Structures reveal details of small molecule binding to cardiac troponin.

In cardiac and skeletal muscle, the troponin complex turns muscle contraction on and off in a calcium-dependent manner. Many small molecules are known to bind to the troponin complex to modulate its calcium binding affinity, and this may be useful in a broad range of conditions in which striated muscle function is compromised, such as congestive heart failure. As a tool for developing drugs specific for the cardiac isoform of troponin, we have designed a chimeric construct (cChimera) consisting of the regulatory N-terminal domain of cardiac troponin C (cNTnC) fused to the switch region of cardiac troponin I (cTnI), mimicking the key binding event that turns on muscle contraction. We demonstrate by solution NMR spectroscopy that cChimera faithfully reproduces the native interface between cTnI and cNTnC. We determined that small molecules based on diphenylamine can bind to cChimera with a KD as low as 10µM. Solution NMR structures show that minimal structural perturbations in cChimera are needed to accommodate 3-methyldiphenylamine (3-mDPA), which is probably why it binds with higher affinity than previously studied compounds like bepridil, despite its significantly smaller size. The unsubstituted aromatic ring of 3-mDPA binds to an inner hydrophobic pocket adjacent to the central beta sheet of cNTnC. However, the methyl-substituted ring is able to bind in two different orientations, either inserting into the cNTnC-cTnI interface or "flipping out" to form contacts primarily with helix C of cNTnC. Our work suggests that preservation of the native interaction between cNTnC and cTnI is key to the development of a high affinity cardiac troponin-specific drug.

Reversible Covalent Binding to Cardiac Troponin C by the Ca2+-Sensitizer Levosimendan.

The binding of Ca2+ to cardiac troponin C (cTnC) triggers contraction in heart muscle. In the diseased heart, the myocardium is often desensitized to Ca2+, which leads to impaired contractility. Therefore, compounds that sensitize cardiac muscle to Ca2+ (Ca2+-sensitizers) have therapeutic promise. The only Ca2+-sensitizer used regularly in clinical settings is levosimendan. While the primary target of levosimendan is thought to be cTnC, the molecular details of this interaction are not well understood. In this study, we used mass spectrometry, computational chemistry, and nuclear magnetic resonance spectroscopy to demonstrate that levosimendan reacts specifically with cysteine 84 of cTnC to form a reversible thioimidate bond. We also showed that levosimendan only reacts with the active, Ca2+-bound conformation of cTnC. Finally, we propose a structural model of levosimendan bound to cTnC, which suggests that the Ca2+-sensitizing function of levosimendan is due to stabilization of the Ca2+-bound conformation of cTnC.

Probing the mechanism of cardiovascular drugs using a covalent levosimendan analog.

One approach to improve contraction in the failing heart is the administration of calcium (Ca(2+)) sensitizers. Although it is known that levosimendan and other sensitizers bind to troponin C (cTnC), their in vivo mechanism is not fully understood. Based on levosimendan, we designed a covalent Ca(2+) sensitizer (i9) that targets C84 of cTnC and exchanged this complex into cardiac muscle. The NMR structure of the covalent complex showed that i9 binds deep in the hydrophobic pocket of cTnC. Despite slightly reducing troponin I affinity, i9 enhanced the Ca(2+) sensitivity of cardiac muscle. We conclude that i9 enhances Ca(2+) sensitivity by stabilizing the open conformation of cTnC. These findings provide new insights into the in vivo mechanism of Ca(2+) sensitization and demonstrate that directly targeting cTnC has significant potential in cardiovascular therapy.

Structure and Dynamics of the Acidosis-Resistant A162H Mutant of the Switch Region of Troponin I Bound to the Regulatory Domain of Troponin C.

Intracellular acidosis lowers the Ca²âº sensitivity of cardiac muscle, which results in decreased force generation, decreased cardiac output, and, eventually, heart failure. The A162H mutant of cardiac troponin I in the thin filament turns the heart acidosis-resistant. Physiological and structural studies have provided insights into the mechanism of protection by the A162H substitution; however, the effect of other native residues of cardiac troponin I is not fully understood. In this study, we determined the structure of the A162H mutant of the switch region of cardiac troponin I bound to the regulatory domain of troponin C at pH 6.1, and the dynamics as a function of pH, by NMR spectroscopy to evaluate the changes induced by protonation of A162H. The results indicate that A162H induces a transitory curved conformation on troponin I that promotes contraction, but it is countered by residue E164 to ensure proper relaxation. Our model explains the absence of diastolic impairment in the gain-of-function phenotype induced by the A162H substitution as well as the effects of a variety of mutants studied previously. The description of this mechanism underlines the fine quality of regulation on cardiac muscle contraction and anticipates pharmacological agents that induce modest changes in the contraction-relaxation equilibrium to produce marked effects in cardiac performance.

