Solution structure of the regulatory domain of human cardiac troponin C in complex with the switch region of cardiac troponin I and W7: The basis of W7 as an inhibitor of cardiac muscle contraction

Oleszczuk, Marta; Robertson, Ian M.; Li, Monica X.; Sykes, Brian D.

doi:10.1016/j.yjmcc.2010.01.016

Cited by 37 publications

(66 citation statements)

References 35 publications

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“…Since neither the structure of cTnI 144–163 , nor the position of cTnI 144–163 is significantly perturbed by the presence of dfbp-o, it appears that dfbp-o is not competing for the same binding site on cNTnC. This is in contrast to W7[57] and bepridil[21], which sterically clash with cTnI and subsequently perturb the position of cTnI on cNTnC.…”

Section: Resultsmentioning

confidence: 99%

“…We have overlaid the secondary structures of these complexes with cNTnC•Ca 2+ •cTnI 144–163 •dfbp-o (figure 6) and found that the most similar was the sNTnC•sTnI 112–131 •anapoe (1ytz.pdb) X-ray crystal structure [59], with a backbone rmsd of 1.253 A. The overlay of cNTnC•cTnI 144–163 •dfbp-o with cNTnC•cTnI 147–163 •bepridil (1lxf.pdb) [21] and cNTnC•cTnI 147–163 •W7 (2krd.pdb) [57] yielded much larger rmsds (2.459 Å and 3.061 Å, respectively). These ligands all bind at the interface formed between cNTnC and cTnI, but only the Ca 2+ -sensitizing agents, dfbp-o and anapoe do not perturb the quaternary structure of the troponin complex.…”

Section: Resultsmentioning

confidence: 99%

“…However, several structural studies have indicated that simply opening cNTnC does not necessarily promote binding of cTnI 147–163 . W7 and bepridil were found to bind to the hydrophobic surface of cNTnC and promote an opening similar to that induced by cTnI 147–163 [17, 54], yet they decreased the affinity of cTnI 147–163 [21, 57]. Both W7 and bepridil have positively charged moieties that repel the positively charged N-terminal Arg147 of cTnI 147–163 .…”

Section: Discussionmentioning

confidence: 99%

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A structural and functional perspective into the mechanism of Ca2+-sensitizers that target the cardiac troponin complex

Robertson

Sun

et al. 2010

Journal of Molecular and Cellular Cardiology

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102

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The Ca 2+ dependant interaction between troponin I (cTnI) and troponin C (cTnC) triggers contraction in heart muscle. Heart failure is characterized by a decrease in cardiac output, and compounds that increase the sensitivity of cardiac muscle to Ca 2+ have therapeutic potential. The Ca 2+ -sensitizer, levosimendan, targets cTnC; however, detailed understanding of its mechanism has been obscured by its instability. In order to understand how this class of positive inotropes function, we investigated the mode of action of two fluorine containing novel analogues of levosimendan; 2' ,4'-difluoro(1,1'-biphenyl)-4-yloxy acetic acid (dfbp-o) and 2' ,4'-difluoro(1,1'-biphenyl)-4-yl acetic acid (dfbp). The affinities of dfbp and dfbp-o for the regulatory domain of cTnC were measured in the absence and presence of cTnI by NMR spectroscopy, and dfbp-o was found to bind more strongly than dfbp. Dfbp-o also increased the affinity of cTnI for cTnC. Dfbp-o increased the Ca 2+ -sensitivity of demembranated cardiac trabeculae in a manner similar to levosimendan. The high resolution NMR solution structure of the cTnC-cTnI-dfbp-o ternary complex showed that dfbp-o bound at the hydrophobic interface formed by cTnC and cTnI making critical interactions with residues such as Arg147 of cTnI. In the absence of cTnI, docking localized dfbp-o to the same position in the hydrophobic groove of cTnC. The structural and functional data reveal that the levosimendan class of Ca 2+ -sensitizers work by binding to the regulatory domain of cTnC and stabilizing the pivotal cTnC-cTnI regulatory unit via a network of hydrophobic and electrostatic interactions, in contrast to the destabilizing effects of antagonists such as W7 at the same interface.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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A structural and functional perspective into the mechanism of Ca2+-sensitizers that target the cardiac troponin complex

Robertson

Sun

et al. 2010

Journal of Molecular and Cellular Cardiology

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102

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“…W7 is an excellent molecule to begin these investigations because of the wealth of biochemical data demonstrating W7 directly binds to cTnC to alter its Ca 2ϩ -binding properties (23). Specifically, W7 binds to the NH 2 -terminal regulatory domain of cTnC, alters Ca 2ϩ affinity, and decreases cTnI switch peptide affinity for cTnC's hydrophobic patch (23,29).…”

Section: Discussionmentioning

confidence: 99%

“…W7 has only been characterized at the biophysical and biochemical level and yet to be studied in disease models. W7 has been shown to bind to cTnC and directly decrease Ca 2ϩ binding affinity (23). In addition, W7 has been shown to decrease Ca 2ϩ sensitivity of force development in membrane-permeabilized myocytes (1) and to reduce contractile amplitude in intact myocytes (11).…”

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confidence: 99%

Sarcomere neutralization in inherited cardiomyopathy: small-molecule proof-of-concept to correct hyper-Ca²⁺-sensitive myofilaments

