The C-Terminus of Troponin T Is Essential for Maintaining the Inactive State of Regulated Actin

Franklin, Andrew; Baxley, Tamatha; Kobayashi, Tomoyoshi; Chalovich, Joseph M.

doi:10.1016/j.bpj.2012.04.037

Cited by 37 publications

(80 citation statements)

References 44 publications

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“…Takeda et al [21] speculated that hcTnT R278 might be located within a Ca 2+ -sensitive interface between TnT and Tm, although experimental evidence indicates that removal of TnT’s C-terminus does not affect TnT-Tm binding [70] and that the two Tm-binding regions of TnT are located elsewhere [71]. The C-terminus of cTnT is involved in some form of interaction with other components of the thin filament as removal of the last fourteen amino acids—including R278—negatively affects Ca 2+ regulation of actomyosin function [72,73] and leads to FHC [74]. An alternative hypothesis is that TnT (residues 272–288) normally interacts with actin in the Ca 2+ free state.…”

Section: Discussionmentioning

confidence: 99%

Ca2+-regulatory function of the inhibitory peptide region of cardiac troponin I is aided by the C-terminus of cardiac troponin T: Effects of familial hypertrophic cardiomyopathy mutations cTnI R145G and cTnT R278C, alone and in combination, on filament sliding

Brunet¹,

Chase²,

Mihajlović³

et al. 2014

Archives of Biochemistry and Biophysics

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Investigations of cardiomyopathy mutations in Ca2+ regulatory proteins troponin and tropomyosin provide crucial information about cardiac disease mechanisms, and also provide insights into functional domains in the affected polypeptides. Hypertrophic cardiomyopathy-associated mutations TnI R145G, located within the inhibitory peptide (Ip) of human cardiac troponin I (hcTnI), and TnT R278C, located immediately C-terminal to the IT arm in human cardiac troponin T (hcTnT), share some remarkable features: structurally, biochemically, and pathologically. Using bioinformatics, we find compelling evidence that TnI and TnT, and more specifically the affected regions of hcTnI and hcTnT, may be related not just structurally but also evolutionarily. To test for functional interactions of these mutations on Ca2+-regulation, we generated and characterized Tn complexes containing either mutation alone, or both mutations simultaneously. The most important results from in vitro motility assays (varying [Ca2+], temperature or HMM density) show that the TnT mutant “rescued” some deleterious effects of the TnI mutant at high Ca2+, but exacerbated the loss of function, i.e., switching off the actomyosin interaction, at low Ca2+. Taken together, our experimental results suggest that the C-terminus of cTnT aids Ca2+-regulatory function of cTnI Ip within the troponin complex.

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Section: Discussionmentioning

confidence: 99%

Ca2+-regulatory function of the inhibitory peptide region of cardiac troponin I is aided by the C-terminus of cardiac troponin T: Effects of familial hypertrophic cardiomyopathy mutations cTnI R145G and cTnT R278C, alone and in combination, on filament sliding

Brunet¹,

Chase²,

Mihajlović³

et al. 2014

Archives of Biochemistry and Biophysics

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show abstract

“…By contrast, there is a controversy on the definition of the T2 domain (Katrukha, 2013). One group claims that the T2 domain spans residues 197-239 of TnT, corresponding to part of the IT arm (Jin and Chong, 2010), whereas other groups designate the last 16 residues of TnT as T2 (Franklin et al, 2012;Tanokura et al, 1982;Morris and Lehrer, 1984). Although the former idea is consistent with the "inverted" model, we argue that the last 16 residues of TnT forms the T2/TnT hook, which strongly binds to tropomyosin as it runs between the two helices of tropomyosin ( Fig.…”

Section: Comparison With the Previous Model Of Troponinmentioning

confidence: 99%

Cryo-EM structures of cardiac thin filaments reveal the 3D architecture of troponin

