Journal of Molecular Biology
Phosphorylation-dependent Conformational Transition of the Cardiac Specific N-Extension of Troponin I in Cardiac Troponin
Introduction
The Ca2+ -sensitive switch of cardiac sarcomeres has structural and functional properties that appear specialized for the regulation of the heartbeat. In both skeletal and cardiac muscle, the switch consists of troponin (Tn), a ternary complex of three proteins (TnC, TnT and TnI) and tropomyosin (Tm). Ca2+ binding to TnC signals a movement of Tm releasing the thin filament from a prevailing inhibition by TnT and TnI, and promoting strong force generating reactions of cross-bridges. Ca2+ signaling through cardiac TnC and transduction of the Ca2+ -binding signal through TnT and TnI differs significantly between cardiac and fast skeletal muscle. Although crystal structures of both isoforms of TnC show a dumbbell-shaped, highly helical protein, the N terminal lobe of cTnC has a single functional regulatory Ca2+ -binding site (site II), which triggers contraction,1., 2., [3] whereas fsTnC has two (site I and site II). Both cTnC and fsTnC have two active metal binding sites (sites III and IV) in the C-terminal lobe, which forms the core of the troponin complex throughout the contraction cycle.
Structural studies on the regulatory domains of skeletal and cardiac TnC reveal important differences in their conformational response to Ca2+ binding. Calcium binding at site I and site II of skeletal (sk)TnC results in an “opening” of the N-terminal regulatory domain via the concerted opening of the EF-hand motifs that exposes a hydrophobic cleft for binding of the switch region of skTnI.4., 5., 6. Calcium binding alone at site II of cTnC does not induce such an opening.[7], 8.
Troponin I, the inhibitory component of the troponin complex, make multiple Ca2+-dependent interactions with TnC, TnT, Tm, and actin. The inhibitory region, residues 137 to 148 (Figure 1), plays a central role in restraining Tm in a blocking position and comprises the minimum sequence necessary for inhibition of actomyosin Mg2+ATPase activity. Thus, the Ca2+-dependent switch between relaxation and contraction involves movement of the inhibitory region on actin-Tm. In the presence of Ca2+, the inhibitory region of cardiac (c)TnI is located alongside the linker region in cTnC.9., 10. Similarly, the inhibitory region of skTnI has recently been shown to lie alongside the interconnecting central helix of skTnC in the skeletal Tn complex.11., 12. In the absence of Ca2+, the inhibitory region of TnI moves away from TnC and interacts with actin.11., 13., [14], 15., 16., 17.
Cardiac TnI has an N-terminal extension of ∼27 to 33 residues containing two cAMP-dependent protein kinase A (PKA) phosphorylation sites, serine residues 23 and 24 in the mouse isoform (Figure 1).18 The phosphorylation motifs are adjacent to a conserved Xaa-Pro region (residues 12–18) of unknown function. In addition, a conserved acidic region is located proximal to the Xaa-Pro region (Figure 1).
Phosphorylation of the cardiac N-extension of cTnI, decreases the Ca2+-sensitivity of muscle contraction, increases the off-rate of Ca2+ dissociation from the regulatory domain of cTnC, increases relaxation and cross-bridge cycling, and contributes to the β-adrenergic agonist induced acceleration of cardiac relaxation (lusitropy).[19], 20., 21., 22., 23., 24.
We have shown that the cardiac N-extension interacts weakly with the N-lobe of cTnC and alters regulatory domain conformational substates, presumably toward more open/active conformations.25., 26., 27., 28. Residues 22 to 34 of the cardiac N-extension provide the primary binding region with the N-lobe of cTnC.29 Phosphorylation at Ser23/24 weakens interactions between the cardiac N- extension of cTnI and the N-lobe of cTnC.25., 27., 28., [30]
To further explore structural relationships within the cardiac specific N-extension, we have completed solution NMR studies and bioinformatics analyses on cTnI(1-32), cTnI(1-32) phosphorylated at Ser23/24 (cTnI(1-32)pp), and cTnI(1-32) having Ser23/24 substituted with Asp (cTnI(1-32)DD), a suitable stable mimetic for examining the structural and dynamic consequences of phosphorylation.27 We determined the structure of the bisphosphorylated form (at Ser23/24) and found evidence for a less structured N-extension in absence of bisphosphorylation. A model for the conformational transition, induced by bisphosphorylation, was obtained by docking the non-phosphorylated and bisphosphorylated cardiac specific N-extensions onto the core structure of cardiac troponin.31 A key feature of our model is that bisphosphorylation extends and stabilizes the C-terminal helix in the cardiac N-extension, weakening interactions with the N-lobe of cTnC and resulting in a bending of cTnI and positioning of the acidic N terminus for electrostatic interactions with the basic inhibitory region of cTnI. This model aids in understanding the modulation of myofilament sensitivity by phosphorylation.
Section snippets
Characterization of cTnI(1-32), cTnI(1-32)DD, and cTnI(1-32)pp
To explore structural relationships within the cardiac N-extension, solution NMR studies were carried out on cTnI(1-32), cTnI(1-32)pp, and cTnI(1-32)DD, having Ser23/24 replaced by Asp27. Natural abundance 1H-15N heteronuclear single-quantum coherence (HSQC) spectra for each of the three cTnI(1-32) peptides are shown in Figure 2. The increased dispersion observed in the 1H-15N spectrum of cTnI(1-32)pp results from increased chemical shift perturbations in residues 21–27 as a consequence of
Discussion
We present here the first atomic model for the phosphorylation-dependent conformational changes of cTnI, including the N-extension for which there is no atomic resolution data. NMR analyses show that residues 1–20 of the cardiac N-extension, containing the Xaa-Pro region and the conserved acidic N terminus, are not significantly affected by introduction of negative charge at Ser23/24 (Figure 2, Figure 3). These data indicate that the first 20 residues of the cardiac specific N-extension have
Peptides
Synthetic peptides cTnI(1-32) and cTnI(1-32)DD were synthesized by Protein Express (Cincinnati, OH) and the sequence confirmed by mass spectroscopy. The cTnI(1-32) peptide had the following sequence: MADESSDAAGEPQPAPAPVRRRSSANYRAYAT-NH2. The purity of the peptide was confirmed by HPLC. The phosphorylated cTnI(1-32) peptide, cTnI(1-32)pp, was synthesized at the Genetic Engineering Facility of the University of Illinois Biotechnology Center. The extent of phosphorylation at Ser23/24 was greater
Acknowledgements
This research was supported by the United States Department of Defense grant ARO MURI DAAD 19-02-1-0027 and National Institutes of Health grant HL83334 (to P.R.R.), National Institutes of Health grant PO1 HL062426 (Project 1) (to R.J.S.), National Institutes of Health grants AI055338 and GM067823 (to J.M.), and US Department of Energy, grant no. DE-FG02-05ER64026 and ARC Federation Fellowship FF0457488 (to J.T.). We are grateful to Dr Bret Abbott, Dr Geneviève, M. C. Gasmi-Seabrook and Ekram
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