Thompson

Martindale

Metzger

2016

American Journal of Physiology-Heart and Circulatory Physiology

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Thompson BR, Martindale J, Metzger JM. Sarcomere neutralization in inherited cardiomyopathy: small-molecule proof-of-concept to correct hyper-Ca 2ϩ -sensitive myofilaments. Am J Physiol Heart Circ Physiol 311: H36 -H43, 2016. First published May 13, 2016 doi:10.1152/ajpheart.00981.2015.-The sarcomere is the functional unit of the heart. Alterations in sarcomere activation lead to disease states such as hypertrophic and restrictive cardiomyopathy (HCM/ RCM). Mutations in many of the sarcomeric genes are causal for HCM/RCM. In most cases, these mutations result in increased Ca 2ϩ sensitivity of the sarcomere, giving rise to altered systolic and diastolic function. There is emerging evidence that small-molecule sarcomere neutralization is a potential therapeutic strategy for HCM/ RCM. To pursue proof-of-concept, W7 was used here because of its well-known Ca 2ϩ desensitizer biochemical effects at the level of cardiac troponin C. Acute treatment of adult cardiac myocytes with W7 caused a dose-dependent (1-10 M) decrease in contractility in a Ca 2ϩ -independent manner. Alkalosis was used as an in vitro experimental model of acquired heightened Ca 2ϩ sensitivity, resulting in increased live cell contractility and decreased baseline sarcomere length, which were rapidly corrected with W7. As an inherited cardiomyopathy model, R193H cardiac troponin I (cTnI) transgenic myocytes showed significant decreased baseline sarcomere length and slowed relaxation that were rapidly and dose-dependently corrected by W7. Langendorff whole heart pacing stress showed that R193H cTnI transgenic hearts had elevated end-diastolic pressures at all pacing frequencies compared with hearts from nontransgenic mice. Acute treatment with W7 rapidly restored end-diastolic pressures to normal values in R193H cTnI hearts, supporting a sarcomere intrinsic mechanism of dysfunction. The known off-target effects of W7 notwithstanding, these results provide further proof-of-concept that small-molecule-based sarcomere neutralization is a potential approach to remediate hyper-Ca 2ϩ -sensitive sarcomere function.

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Visualizing the principal component of 1H,15N-HSQC NMR spectral changes that reflect protein structural or functional properties: application to troponin C

2011

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Laboratories often repeatedly determine the structure of a given protein under a variety of conditions, mutations, modifications, or in a number of states. This approach can be cumbersome and tedious. Given then a database of structures, identifiers, and corresponding (1)H,(15)N-HSQC NMR spectra for homologous proteins, we investigated whether structural information could be ascertained for a new homolog solely from its (1)H,(15)N-HSQC NMR spectrum. We addressed this question with two different approaches. First, we used a semi-automated approach with the program, ORBplus. ORBplus looks for patterns in the chemical shifts and correlates these commonalities to the explicit property of interest. ORBplus ranks resonances based on consistency of the magnitude and direction of the chemical shifts within the database, and the chemical shift correlation of the unknown protein with the database. ORBplus visualizes the results by a histogram and a vector diagram, and provides residue specific predictions on structural similarities with the database. The second method we used was partial least squares (PLS), which is a multivariate statistical technique used to correlate response and predictor variables. We investigated the ability of these methods to predict the tertiary structure of the contractile regulatory protein troponin C. Troponin C undergoes a closed-to-open conformational change, which is coupled to its function in muscle. We found that both ORBplus and PLS were able to identify patterns in the (1)H,(15)N-HSQC NMR data from different states of troponin C that correlated to its conformation.

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Solution structure of the regulatory domain of human cardiac troponin C in complex with the switch region of cardiac troponin I and W7: The basis of W7 as an inhibitor of cardiac muscle contraction

Cited by 37 publications

References 35 publications

A structural and functional perspective into the mechanism of Ca2+-sensitizers that target the cardiac troponin complex

A structural and functional perspective into the mechanism of Ca2+-sensitizers that target the cardiac troponin complex

Sarcomere neutralization in inherited cardiomyopathy: small-molecule proof-of-concept to correct hyper-Ca²⁺-sensitive myofilaments

Visualizing the principal component of 1H,15N-HSQC NMR spectral changes that reflect protein structural or functional properties: application to troponin C

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Solution structure of the regulatory domain of human cardiac troponin C in complex with the switch region of cardiac troponin I and W7: The basis of W7 as an inhibitor of cardiac muscle contraction

Cited by 37 publications

References 35 publications

A structural and functional perspective into the mechanism of Ca2+-sensitizers that target the cardiac troponin complex

A structural and functional perspective into the mechanism of Ca2+-sensitizers that target the cardiac troponin complex

Sarcomere neutralization in inherited cardiomyopathy: small-molecule proof-of-concept to correct hyper-Ca2+-sensitive myofilaments

Visualizing the principal component of 1H,15N-HSQC NMR spectral changes that reflect protein structural or functional properties: application to troponin C

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Sarcomere neutralization in inherited cardiomyopathy: small-molecule proof-of-concept to correct hyper-Ca²⁺-sensitive myofilaments