Computational studies of the effect of the S23D/S24D troponin I mutation on cardiac troponin structural dynamics.

During ß-adrenergic stimulation, cardiac troponin I (cTnI) is phosphorylated by protein kinase A (PKA) at sites S23/S24, located at the N-terminus of cTnI. This phosphorylation has been shown to decrease KCa and pCa50, and weaken the cTnC-cTnI (C-I) interaction. We recently reported that phosphorylation results in an increase in the rate of early, slow phase of relaxation (kREL,slow) and a decrease in its duration (tREL,slow), which speeds up the overall relaxation. However, as the N-terminus of cTnI (residues 1-40) has not been resolved in the whole cardiac troponin (cTn) structure, little is known about the molecular-level behavior within the whole cTn complex upon phosphorylation of the S23/S24 residues of cTnI that results in these changes in function. In this study, we built up the cTn complex structure (including residues cTnC 1-161, cTnI 1-172, and cTnT 236-285) with the N-terminus of cTnI. We performed molecular-dynamics (MD) simulations to elucidate the structural basis of PKA phosphorylation-induced changes in cTn structure and Ca(2+) binding. We found that introducing two phosphomimic mutations into sites S23/S24 had no significant effect on the coordinating residues of Ca(2+) binding site II. However, the overall fluctuation of cTn was increased and the C-I interaction was altered relative to the wild-type model. The most significant changes involved interactions with the N-terminus of cTnI. Interestingly, the phosphomimic mutations led to the formation of intrasubunit interactions between the N-terminus and the inhibitory peptide of cTnI. This may result in altered interactions with cTnC and could explain the increased rate and decreased duration of slow-phase relaxation seen in myofibrils.

Phosphorylation modulates the mechanical stability of the cardiac myosin-binding protein C motif.

Cardiac myosin-binding protein C (cMyBP-C) is a thick-filament-associated protein that modulates cardiac contractility through interactions of its N-terminal immunoglobulin (Ig)-like C0-C2 domains with actin and/or myosin. These interactions are modified by the phosphorylation of at least four serines located within the motif linker between domains C1 and C2. We investigated whether motif phosphorylation alters its mechanical properties by characterizing force-extension relations using atomic force spectroscopy of expressed mouse N-terminal cMyBP-C fragments (i.e., C0-C3). Protein kinase A phosphorylation or serine replacement with aspartic acids did not affect persistence length (0.43 ± 0.04 nm), individual Ig-like domain unfolding forces (118 ± 3 pN), or Ig extension due to unfolding (30 ± 0.38 nm). However, phosphorylation did significantly decrease the C0-C3 mean contour length by 24 ± 2 nm. These results suggest that upon phosphorylation, the motif, which is freely extensible in the nonphosphorylated state, adopts a more stable and/or different structure. Circular dichroism and dynamic light scattering data for shorter expressed C1-C2 fragments with all four serines replaced by aspartic acids confirmed that the motif did adopt a more stable structure that was not apparent in the nonphosphorylated motif. These biophysical data provide both a mechanical and structural basis for cMyBP-C regulation by motif phosphorylation.

Structural insight into unique cardiac myosin-binding protein-C motif: a partially folded domain.

The structural role of the unique myosin-binding motif (m-domain) of cardiac myosin-binding protein-C remains unclear. Functionally, the m-domain is thought to directly interact with myosin, whereas phosphorylation of the m-domain has been shown to modulate interactions between myosin and actin. Here we utilized NMR to analyze the structure and dynamics of the m-domain in solution. Our studies reveal that the m-domain is composed of two subdomains, a largely disordered N-terminal portion containing three known phosphorylation sites and a more ordered and folded C-terminal portion. Chemical shift analyses, d(NN)(i, i + 1) NOEs, and (15)N{(1)H} heteronuclear NOE values show that the C-terminal subdomain (residues 315-351) is structured with three well defined helices spanning residues 317-322, 327-335, and 341-348. The tertiary structure was calculated with CS-Rosetta using complete (13)C(α), (13)C(ß), (13)C', (15)N, (1)H(α), and (1)H(N) chemical shifts. An ensemble of 20 acceptable structures was selected to represent the C-terminal subdomain that exhibits a novel three-helix bundle fold. The solvent-exposed face of the third helix was found to contain the basic actin-binding motif LK(R/K)XK. In contrast, (15)N{(1)H} heteronuclear NOE values for the N-terminal subdomain are consistent with a more conformationally flexible region. Secondary structure propensity scores indicate two transient helices spanning residues 265-268 and 293-295. The presence of both transient helices is supported by weak sequential d(NN)(i, i + 1) NOEs. Thus, the m-domain consists of an N-terminal subdomain that is flexible and largely disordered and a C-terminal subdomain having a three-helix bundle fold, potentially providing an actin-binding platform.

