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 10 μM. 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.

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][149][150][151][152][153][154][155][156][157][158] for cNTnC and cTnI for cCTnC. We used multinuclear multidimensional solution NMR spectroscopy to study cTnI in complex with cTnC. cTnI binds to the hydrophobic face of cCTnC, stabilizing an alpha helix in cTnI and a type VIII turn in cTnI [38][39][40][41]. In contrast, cTnI[1-37] remains disordered, although cTnI [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][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][149][150][151][152][153][154][155][156][157][158] presented to cNTnC, and this is how cTnI [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][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. heart failure | dilated cardiomyopathy | hypertrophic cardiomyopathy | post-translational modification | fuzzy complex T he balance between contraction and relaxation must be carefully regulated in the heart. Impaired relaxation can lead to diastolic heart failure, whereas systolic failure is characterized by insufficient contractility. Despite having different etiologies, both forms of heart failure are similar in terms of prevalence, symptoms, and mortality (1). Of all of the signaling pathways that regulate contractile function, the best studied is sympathetic β 1 -adrenergic stimulation (2), which leads to cardiomyocyte cAMP production and activation of protein kinase A (PKA). Downstream phosphorylation of L-type calcium channels and phospholamban increases calcium fluxes, whereas phosphorylation of sarcomeric proteins, cardiac troponin I (cTnI), cardiac myosin binding protein-C, and titin (3) regulates the calciuminduced mechanical response.In human cTnI, Ser22 and Ser23 are the residues most consistently phosphorylated (4, 5). (There are some numbering inconsistencies in the literature, and we will refer to Ser22/23 in...

The binding of Ca to cardiac troponin C (cTnC) triggers contraction in heart muscle. In the diseased heart, the myocardium is often desensitized to Ca, which leads to impaired contractility. Therefore, compounds that sensitize cardiac muscle to Ca (Ca-sensitizers) have therapeutic promise. The only Ca-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, Ca-bound conformation of cTnC. Finally, we propose a structural model of levosimendan bound to cTnC, which suggests that the Ca-sensitizing function of levosimendan is due to stabilization of the Ca-bound conformation of cTnC.

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.

One approach to improve contraction in the failing heart is the administration of calcium (Ca2 +) 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 Ca2 + 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 Ca2 + sensitivity of cardiac muscle. We conclude that i9 enhances Ca2 + sensitivity by stabilizing the open conformation of cTnC. These findings provide new insights into the in vivo mechanism of Ca2 + sensitization and demonstrate that directly targeting cTnC has significant potential in cardiovascular therapy.