilize the active M state, A8V TnC and Δ14 TnT, appeared to be completely in the active state in the presence of only Ca2+. Because Ca2+ gives full activation, in this situation, troponin must be capable of positioning tropomyosin in the active M state without the need for rigor myosin binding.

Thermodynamics and molecular dynamics simulations of calcium binding to the regulatory site of human cardiac troponin C: evidence for communication with the structural calcium binding sites.

Human cardiac troponin C (HcTnC), a member of the EF hand family of proteins, is a calcium sensor responsible for initiating contraction of the myocardium. Ca(2+) binding to the regulatory domain induces a slight change in HcTnC conformation which modifies subsequent interactions in the troponin-tropomyosin-actin complex. Herein, we report a calorimetric study of Ca(2+) binding to HcTnC. Isotherms obtained at 25 °C (10 mM 2-morpholinoethanesulfonic acid, 50 mM KCl, pH 7.0) provided thermodynamic parameters for Ca(2+) binding to both the high-affinity and the low-affinity domain of HcTnC. Ca(2+) binding to the N-domain was shown to be endothermic in 2-morpholinoethanesulfonic acid buffer and allowed us to extract the thermodynamics of Ca(2+) binding to the regulatory domain. This pattern stems from changes that occur at the Ca(2+) site rather than structural changes of the protein. Molecular dynamics simulations performed on apo and calcium-bound HcTnC(1-89) support this claim. The values of the Gibbs free energy for Ca(2+) binding to the N-domain in the full-length protein and to the isolated domain (HcTnC(1-89)) are similar; however, differences in the entropic and enthalpic contributions to the free energy provide supporting evidence for the cooperativity of the C-domain and the N-domain. Thermograms obtained at two additional temperatures (10 and 37 °C) revealed interesting trends in the enthalpies and entropies of binding for both thermodynamic events. This allowed the determination of the change in heat capacity (∆C(p)) from a plot of ∆H verses temperature and may provide evidence for positive cooperativity of Ca(2+) binding to the C-domain.

The C-terminus of troponin T is essential for maintaining the inactive state of regulated actin.

Striated muscle contraction is regulated by the actin binding proteins tropomyosin and troponin. Defects in these proteins lead to myopathies and cardiomyopathies. Deletion of the 14 C-terminal residues of cardiac troponin T leads to hypertrophic cardiomyopathy. We showed earlier that regulated actin containing Δ14 TnT was more readily activated than wild-type regulated actin. We suggested that the equilibria among the inactive (blocked), intermediate (closed or calcium), and active (open or myosin) states was shifted to the active state. We now show that, in addition, such regulated actin filaments cannot enter the inactive or blocked state. Regulated actin containing Δ14 TnT had ATPase activities in the absence of Ca2+ that were higher than wild-type filaments but far below the fully active rate. The rapid dissociation of S1-ATP from regulated actin filaments containing Δ14 TnT and acrylodan-labeled tropomyosin did not show the fluorescence increase characteristic of moving to the inactive state. Replacing wild-type TnI with S45E TnI, that favors the inactive state, did not restore the fluorescence change. We conclude that TnT has a previously unrecognized role in forming the inactive state of regulated actin.

Acrylodan-labeled smooth muscle tropomyosin reports differences in the effects of troponin and caldesmon in the transition from the active state to the inactive state.

Changes in the orientation of tropomyosin on actin are important for the regulation of striated muscle contraction and could also be important for smooth muscle regulation. We showed earlier that acrylodan-labeled skeletal muscle tropomyosin reports the kinetics of the reversible transitions among the active, intermediate, and inactive states when S1 is rapidly detached from actin-tropomyosin. We now show that acrylodan-labeled smooth muscle tropomyosin reports similar transitions among states of actin-tropomyosin. When S1 was rapidly detached from actin-smooth muscle tropomyosin, there was a rapid decrease in acrylodan-tropomyosin fluorescence as the intermediate state became populated. The rate constant for this process was >600 s(-1) at temperatures near 5 °C. In the presence of skeletal troponin and EGTA, the decrease in fluorescence was followed by the redevelopment of fluorescence as the inactive state became populated. The apparent rate constant for the fluorescence increase was 14 s(-1) at 5 °C. Substituting smooth muscle caldesmon for skeletal muscle troponin produced a similar decrease and re-increase in fluorescence, but the apparent rate constant for the increase was >10 times that observed with troponin. Furthermore, the fluorescence increase was correlated with an increase in the extent of caldesmon attachment as S1-ATP dissociated. Although the measured rate constant appeared to reflect the rate-limiting transition for inactivation, it is unclear if the fluorescence change resulted from caldesmon binding, the movement of tropomyosin over actin, or both.

A computational and experimental approach to investigate bepridil binding with cardiac troponin.

Cardiac troponin is a Ca(2+)-dependent switch for the contraction in heart muscle and a potential target for drugs in the therapy of heart failure. Bepridil is a drug that binds to troponin and increases calcium sensitivity of muscle contraction. Because bepridil has been well studied, it is a good model for analysis by computational and experimental methods. Molecular dynamics (MD) simulations were performed on troponin complexes of different sizes in the presence and absence of bepridil bound within the hydrophobic pocket at the N-terminal domain of troponin C. About 100 ns of simulation trajectory data were generated, which were analyzed using cross-correlation analyses and MMPBSA and MMGBSA techniques. The results indicated that bepridil binding within the hydrophobic pocket of cardiac TnC decreases the interaction of TnC with TnI at both the N-domain of TnC and the C-domain of TnC, and decreases the correlations of motions among the segments of the troponin subunits. The estimated calcium-binding affinities using MMPBSA showed that bepridil has a sensitizing effect for the isolated system of TnC, but loses this effect for the complex. Our experimental measurements of calcium dissociation rates were consistent with that prediction. We also observed that while bepridil enhanced the troponin-tropomyosin-actin-activated ATPase activity of myosin S1 at low calcium concentrations it was slightly inhibitory at high calcium concentrations. Bepridil increases the ATPase activity and force generation in muscle fibers, but its effects appear to depend on the concentration of calcium.