The interaction of the bisphosphorylated N‐terminal arm of cardiac troponin I‐A <sup>31</sup>P‐NMR study

Schmidtmann, Anja; Lohmann, Karin; Jaquet, Kornélia

doi:10.1016/s0014-5793(02)02340-2

Cited by 12 publications

(11 citation statements)

References 32 publications

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“…This change may affect the Ca 2ϩ sensitivity of the cTnC N-domain. Schmidtmann et al (47) indicated in their 31 P-NMR study that the PKA phosphorylation signal is transmitted to the N-domain of cTnC by an indirect propagation through cTnI itself and interaction with cTnT. Because the region of Gly-159 is known to interact with the C-terminal end of the N-terminal extension of cTnI, the effect of Gly-159 we observed here may be due to global conformational changes in cTnI and cTnT as previously reported (15).…”

Section: Discussionsupporting

confidence: 83%

Structural Kinetics of Cardiac Troponin C Mutants Linked to Familial Hypertrophic and Dilated Cardiomyopathy in Troponin Complexes

Dong

Xing

Ouyang

et al. 2008

Journal of Biological Chemistry

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The key events in regulating cardiac muscle contraction involve Ca 2؉ binding to and release from cTnC (troponin C) and structural changes in cTnC and other thin filament proteins triggered by Ca 2؉ movement. Single mutations L29Q and G159D in human cTnC have been reported to associate with familial hypertrophic and dilated cardiomyopathy, respectively. We have examined the effects of these individual mutations on structural transitions in the regulatory N-domain of cTnC triggered by Ca 2؉ binding and dissociation. This study was carried out with a double mutant or triple mutants of cTnC, reconstituted into troponin with tryptophanless cTnI and cTnT. The double mutant, cTnC(L12W/N51C) labeled with 1,5-IAEDANS at Cys-51, served as a control to monitor Ca 2؉ -induced opening and closing of the N-domain by Förster resonance energy transfer (FRET). The triple mutants contained both L12W and N51C labeled with 1,5-IAEDANS, and either L29Q or G159D. Both mutations had minimal effects on the equilibrium distance between Trp-12 and Cys-51-AEDANS in the absence or presence of bound Ca 2؉ . L29Q had no effect on the closing rate of the N-domain triggered by release of Ca 2؉ , but reduced the Ca 2؉ -induced opening rate. G159D reduced both the closing and opening rates. Previous results showed that the closing rate of cTnC N-domain triggered by Ca 2؉ dissociation was substantially enhanced by PKA phosphorylation of cTnI. This rate enhancement was abolished by L29Q or G159D. These mutations alter the kinetics of structural transitions in the regulatory N-domain of cTnC that are involved in either activation (L29Q) or deactivation (G159D). Both mutations appear to be antagonistic toward phosphorylation signaling between cTnI and cTnC.Contraction of cardiac muscle is activated by the binding of Ca 2ϩ to the Ca 2ϩ -binding subunit troponin C (cTnC) 2 of the trimeric troponin complex. cTnC, the other two troponin subunits (troponin I (cTnI) and troponin T (cTnT)) and tropomyosin form the regulatory system of the contractile apparatus. These proteins are located in the thin filament. Contraction occurs when the myosin head in the thick filament interacts with actin causing the two filaments to slide past each other. The troponin complex in the thin filament regulates the actinmyosin interaction. Ca 2ϩ binding to the single site in the N-domain of cTnC initiates the activation process. cTnC has two globular regions, an N-terminal domain and a C-terminal domain. The N-domain has only one Ca 2ϩ binding site (site II) and the C-domain contains two binding site (binding sites III and IV). Ca 2ϩ binding to site II in the presence of cTnI induces an open conformation of the N-domain to expose a hydrophobic patch in the domain (1-3). Once exposed, the hydrophobic patch binds strongly with the regulatory region of cTnI. This strong interaction pulls cTnI away from actin and relieves the inhibition of actomyosin ATPase and muscle contraction. This regulation process is further fine-tuned with phosphorylation of cTnI serine 23 and serine 24...

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Section: Discussionsupporting

confidence: 83%

Structural Kinetics of Cardiac Troponin C Mutants Linked to Familial Hypertrophic and Dilated Cardiomyopathy in Troponin Complexes

Dong

Xing

Ouyang

et al. 2008

Journal of Biological Chemistry

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“…This interaction is nearly abolished upon bisphosphorylation of cTnI at serine 22 and serine 23. Furthermore, previous 31 P‐NMR measurements indicated that an interaction of the bisphosphorylated N terminus occurs within the cardiac troponin complex [12,17]. An interaction of the bisphosphorylated N‐teminal arm with cTnC can be excluded, but may occur within cTnI itself [17].…”

Section: Discussionmentioning

confidence: 99%

Cardiac troponin C‐L29Q, related to hypertrophic cardiomyopathy, hinders the transduction of the protein kinase A dependent phosphorylation signal from cardiac troponin I to C

