Ca2+-induced Conformational Transition in the Inhibitory and Regulatory Regions of Cardiac Troponin I

Dong, Wen‐Ji; Robinson, John M.; Stagg, Scott M.; Xing, Jun; Cheung, Herbert C.

doi:10.1074/jbc.m212886200

Cited by 44 publications

(49 citation statements)

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“…Based on their crystal structures of fsTn complex in the presence and absence of Ca 2+ , Vinogradova et al [36] proposed a model for Tn activation and relaxation, in which the central linker region of TnC undergoes disordered to helical structure upon activation, whereas the inhibitory region undergoes short helix to flexible extended conformation. Consistent with their proposal, experimental results suggest that the inhibitory region undergoes a conformational switch from a β-hairpin type structure when bound to actin to a more elongated structure having a propensity to form a helix [36][37][38]. The elongated inhibitory region interacts with the central helical region of TnC to stabilize the active form of the Tn complex.…”

Section: Molecular Mechanisms Of Ctni Function In Ca2+ and Crossbridgsupporting

confidence: 74%

The unique functions of cardiac troponin I in the control of cardiac muscle contraction and relaxation

Solaro

Rosevear

Kobayashi

2008

Biochemical and Biophysical Research Communications

104

119

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We review development of evidence and current perceptions of the multiple and significant functions of cardiac troponin I in regulation and modulation of cardiac function. Our emphasis is on the unique structure function relations of the cardiac isoform of troponin I, especially regions containing sites of phosphorylation. The data indicate that modifications of specific regions cardiac troponin I by phosphorylations either promote or reduce cardiac contractility. Thus, a homeostatic balance in these phosphorylations is an important aspect of control of cardiac function. A new concept is the idea that the homeostatic mechanisms may involve modifications of intra-molecular interactions in cardiac troponin I. KeywordsCardiac; Sarcomeres; Adrenergic stimulation; Mechanics; Kinases; Phosphatases In the time since the troponin discovery and naming by the laboratory of Setsuro Ebashi [1], there has been a constant stream of evidence indicating the substantial role of modifications in cardiac troponin (cTn) as a critical control element in the body's response to physiological stressors such as the hemodynamic demands of exercise. It is now widely recognized that regulatory processes at the level of the sarcomeres and the hetero-trimeric Tn complex involve not only the reception of Ca 2+ by cTnC, but also transduction of this Ca-binding signal by cTnI and cTnT [2,3]. Multiple interactions of cTnI and cTnT with actin and tropomyosin (Tm) control the number and kinetics of interactions of cross-bridges with the thin filament. Our focus here on cTnI serves to highlight the significant impact of modifications in the function of Tn in working cardiac myocytes and in the integrated physiological responses to exercise, which we have reviewed previously [4,5]. Evidence summarized below indicates that phosphorylation of cTnI by adrenergic signaling cascades is an indispensable control mechanism in matching cardiac output to venous return and myocyte dynamics to heart rate. Specific modifications in troponin I affect the dynamics and intensity of the heart beatElucidation of the function of an N-terminal extension of ~30 amino acids with sites (Ser-23, Ser-24) of phosphorylation by protein kinase A (PKA), not present in the fast skeletal (fsTnI) or slow skeletal isoforms (ssTnI), provided the first evidence that cTnI may have a special role * Corresponding

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Section: Molecular Mechanisms Of Ctni Function In Ca2+ and Crossbridgsupporting

confidence: 74%

The unique functions of cardiac troponin I in the control of cardiac muscle contraction and relaxation

