Phosphorylation-dependent Conformational Transition of the Cardiac Specific N-Extension of Troponin I in Cardiac Troponin
Department of Chemistry, University of Utah, Salt Lake City, Utah, United States Journal of Molecular Biology
(Impact Factor: 4.33).
11/2007; 373(3):706-22. DOI: 10.1016/j.jmb.2007.08.035
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.
Available from: Steven Marston
- "This affects the cTnC interaction with both the regulatory Ca2+ and the TnI switch peptide (144–160) (Li et al., 2008). When TnI is unphosphorylated there is a weak ionic bond between the N terminal and the regulatory Ca2+-binding EF hand of TnC (Howarth et al., 2007). When Ser 22 and 23 are phosphorylated the binding is further weakened (Keane et al., 1990; Ferrieres et al., 2000; Ward et al., 2004a,b; Baryshnikova et al., 2008). "
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ABSTRACT: Contraction in the mammalian heart is controlled by the intracellular Ca(2+) concentration as it is in all striated muscle, but the heart has an additional signaling system that comes into play to increase heart rate and cardiac output during exercise or stress. β-adrenergic stimulation of heart muscle cells leads to release of cyclic-AMP and the activation of protein kinase A which phosphorylates key proteins in the sarcolemma, sarcoplasmic reticulum and contractile apparatus. Troponin I (TnI) and Myosin Binding Protein C (MyBP-C) are the prime targets in the myofilaments. TnI phosphorylation lowers myofibrillar Ca(2+)-sensitivity and increases the speed of Ca(2+)-dissociation and relaxation (lusitropic effect). Recent studies have shown that this relationship between Ca(2+)-sensitivity and TnI phosphorylation may be unstable. In familial cardiomyopathies, both dilated and hypertrophic (DCM and HCM), a mutation in one of the proteins of the thin filament often results in the loss of the relationship (uncoupling) and blunting of the lusitropic response. For familial dilated cardiomyopathy in thin filament proteins it has been proposed that this uncoupling is causative of the phenotype. Uncoupling has also been found in human heart tissue from patients with hypertrophic obstructive cardiomyopathy as a secondary effect. Recently, it has been found that Ca(2+)-sensitizing drugs can promote uncoupling, whilst one Ca(2+)-desensitizing drug Epigallocatechin 3-Gallate (EGCG) can reverse uncoupling. We will discuss recent findings about the role of uncoupling in the development of cardiomyopathies and the molecular mechanism of the process.
Available from: Genaro A Ramirez-Correa
- ", and cTnI N-term model from NMR structure (2JPW.pdb) from Howarth and colleagues , overimposed in a front view. (B) Models from (A) in a 90Њ axis rotation. "
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ABSTRACT: Global cardiac myofilament proteins phosphorylation levels, and their site-specific stoichiometry, are physiologically and clinically relevant for heart function. Unlike myofilament phosphorylation, other post-translational modifications (PTMs) such as O-GlcNAcylation, are just beginning to gain attention due to their potential physiological and clinical implications. This review will focus on what is currently known about cardiac Troponin I (cTnI) phosphorylation, and on the potential physiological and clinical impact of targeted proteomics including new findings on cTnI sites and stoichiometry. We will then discuss the increasing recognition of other myofilament PTMs functional relevance and the potential of targeted mass spectrometry approaches, particularly multiple reaction monitoring (MRM), for accelerating their systematic characterization. In addition, we will broadly discuss the development and application of MRM to quantitatively assess site-specific PTMs. Finally, we will give an overview of expert's consensus on MRM methods design/validation and best practices to develop MRM assays intended to reach clinical application. The unique ability of MRM and similar methods to identify and quantify cardiac myofilament PTMs is likely to become central in answering important biological questions in the field of cardiac integrative physiology.This article is protected by copyright. All rights reserved
Available from: Sachio Morimoto
- "In 2009, Carballo, et al. reported that the mutations of K36Q and N185K of cTnI cause DCM in an autosomal dominant manner. K36Q has been shown to mediate the movement of the N-terminal region in cTnI upon phosphorylation of S22/23 by cAMP- dependent protein kinase. Functional studies of K36Q and N185K have revealed that these DCM mutations of cTnI decrease the maximum activity and Ca2+-sensitivity of actin-myosin S1 ATPase and significantly reduce the Ca2+ binding affinity of the regulatory site of cTnC in the thin filament. "
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ABSTRACT: Genetic investigations of cardiomyopathy in the recent two decades have revealed a large number of mutations in the genes encoding sarcomeric proteins as a cause of inherited hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), or restrictive cardiomyopathy (RCM). Most functional analyses of the effects of mutations on cardiac muscle contraction have revealed significant changes in the Ca(2+)-regulatory mechanism, in which cardiac troponin (cTn) plays important structural and functional roles as a key regulatory protein. Over a hundred mutations have been identified in all three subunits of cTn, i.e., cardiac troponins T, I, and C. Recent studies on cTn mutations have provided plenty of evidence that HCM- and RCM-linked mutations increase cardiac myofilament Ca(2+) sensitivity, while DCM-linked mutations decrease it. This review focuses on the functional consequences of mutations found in cTn in terms of cardiac myofilament Ca(2+) sensitivity, ATPase activity, force generation, and cardiac troponin I phosphorylation, to understand potential molecular and cellular pathogenic mechanisms of the three types of inherited cardiomyopathy.
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