et al. 2005

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We investigated structural and functional aspects of the first mutation in TNNC1, coding for the calcium‐binding subunit (cTnC) of cardiac troponin, which was detected in a patient with hypertrophic cardiomyopathy [ Hoffmann B, Schmidt‐Traub H, Perrot A, Osterziel KJ & Gessner R (2001) Hum Mut17, 524]. This mutation leads to a leucine–glutamine exchange at position 29 in the nonfunctional calcium‐binding site of cTnC. Interestingly, the mutation is located in a putative interaction site for the nonphosphorylated N‐terminal arm of cardiac troponin I (cTnI) [ Finley NL, Abbott MB, Abusamhadneh E, Gaponenko V, Dong W, Seabrook G, Howarth JW, Rana M, Solaro RJ, Cheung HC et al. (1999) EJB Lett453, 107–112]. According to peptide array experiments, the nonphosphorylated cTnI arm interacts with cTnC around L29. This interaction is almost abolished by L29Q, as observed upon protein kinase A‐dependent phosphorylation of cTnI at serine 22 and serine 23 in wild‐type troponin. With CD spectroscopy, minor changes are observed in the backbone of Ca2+‐free and Ca2+‐saturated cTnC upon the L29Q replacement. A small, but significant, reduction in calcium sensitivity was detected upon measuring the Ca2+‐dependent actomyosin subfragment 1 (actoS1)‐ATPase activity and the sliding velocity of thin filaments. The maximum actoS1‐ATPase activity, but not the maximum sliding velocity, was significantly enhanced. In addition, we performed our investigations at different levels of protein kinase A‐dependent phosphorylation of cTnI. The in vitro assays mainly showed that the Ca2+ sensitivity of the actoS1‐ATPase activity, and the mean sliding velocity of thin filaments, were no longer affected by protein kinase A‐dependent phosphorylation of cTnI owing to the L29Q exchange in cTnC. The findings imply a hindered transduction of the phosphorylation signal from cTnI to cTnC.

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“…The phosphorylation-induced enhancement of the Ca 2+ dissociation rate appears to be associated with global conformational changes in cTnI as shown by fluorescence anisotropy [42] and FRET measurements [43]. Additional functional changes induced by cTnI phosphorylation may be related to altered protein-protein interactions within the cTn complex [44–46]. …”

Section: Discussionmentioning

confidence: 99%

Pathogenesis associated with a restrictive cardiomyopathy mutant in cardiac troponin T is due to reduced protein stability and greatly increased myofilament Ca2+ sensitivity

Parvatiyar

Pinto

2015

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background Dilated and hypertrophic cardiomyopathy mutations in troponin can blunt effects of protein kinase A (PKA) phosphorylation of cardiac troponin I (cTnI), decreasing myofilament Ca2+-sensitivity; however this effect has never been tested for restrictive cardiomyopathy (RCM) mutants. This study explores whether an RCM cardiac troponin T mutant (cTnT-ΔE96) interferes with convergent PKA regulation and if TnT instability contributes to greatly enhanced Ca2+-sensitivity in skinned fibers. Methods and Results A decrease of −0.26 and −0.25 pCa units in Ca2+-sensitivity of contraction after PKA incubation was observed for skinned fibers incorporated with WT or cTnT-ΔE96, respectively. To further assess whether cTnT-ΔE96 interferes solely with transmission of cTnI phosphorylation effects, skinned fibers were reconstituted with PKA pseudo-phosphorylated cTnI (cTnI-SS/DD.cTnC). Fibers displaced with cTnT-WT, reconstituted with cTnI-SS/DD.cTnC decreased Ca2+-sensitivity of force (pCa50 = 5.61) compared to control cTnI-WT. cTnC (pCa50 = 5.75), similarly affecting cTnT-ΔE96 (pCa50 = 6.03) compared to control cTnI-WT.cTnC (pCa50 = 6.14). Fluorescence studies measuring cTnCIAANS Ca2+-affinity changes due to cTnT-ΔE96 indicated higher complexity (thin filament) better recapitulates skinned fiber Ca2+ sensitive changes. Circular Dichroism revealed reduced α-helicity and earlier thermal unfolding for cTnT-ΔE96 compared to WT. Conclusions 1) although ineffective in decreasing myofilament Ca2+-sensitivity to normal levels, cTnT-ΔE96 does not interfere with PKA cTnI phosphorylation mediated effects; 2) cTnT-ΔE96 requires actin to increase cTnC Ca2+-affinity; and 3) deletion of E96 reduces cTnT stability, likely disrupting crucial thin filament interactions. General Significance The pathological effect of cTnT-ΔE96 is largely manifested by dramatic myofilament Ca2+-sensitization which still persists even after PKA phosphorylation mediated Ca2+-desensitization.

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The interaction of the bisphosphorylated N‐terminal arm of cardiac troponin I‐A ³¹P‐NMR study

Cited by 12 publications

References 32 publications

Structural Kinetics of Cardiac Troponin C Mutants Linked to Familial Hypertrophic and Dilated Cardiomyopathy in Troponin Complexes

Structural Kinetics of Cardiac Troponin C Mutants Linked to Familial Hypertrophic and Dilated Cardiomyopathy in Troponin Complexes

Cardiac troponin C‐L29Q, related to hypertrophic cardiomyopathy, hinders the transduction of the protein kinase A dependent phosphorylation signal from cardiac troponin I to C

Pathogenesis associated with a restrictive cardiomyopathy mutant in cardiac troponin T is due to reduced protein stability and greatly increased myofilament Ca2+ sensitivity

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The interaction of the bisphosphorylated N‐terminal arm of cardiac troponin I‐A 31P‐NMR study

Cited by 12 publications

References 32 publications

Structural Kinetics of Cardiac Troponin C Mutants Linked to Familial Hypertrophic and Dilated Cardiomyopathy in Troponin Complexes

Structural Kinetics of Cardiac Troponin C Mutants Linked to Familial Hypertrophic and Dilated Cardiomyopathy in Troponin Complexes

Cardiac troponin C‐L29Q, related to hypertrophic cardiomyopathy, hinders the transduction of the protein kinase A dependent phosphorylation signal from cardiac troponin I to C

Pathogenesis associated with a restrictive cardiomyopathy mutant in cardiac troponin T is due to reduced protein stability and greatly increased myofilament Ca2+ sensitivity

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The interaction of the bisphosphorylated N‐terminal arm of cardiac troponin I‐A ³¹P‐NMR study