Role of the acidic N' region of cardiac troponin I in regulating myocardial function.

Cardiac troponin I (cTnI) phosphorylation modulates myocardial contractility and relaxation during beta-adrenergic stimulation. cTnI differs from the skeletal isoform in that it has a cardiac specific N' extension of 32 residues (N' extension). The role of the acidic N' region in modulating cardiac contractility has not been fully defined. To test the hypothesis that the acidic N' region of cTnI helps regulate myocardial function, we generated cardiac-specific transgenic mice in which residues 2-11 (cTnI(Delta2-11)) were deleted. The hearts displayed significantly decreased contraction and relaxation under basal and beta-adrenergic stress compared to nontransgenic hearts, with a reduction in maximal Ca(2+)-dependent force and maximal Ca(2+)-activated Mg(2+)-ATPase activity. However, Ca(2+) sensitivity of force development and cTnI-Ser(23/24) phosphorylation were not affected. Chemical shift mapping shows that both cTnI and cTnI(Delta2-11) interact with the N lobe of cardiac troponin C (cTnC) and that phosphorylation at Ser(23/24) weakens these interactions. These observations suggest that residues 2-11 of cTnI, comprising the acidic N' region, do not play a direct role in the calcium-induced transition in the cardiac regulatory or N lobe of cTnC. We hypothesized that phosphorylation at Ser(23/24) induces a large conformational change positioning the conserved acidic N region to compete with actin for the inhibitory region of cTnI. Consistent with this hypothesis, deletion of the conserved acidic N' region results in a decrease in myocardial contractility in the cTnI(Delta2-11) mice demonstrating the importance of acidic N' region in regulating myocardial contractility and mediating the response of the heart to beta-AR stimulation.

Phosphorylation-dependent conformational transition of the cardiac specific N-extension of troponin I in cardiac troponin.

We present here the solution structure for the bisphosphorylated form of the cardiac N-extension of troponin I (cTnI(1-32)), a region for which there are no previous high-resolution data. Using this structure, the X-ray crystal structure of the cardiac troponin core, and uniform density models of the troponin components derived from neutron contrast variation data, we built atomic models for troponin that show the conformational transition in cardiac troponin induced by bisphosphorylation. In the absence of phosphorylation, our NMR data and sequence analyses indicate a less structured cardiac N-extension with a propensity for a helical region surrounding the phosphorylation motif, followed by a helical C-terminal region (residues 25-30). In this conformation, TnI(1-32) interacts with the N-lobe of cardiac troponin C (cTnC) and thus is positioned to modulate myofilament Ca2+-sensitivity. Bisphosphorylation at Ser23/24 extends the C-terminal helix (residues 21-30) which results in weakening interactions with the N-lobe of cTnC and a re-positioning of the acidic amino terminus of cTnI(1-32) for favorable interactions with basic regions, likely the inhibitory region of cTnI. An extended poly(L-proline)II helix between residues 11 and 19 serves as the rigid linker that aids in re-positioning the amino terminus of cTnI(1-32) upon bisphosphorylation at Ser23/24. We propose that it is these electrostatic interactions between the acidic amino terminus of cTnI(1-32) and the basic inhibitory region of troponin I that induces a bending of cTnI at the end that interacts with cTnC. This model provides a molecular mechanism for the observed changes in cross-bridge kinetics upon TnI phosphorylation.

Derivation of structural restraints using a thiol-reactive chelator.

Recognition and identification of protein folds is a prerequisite for high-throughput structural genomics. Here we demonstrate a simple protocol for covalent attachment of a short and more rigid metal-chelating tag, thiol-reactive EDTA, by chemical modification of the single cysteine residue in barnase(H102C). Conjugation of the metal-chelating tag provides the advantage of allowing a greater range of paramagnetic metal substitutions. Substitution of Yb(3+), Mn(2+), and Co(2+) permitted measurement of metal-amide proton distances, dipolar shifts, and residual dipolar couplings. Paramagnetic-derived restraints are advantageous in the NMR structure elucidation of large protein complexes and are shown sufficient for validation of homology-based fold predictions.

Stereoselective binding of levosimendan to cardiac troponin C causes Ca2+-sensitization.