Solaro

Rosevear

Kobayashi

2008

Biochemical and Biophysical Research Communications

104

119

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“…Upon Ca 2+ binding to the single Ca 2+ binding site at the N-domain of cTnC, the regulatory processes occurring on the thin filament can be characterized by a series of structural changes in the thin filament proteins. These changes include an opening of the N-domain of cTnC (4,5), changes in the conformations of the inhibitory region (Ir) and the regulatory region (Rr) of cTnI (6,7), a switch of the Ir/Rr of cTnI from interacting with actin to interacting with cTnC (8), and movement of Tm on the actin surface (9). These transitions are the structural basis of thin filament regulation and result in force generation via strong interactions between actin and myosin head (S1).…”

mentioning

confidence: 99%

Effects of PKA Phosphorylation of Cardiac Troponin I and Strong Crossbridge on Conformational Transitions of the N-Domain of Cardiac Troponin C in Regulated Thin Filaments

Dong

Jayasundar

et al. 2007

Biochemistry

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Regulation of cardiac muscle function is initiated by binding of Ca 2+ to troponin C (cTnC) which induces a series of structural changes in cTnC and other thin filament proteins. These structural changes are further modulated by crossbridge formation and fine tuned by phosphorylation of cTnI. The objective of the present study is to use a new Förster Resonance Energy Transfer-based structural marker to distinguish structural and kinetic effects of Ca 2+ binding, crossbridge interaction and protein kinase A phosphorylation of cTnI on the conformational changes of the cTnC N-domain. The FRET-based structural marker was generated by attaching AEDANS to one cysteine of a doublecysteine mutant cTnC(13C/51C) as a FRET donor and attaching DDPM to the other cysteine as the acceptor. The doubly labeled cTnC mutant was reconstituted into the thin filament by adding cTnI, cTnT, tropomyosin and actin. Changes in the distance between Cys13 and Cys51 induced by Ca 2+ binding/dissociation were determined by FRET-sensed Ca 2+ titration and stopped-flow studies, and time-resolved fluorescence measurements. The results showed that the presence of both Ca 2+ and strong binding of myosin head to actin was required to achieve a fully open structure of the cTnC N-domain in regulated thin filaments. Equilibrium and stopped-flow studies suggested that strongly bound myosin head significantly increased the Ca 2+ sensitivity and changed the kinetics of the structural transition of the cTnC N-domain. PKA phosphorylation of cTnI impacted the Ca 2+ sensitivity and kinetics of the structural transition of the cTnC N-domain but showed no global structural effect on cTnC opening. These results provide an insight into the modulation mechanism of strong crossbridge and cTnI phosphorylation in cardiac thin filament activation/relaxation processes. Keywordsphosphorylation; cardiac troponin C; thin filament; FRET; Ca 2+ activation; kinetics Force development during cardiac muscle contraction is dependent upon the strong interactions between myosin and the actin filament. These interactions are governed by the regulatory

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“…In this case, NMR structural and relaxation data of a slightly different CLD construct have shown that both the N-and C-terminal lobes exhibit significant chemical exchange, 3 a situation unlikely to produce highly anisotropic orientations especially between D-A combinations between the two lobes. Lackowicz and co-workers (17,25) have also shown experimentally using anisotropy measurements that this assumption produces negligible error in similar studies of the related TnC protein, and Cheung and co-workers (13,14,16) have confirmed this result with complexed TnC. It is also important to note that in steady-state FRET measurements, the range of validity for R is Ϯ50% of R 0 (23).…”

Section: Methodsmentioning

confidence: 52%

“…Effect of Conformational Averaging-The approach used in the current study for obtaining spatial interdomain information has been employed extensively recently to characterize proteins involved in cardiac muscle contraction (13,14,16,17) including the closely related TnC helix-loop-helix Ca 2ϩ -binding regulatory protein, which has Ͼ37% identity to CLD. These studies have measured intramolecular and intermolecular distances between TnC and the inhibitory subunit troponin I, which interact in a manner similar to JD-CLD.…”

Section: Discussionmentioning

confidence: 99%

“…Selectively mutating the N-terminal residues to Trp and labeling the Cys with IAEDANS allowed us to obtain interdomain FRET measurements. Similar FRET measurements have been reported for studies of the related calcium-regulatory protein troponin C (TnC) and its interaction with troponin I (13)(14)(15)(16)(17). The validity of the interdomain distances obtained for CLD was assessed by using triple mutants to give N-terminal intradomain measurements and compared with the known NMR structural data.…”

