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

Abstract

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...

Cardiac Myosin-binding Protein C and Troponin-I Phosphorylation Independently Modulate Myofilament Length-dependent Activation

“…long) of ϩ16.7 mN/mm 2 (p Ͻ 0.001), whereas in contrast, the impact of TnI-DD versus TnI-AA on F max was not significant (p ϭ 0.1). For all groups, this analysis revealed a significant (p Ͻ 0.001) impact of SL on EC 50 . There were no significant differences for the n H .…”

“…The mechanism underlying cTnI PKA phosphorylation may be related to a molecular conformational change upon introduction of the negatively charged phosphates into the intrinsically disordered NЈ domain of the molecule (50). This rearrangement could involve new intramolecular interactions between the NЈ and the inhibitory peptide (IP) domain of cTnI (34).…”

“…͔ at half-maximal force development, and the Hill coefficient (n H ), a measure of cooperative activation. Data were collected at short and long SLs, allowing for the calculation of SL⌬EC 50 and the difference in EC 50 between the short and long SL. Passive force (SL⌬F pass ) was indexed as the difference in passive myocyte force in relaxing solution between the short and the long SLs.…”

“…p Ͻ 0.05 (***) AAA versus DDD and p Ͻ 0.05 (**) AA versus DD using two-way analysis of variance for the SL⌬ parameters (SL⌬EC 50 , SL⌬F max , ⌬SLF pass ); this analysis furthermore revealed that the average impact on SL⌬EC 50 of cMyBP-C-DDD versus cMyBP-C-AAA was Ϫ0.48 M and that of recombinant TnI (rTnI)-DD versus rTnI-AA was Ϫ0.24 M. *, p ϭ 0.01 AAA/AA versus all other groups. Three-way (repeated measures for SL) analysis of variance was used to test for the parameters F max , EC 50 , and n Hill (Hill parameter, index of activation cooperativity). For F max , this analysis revealed an overall impact for cMyBP-C-DDD versus cMyBP-C-AAA of ϩ6.2 mN/mm 2 (p Ͻ 0.001) and an overall impact in all groups for SL (short versus.…”

Structure and function of cardiac troponin C (TNNC1): Implications for heart failure, cardiomyopathies, and troponin modulating drugs

“…Recently, we have used solution NMR spectroscopy to study cTnI 1- 73 in complex with cTnC •3Ca 2+68 . In this complex, cTnI 39 -60 binds to cCTnC, stabilizing an α helix in cTnI 41 -67 and a type VIII turn in cTnI 38 -41 that brings cTnI1 9 - 3 7 into close proximity with the negatively charged surface of cNTnC (opposite the hydrophobic surface that binds the switch peptide, cTnI 148 -158).…”