Oda

Yanagisawa

Wakabayashi

2020

Preprint

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Troponin is an essential component of striated muscle and it regulates the sliding of actomyosin system in a calcium-dependent manner. Despite its importance, the structure of troponin has been elusive due to its high structural heterogeneity. In this study, we analyzed the 3D structures of murine cardiac thin filaments using a cryo-electron microscope equipped with a Volta phase plate (VPP). Contrast enhancement by a VPP enabled us to reconstruct the entire repeat of the thin filament. We determined the orientation of troponin relative to F-actin and tropomyosin, and characterized the interactions between troponin and tropomyosin. This study provides a structural basis for understanding the molecular mechanism of actomyosin system. al., 2017). Although heavy-metal staining increases the contrast of troponin, resolution of the negatively-stained troponin map (~27 Å) is insufficient to make a meaningful comparison with crystal/NMR structures. To enhance the contrast of frozen-hydrated troponin and improve the accuracy of alignment, we utilized the VPP technology (Danev and Baumeister, 2016) for the cryo-EM of cardiac thin filaments. Focused 3D classification and multi-body refinement (Nakane et al., 2018) successfully solved the structures of thin filaments in sub-nanometer resolution and revealed calcium-dependent changes in the structural relationship between troponin and tropomyosin. This study has uncovered the long-sought piece of the puzzle in understanding the regulatory mechanism of actomyosin system. Results Cryo-EM of cardiac thin filamentsWe isolated thin and thick filaments from murine myocardium and reconstructed the 3D structures of the thin filaments in low-and high-calcium states. We initially failed to visualize the troponin structure on native thin filaments probably due to detachment of troponin upon blotting and freezing. Thus, we fixed the filaments using 0.25% glutaraldehyde (Risi et al., 2017), which greatly improved the occupancy of troponin on the filaments (Fig. 1A). Fortunately, it has been reported that glutaraldehyde-fixation does not inhibit the calcium-dependent activation of myosin by thin filaments, although the efficiency 5 of myosin-activation and mobility of tropomyosin were slightly altered (Risi et al., 2017).

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“…Binding studies of TnT2 have shown that both 51,52 the region N-terminal 53 and C-terminal 54 to the IT arm (cTnT 225 - 276 ) contribute to the interaction with tropomyosin, suggesting that the IT arm is rigidly fixed to tropomyosin. The C-terminus of cTnT, cTnT 275 - 288 , is also immediately adjacent to the cTnI1 28–147 inhibitory region, and both are essential for maintaining the blocked state of actin-tropomyosin 55 . On the other hand, one group has reported that the region corresponding to cTnT1 82 - 2 1 5 interacts with actin and contributes to the activation of actomyosin ATPase 56 .…”

Section: Structure and Function Of Cardiac Troponin C Within The Tropmentioning

confidence: 99%

Structure and function of cardiac troponin C (TNNC1): Implications for heart failure, cardiomyopathies, and troponin modulating drugs

Hwang

2015

Gene

106

105

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In striated muscle, the protein troponin complex turns contraction on and off in a calcium-dependent manner. The calcium-sensing component of the complex is troponin C, which is expressed from the TNNC1 gene in both cardiac muscle and slow-twitch skeletal muscle (identical transcript in both tissues) and the TNNC2 gene in fast-twitch skeletal muscle. Cardiac troponin C (cTnC) is made up of two globular EF-hand domains connected by a flexible linker. The structural C-domain (cCTnC) contains two high affinity calcium-binding sites that are always occupied by Ca2+ or Mg2+ under physiologic conditions, stabilizing an open conformation that remains anchored to the rest of the troponin complex. In contrast, the regulatory N-domain (cNTnC) contains a single low affinity site that is largely unoccupied at resting calcium concentrations. During muscle activation, calcium binding to cNTnC favors an open conformation that binds to the switch region of troponin I, removing adjacent inhibitory regions of troponin I from actin and allowing muscle contraction to proceed. Regulation of the calcium binding affinity of cNTnC is physiologically important, because it directly impacts the calcium sensitivity of muscle contraction. Calcium sensitivity can be modified by drugs that stabilize the open form of cNTnC, post-translational modifications like phosphorylation of troponin I, or downstream thin filament protein interactions that impact the availability of the troponin I switch region. Recently, mutations in cTnC have been associated with hypertrophic or dilated cardiomyopathy. A detailed understanding of how calcium sensitivity is regulated through the troponin complex is necessary for explaining how mutations perturb its function to promote cardiomyopathy and how post-translational modifications in the thin filament affect heart function and heart failure. Troponin modulating drugs are being developed for the treatment of cardiomyopathies and heart failure.

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The C-Terminus of Troponin T Is Essential for Maintaining the Inactive State of Regulated Actin

Cited by 37 publications

References 44 publications

Ca2+-regulatory function of the inhibitory peptide region of cardiac troponin I is aided by the C-terminus of cardiac troponin T: Effects of familial hypertrophic cardiomyopathy mutations cTnI R145G and cTnT R278C, alone and in combination, on filament sliding

Ca2+-regulatory function of the inhibitory peptide region of cardiac troponin I is aided by the C-terminus of cardiac troponin T: Effects of familial hypertrophic cardiomyopathy mutations cTnI R145G and cTnT R278C, alone and in combination, on filament sliding

Cryo-EM structures of cardiac thin filaments reveal the 3D architecture of troponin

Structure and function of cardiac troponin C (TNNC1): Implications for heart failure, cardiomyopathies, and troponin modulating drugs

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