Phosphorylation-dependent Conformational Transition of the Cardiac Specific N-Extension of Troponin I in Cardiac Troponin

Abstract

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.

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.