The effects of the Ca(2+) sensitizer levosimendan and that of its stereoisomer dextrosimendan on the cardiac contractile apparatus were studied using skinned fibers obtained from guinea pig hearts. Levosimendan was found to be more effective than dextrosimendan in this model. The respective concentrations of levosimendan and dextrosimendan at EC(50) were 0.3 and 3 microM. In order to explain the difference in efficacy as Ca(2+) sensitizers, the binding of the two stereoisomers on cardiac troponin C was studied by nuclear magnetic resonance in the absence and presence of two peptides of cardiac troponin I. The two stereoisomers interacted with both domains of cardiac troponin C in the absence of cardiac troponin I. In the presence of cardiac troponin I-(32-79) and cardiac troponin I-(128-180), the binding of both levosimendan and dextrosimendan to the C-terminal domain of cardiac troponin C was blocked and only the binding to the N-terminal domain was observable. Differences in the overall binding behavior of the two isomers to cardiac troponin C were highlighted in order to discuss their structure to activity relation. Our data are consistent with the notion that the action of levosimendan as a Ca(2+) sensitizer and positive inotrope relates to its stereoselective binding to Ca(2+)-saturated cardiac troponin C.

In vivo and in vitro analysis of cardiac troponin I phosphorylation.

Adrenergic stimulation induces positive changes in cardiac contractility and relaxation. Cardiac troponin I is phosphorylated at different sites by protein kinase A and protein kinase C, but the effects of these post-translational modifications on the rate and extent of contractility and relaxation during beta-adrenergic stimulation in the intact animal remain obscure. To investigate the effect(s) of complete and chronic cTnI phosphorylation on cardiac function, we generated transgenic animals in which the five possible phosphorylation sites were replaced with aspartic acid, mimicking a constant state of complete phosphorylation (cTnI-AllP). We hypothesized that chronic and complete phosphorylation of cTnI might result in increased morbidity or mortality, but complete replacement with the transgenic protein was benign with no detectable pathology. To differentiate the effects of the different phosphorylation sites, we generated another mouse model, cTnI-PP, in which only the protein kinase A phosphorylation sites (Ser(23)/Ser(24)) were mutated to aspartic acid. In contrast to the cTnIAllP, the cTnI-PP mice showed enhanced diastolic function under basal conditions. The cTnI-PP animals also showed augmented relaxation and contraction at higher heart rates compared with the nontransgenic controls. Nuclear magnetic resonance amide proton/nitrogen chemical shift analysis of cardiac troponin C showed that, in the presence of cTnI-AllP and cTnI-PP, the N terminus exhibits a more closed conformation, respectively. The data show that protein kinase C phosphorylation of cTnI plays a dominant role in depressing contractility and exerts an antithetic role on the ability of protein kinase A to increase relaxation.

Introduction of negative charge mimicking protein kinase C phosphorylation of cardiac troponin I. Effects on cardiac troponin C.

Protein kinase C phosphorylation of cardiac troponin, the Ca(2+)-sensing switch in muscle contraction, is capable of modulating the response of cardiac muscle to a Ca(2+) ion concentration. The N-domain of cardiac troponin I contains two protein kinase C phosphorylation sites. Although the physiological consequences of phosphorylation at Ser(43)/Ser(45) are known, the molecular mechanisms responsible for these functional changes have yet to be established. In this work, NMR was used to identify conformational and dynamic changes in cardiac troponin C upon binding a phosphomimetic troponin I, having Ser(43)/Ser(45) mutated to Asp. Chemical shift perturbation mapping indicated that residues in helix G were most affected. Smaller chemical shift changes were observed in residues located in the Ca(2+)/Mg(2+)-binding loops. Amide hydrogen/deuterium exchange rates in the C-lobe of troponin C were compared in complexes containing either the wild-type or phosphomimetic N-domain of troponin I. In the presence of a phosphomimetic domain, exchange rates in helix G increased, whereas a decrease in exchange rates for residues mapping to Ca(2+)/Mg(2+)-binding loops III and IV was observed. Increased exchange rates are consistent with destabilization of the Thr(129)-Asp(132) helix capping box previously characterized in helix G. The perturbation of helix G and metal binding loops III and IV suggests that phosphorylation alters metal ion affinity and inter-subunit interactions. Our studies support a novel mechanism for protein kinase C signal transduction, emphasizing the importance of C-lobe Ca(2+)/Mg(2+)-dependent troponin interactions.

Structure of the Mg2+-loaded C-lobe of cardiac troponin C bound to the N-domain of cardiac troponin I: comparison with the Ca2+-loaded structure.