The cardiac-specific N-terminal region of troponin I positions the regulatory domain of troponin C.

The cardiac isoform of troponin I (cTnI) has a unique 31-residue N-terminal region that binds cardiac troponin C (cTnC) to increase the calcium sensitivity of the sarcomere. The interaction can be abolished by cTnI phosphorylation at Ser22 and Ser23, an important mechanism for regulating cardiac contractility. cTnC contains two EF-hand domains (the N and C domain of cTnC, cNTnC and cCTnC) connected by a flexible linker. Calcium binding to either domain favors an "open" conformation, exposing a large hydrophobic surface that is stabilized by target binding, cTnI[148-158] for cNTnC and cTnI[39-60] for cCTnC. We used multinuclear multidimensional solution NMR spectroscopy to study cTnI[1-73] in complex with cTnC. cTnI[39-60] binds to the hydrophobic face of cCTnC, stabilizing an alpha helix in cTnI[41-67] and a type VIII turn in cTnI[38-41]. In contrast, cTnI[1-37] remains disordered, although cTnI[19-37] is electrostatically tethered to the negatively charged surface of cNTnC (opposite its hydrophobic surface). The interaction does not directly affect the calcium binding affinity of cNTnC. However, it does fix the positioning of cNTnC relative to the rest of the troponin complex, similar to what was previously observed in an X-ray structure [Takeda S, et al. (2003) Nature 424(6944):35-41]. Domain positioning impacts the effective concentration of cTnI[148-158] presented to cNTnC, and this is how cTnI[19-37] indirectly modulates the calcium affinity of cNTnC within the context of the cardiac thin filament. Phosphorylation of cTnI at Ser22/23 disrupts domain positioning, explaining how it impacts many other cardiac regulatory mechanisms, like the Frank-Starling law of the heart.

Versatile cardiac troponin chimera for muscle protein structural biology and drug discovery.

Investigation of the molecular interactions within and between subunits of the heterotrimeric troponin complex, and with other proteins in the sarcomere, has revealed salient structural elements involved in regulation of muscle contraction. The discovery of new cardiotonic drugs and structural studies utilizing intact troponin, or regulatory complexes formed between the key regions identified in troponin C and troponin I, face intrinsic and technical difficulties associated with weak protein-protein interactions and with solubility, aggregation, stability of the overall architecture, isotope labeling, and size, respectively. We have designed and characterized a chimeric troponin C-troponin I hybrid protein with a cleavable linker that is useful for producing isotopically labeled troponin peptides, stabilizes their interaction, and has proven to be a faithful representation of the original complex in the systolic state, but lacking its disadvantages, making it particularly suitable for drug screening and structural studies.

Conformation of the critical pH sensitive region of troponin depends upon a single residue in troponin I.

The calcium sensitivity of cardiac and skeletal muscle is reduced during cytosolic acidosis, and this inhibition is more pronounced in cardiac muscle. Replacing cardiac troponin I with skeletal troponin I reduces the pH sensitivity of cardiac muscle. This diminished pH sensitivity depends on a single amino acid difference in troponin I: an alanine in cardiac and a histidine in skeletal. Studies suggested that when this histidine is protonated, it forms an electrostatic interaction with glutamate 19 on the surface of cardiac troponin C. Structures of the skeletal and cardiac troponin complexes show very different conformations for the region of troponin I surrounding this residue. In this study, we determined the structure of skeletal troponin I bound to cardiac troponin C. Skeletal troponin I is found to bind to cardiac troponin C with histidine 130 in close proximity to glutamate 19. This conformation is homologous to the crystal structure of the skeletal troponin complex; but different than in the cardiac complex. We show that an A162H variant of cardiac troponin I adopts a conformation similar to the skeletal structure. The implications of these structural differences in the context of cardiac muscle regulation are discussed.