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confidence: 90%

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Conformational Changes in the Ca2+-regulatory Region from Soybean Calcium-dependent Protein Kinase-α

Weljie¹,

Robertson

Vogel

2003

Journal of Biological Chemistry

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Calcium-dependent protein kinases are key proteins involved in plant and protozoal Ca 2؉ signaling. These unique molecules include a calcium regulatory calmodulin-like domain (CLD), which binds to another small regulatory domain named the junction domain (JD). Both CLD and JD are part of the same polypeptide as the protein kinase domain. The CLD consists of N-and C-terminal lobes, each having two helix-loop-helix Ca 2؉ -binding motifs. In this study, fluorescence resonance energy transfer using a series of Trp and Cys site-directed mutants was undertaken to probe the relative motions of the two lobes of CLD between the apo-and Ca 2؉ -saturated forms, as well as bound to a peptide encoding the JD sequence. Using an IAEDANS-modified Cys, a total of 15 Trp 3 Cys distances were measured in these three states from the five donor-acceptor combinations F334W-Cys 436 , L371W-Cys 436 , L403W-Cys 436 , F334W-L403C, and L371W-L403C. Consistent with recently reported NMR diffusion measurements and with 1 H, 15 N heteronuclear correlation NMR spectra, the distances derived from fluorescence resonance energy transfer measurements in apoCLD indicate partial unfolding and a subsequent contraction on binding Ca 2؉ , which is maintained on addition of the JD peptide. Interpretation of the distances suggests that the Ca 2؉ -saturated form is open with the two lobes sitting side-by-side although highly flexible. The transition to the JD-CLD state appears to be accompanied by a rotation of the N-and C-terminal domains with respect to each other inducing a slightly more closed overall complex. The observed differences between the behavior of CLD and the well studied related protein calmodulin are likely because of different physiological requirements for activation in vivo.Calcium-dependent protein kinases (CDPKs) 1 play a crucial role in the Ca 2ϩ -induced signaling pathways of both plants and protists (1-3). The CDPK response is calcium-dependent but calmodulin (CaM)-independent, presumably because of the presence of an internal Ca 2ϩ -regulatory region (calmodulinlike domain, or CLD) near the C terminus of CDPK. The CLD is very similar (Ͼ40% identity) to the ubiquitous calcium-regulatory protein CaM. The target for the CLD is another short domain found in CDPK entitled the junction domain (JD), which connects the CLD with the N-terminal protein kinase domain (4, 5).In vivo, increases in the cytosolic [Ca 2ϩ ] presumably are thought to induce a structural change directly from the apo state of the CLD to the activated (JD-CLD) form. Bimolecular studies (2, 6, 7) demonstrate that significant activity can be reconstituted using truncated CDPK constructs lacking the CLD with exogenous CLD in the presence of Ca 2ϩ . Although recent structural work demonstrates that the CLD shares the same global fold as CaM, it is clear that functionally (2, 6, 7) and structurally there are also some significant distinctions. 2 Like CaM (8 -10), CLD is comprised of two domains, each consisting of two helix-loop-helix EF-hand Ca 2ϩ binding loops (see Fi...

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Ca2+-induced Conformational Transition in the Inhibitory and Regulatory Regions of Cardiac Troponin I

Cited by 44 publications

References 32 publications

The unique functions of cardiac troponin I in the control of cardiac muscle contraction and relaxation

The unique functions of cardiac troponin I in the control of cardiac muscle contraction and relaxation

Effects of PKA Phosphorylation of Cardiac Troponin I and Strong Crossbridge on Conformational Transitions of the N-Domain of Cardiac Troponin C in Regulated Thin Filaments

Conformational Changes in the Ca2+-regulatory Region from Soybean Calcium-dependent Protein Kinase-α

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