Cardiac troponin C (cTnC) is the Ca(2+)-binding component of the troponin complex and, as such, is the Ca(2+)-dependent switch in muscle contraction. This protein consists of two globular lobes, each containing a pair of EF-hand metal-binding sites, connected by a linker. In the N lobe, Ca(2+)-binding site I is inactive and Ca(2+)-binding site II is primarily responsible for initiation of muscle contraction. The C lobe contains Ca(2+)/Mg(2+)-binding sites III and IV, which bind Mg(2+) with lower affinity and play a structural as well as a secondary role in modulating the Ca(2+) signal. To understand the structural consequences of Ca(2+)/Mg(2+) exchange in the C lobe, we have determined the NMR solution structure of the Mg(2+)-loaded C lobe, cTnC(81-161), in a complex with the N domain of cardiac troponin I, cTnI(33-80), and compared it with a refined Ca(2+)-loaded structure. The overall tertiary structure of the Mg(2+)-loaded C lobe is very similar to that of the refined Ca(2+)-loaded structure as evidenced by the root-mean-square deviation of 0.94 A for all backbone atoms. While metal-dependent conformational changes are minimal, substitution of Mg(2+) for Ca(2+) is characterized by condensation of the C-terminal portion of the metal-binding loops with monodentate Mg(2+) ligation by the conserved Glu at position 12 and partial closure of the cTnI hydrophobic binding cleft around site IV. Thus, conformational plasticity in the Ca(2+)/Mg(2+)-dependent binding loops may represent a mechanism to modulate C-lobe cTnC interactions with the N domain of cTnI.

Solution structure of calcium-saturated cardiac troponin C bound to cardiac troponin I.

Cardiac troponin C (TnC) is composed of two globular domains connected by a flexible linker. In solution, linker flexibility results in an ill defined orientation of the two globular domains relative to one another. We have previously shown a decrease in linker flexibility in response to cardiac troponin I (cTnI) binding. To investigate the relative orientation of calcium-saturated TnC domains when bound to cTnI, (1)H-(15)N residual dipolar couplings were measured in two different alignment media. Similarity in alignment tensor orientation for the two TnC domains supports restriction of domain motion in the presence of cTnI. The relative spatial orientation of TnC domains bound to TnI was calculated from measured residual dipolar couplings and long-range distance restraints utilizing a rigid body molecular dynamics protocol. The relative domain orientation is such that hydrophobic pockets face each other, forming a latch to constrain separate helical segments of TnI. We have utilized this structure to successfully explain the observed functional consequences of linker region deletion mutants. Together, these studies suggest that, although linker plasticity is important, the ability of TnC to function in muscle contraction can be correlated with a preferred domain orientation and interdomain distance.

The solution structure of a cardiac troponin C-troponin I-troponin T complex shows a somewhat compact troponin C interacting with an extended troponin I-troponin T component.

We have investigated the structure of the cTnC-cTnI-cTnT(198-298) calcium-saturated, ternary cardiac troponin complex by small-angle scattering with contrast variation. Shape restoration was also applied to the scattering information resulting from the deuterated cTnC subunit, the unlabeled cTnI-cTnT(198-298) subunits, and the entire complex. The experimental results and modeling indicate that cTnC adopts a partially collapsed conformation, while the cTnI-cTnT(198-298) components have an extended, rod-like structure. Shape restoration applied to the X-ray scattering data and the entire contrast variation series suggest that cTnC and the cTnI-cTnT(198-298) component lie with their long axes roughly parallel to one another with a relatively small surface area for interaction. Our findings indicate that the nature of the interactions between TnC and the TnI-TnT component differs significantly between the cardiac and skeletal isoforms as evidenced by the different degrees of compactness between the cardiac TnC and skeletal TnC in their respective ternary complexes and the fact that the cTnC subunit is not highly intertwined with the other subunits, as observed in the binary complex of the skeletal isoforms [Olah, G. A., and Trewhella, J. (1994) Biochemistry 33, 12800-12806].

Small-angle neutron scattering with contrast variation reveals spatial relationships between the three subunits in the ternary cardiac troponin complex and the effects of troponin I phosphorylation.

Small-angle neutron scattering with contrast variation has been used to determine the shapes and dispositions of the three subunits of cardiac troponin and to study the influence of phosphorylation on the structure. Three contrast variation series were collected on three different isotopically labeled variants of the cTnC/cTnI/cTnT(198-298) complex, one of which contained deuterated and bisphosphorylated cTnI. Analysis of the scattering data shows cTnT(198-298) interacting with a single lobe of a somewhat compacted cTnC that sits at one end of an elongated rodlike cTnI, covering about one-third of its length. The cTnT(198-298) sits near the center of the long cTnI axis. The components undergo significant conformational changes and reorientations in response to protein kinase A phosphorylation of cTnI. The rodlike cTnI bends sharply at the end interacting with the cTnC/cTnT(198-298) component, which reorients so as to maintain its contacts with cTnI while undergoing only a relatively small change in shape.