Interaction between the regulatory domain of cardiac troponin C and the acidosis-resistant cardiac troponin I A162H.

AIMS: Ischaemic heart disease is the leading cause of mortality worldwide. Acidosis is the main mediator of ischaemia and shielding against it might be possible. In this study, we characterize the nature of interaction between the regulatory domain of cardiac troponin C and the A162H-substituted cardiac troponin I (cTnI) that confers protection against acidosis. METHODS AND RESULTS: We used nuclear magnetic resonance spectroscopy to study the interaction of the Ca(2+)-saturated N-domain of cardiac troponin C with the switch region of cTnI containing the A162H substitution under normal and acidic conditions. Our results show that H162 increases the affinity of TnI for troponin C at pH 7 and this affinity is further enhanced at pH 6. To investigate the nature of the interactions responsible for such improvement, we determined the acid dissociation constants of the glutamate residues in troponin C. The results show that E15 and E19 exhibit deviations in their acid dissociation constant (pK(a)) profiles and reflect a common high pK(a) value of 6.8, indicating electrostatic interactions with H162. Residue H171 in wild-type cTnI does not play a similar role. CONCLUSION: This work provides evidence for the mechanism by which cTnI A162H improves myocardial performance during acidosis. The electrostatic interaction between residues E15 and E19 in troponin C and H162 in TnI at low pH is responsible for stabilizing the conformation of troponin C that leads to contraction, thus partially ablating the decreased Ca(2+)-sensitivity caused by acidosis.

Elucidation of isoform-dependent pH sensitivity of troponin i by NMR spectroscopy.

Myocardial ischemia is characterized by reduced blood flow to cardiomyocytes, which can lead to acidosis. Acidosis decreases the calcium sensitivity and contractile efficiency of cardiac muscle. By contrast, skeletal and neonatal muscles are much less sensitive to changes in pH. The pH sensitivity of cardiac muscle can be reduced by replacing cardiac troponin I with its skeletal or neonatal counterparts. The isoform-specific response of troponin I is dictated by a single histidine, which is replaced by an alanine in cardiac troponin I. The decreased pH sensitivity may stem from the protonation of this histidine at low pH, which would promote the formation of electrostatic interactions with negatively charged residues on troponin C. In this study, we measured acid dissociation constants of glutamate residues on troponin C and of histidine on skeletal troponin I (His-130). The results indicate that Glu-19 comes in close contact with an ionizable group that has a pK(a) of ∼6.7 when it is in complex with skeletal troponin I but not when it is bound to cardiac troponin I. The pK(a) of Glu-19 is decreased when troponin C is bound to skeletal troponin I and the pK(a) of His-130 is shifted upward. These results strongly suggest that these residues form an electrostatic interaction. Furthermore, we found that skeletal troponin I bound to troponin C tighter at pH 6.1 than at pH 7.5. The data presented here provide insights into the molecular mechanism for the pH sensitivity of different muscle types.

Structure of trans-resveratrol in complex with the cardiac regulatory protein troponin C.

Cardiac troponin, a heterotrimeric protein complex that regulates heart contraction, represents an attractive target for the development of drugs for treating heart disease. Cardiovascular diseases are one of the chief causes of morbidity and mortality worldwide. In France, however, the death rate from heart disease is remarkably low relative to fat consumption. This so-called "French paradox" has been attributed to the high level of consumption of wine in France, and the antioxidant trans-resveratrol is thought to be the primary basis for wine's cardioprotective nature. It has been demonstrated that trans-resveratrol increases the myofilament Ca(2+) sensitivity of guinea pig myocytes [Liew, R., Stagg, M. A., MacLeod, K. T., and Collins, P. (2005) Eur. J. Pharmacol. 519, 1-8]; however, the specific mode of its action is unknown. In this study, the structure of trans-resveratrol free and bound to the calcium-binding protein, troponin C, was determined by nuclear magnetic resonance spectroscopy. The results indicate that trans-resveratrol undergoes a minor conformational change upon binding to the hydrophobic pocket of the C-domain of troponin C. The location occupied by trans-resveratrol coincides with the binding site of troponin I, troponin C's natural binding partner. This has been seen for other troponin C-targeting inotropes and implicates the modulation of the troponin C-troponin I interaction as a possible mechanism of action for trans-resveratrol.