FRET-based analysis of the cardiac troponin T linker region reveals the structural basis of the hypertrophic cardiomyopathy-causing Δ160E mutation

Abdullah, Salwa; Lynn, Melissa L.; McConnell, Mark T.; Klass, Matthew M.; Baldo, Anthony P.; Schwartz, Steven D.; Tardiff, Jil C.

doi:10.1074/jbc.ra118.005098

Cited by 11 publications

(22 citation statements)

References 64 publications

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“…Previously proposed, short helical fragments may define the TnT1-TnT2 junction near tropomyosin residue 175 (33,35,53). It follows that TnT makes secondary contact with tropomyosin near to this junction and, then, via TnT2 connects to the troponin core (20,24,25,27,56).…”

Section: Discussionmentioning

confidence: 93%

Docking Troponin T onto the Tropomyosin Overlapping Domain of Thin Filaments

Pavadai

Rynkiewicz

Ghosh

et al. 2020

Biophysical Journal

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Complete description of thin filament conformational transitions accompanying muscle regulation requires ready access to atomic structures of actin-bound tropomyosin-troponin. To date, several molecular-docking protocols have been employed to identify troponin interactions on actin-tropomyosin because high-resolution experimentally determined structures of filamentassociated troponin are not available. However, previously published all-atom models of the thin filament show chain separation and corruption of components during our molecular dynamics simulations of the models, implying artifactual subunit organization, possibly due to incorporation of unorthodox tropomyosin-TnT crystal structures and complex FRET measurements during model construction. For example, the recent Williams et al. ( 2016) atomistic model of the thin filament displays a paucity of salt bridges and hydrophobic complementarity between the TnT tail (TnT1) and tropomyosin, which is difficult to reconcile with the high, 20 nM K d binding of TnT onto tropomyosin. Indeed, our molecular dynamics simulations show the TnT1 component in their model partially dissociates from tropomyosin in under 100 ns, whereas actin-tropomyosin and TnT1 models themselves remain intact. We therefore revisited computational work aiming to improve TnT1-thin filament models by employing unbiased docking methodologies, which test billions of trial rotations and translations of TnT1 over three-dimensional grids covering end-to-end bonded tropomyosin alone or tropomyosin on F-actin. We limited conformational searches to the association of well-characterized TnT1 helical domains and either isolated tropomyosin or actin-tropomyosin yet avoided docking TnT domains that lack known or predicted structure. The docking programs PIPER and ClusPro were used, followed by interaction energy optimization and extensive molecular dynamics. TnT1 docked to either side of isolated tropomyosin but uniquely onto one location of actin-bound tropomyosin. The antiparallel interaction with tropomyosin contained abundant salt bridges and intimately integrated hydrophobic networks joining TnT1 and the tropomyosin N-/C-terminal overlapping domain. The TnT1-tropomyosin linkage yields well-defined molecular crevices. Interaction energy measurements strongly favor this TnT1-tropomyosin design over previously proposed models.

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Section: Discussionmentioning

confidence: 93%

Docking Troponin T onto the Tropomyosin Overlapping Domain of Thin Filaments

Pavadai

Rynkiewicz

Ghosh

et al. 2020

Biophysical Journal

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“…Although the effects of the L48Q cTnC mutation on the molecular structure of Tn have been previously investigated ( 16 , 19 ), it is not known if the L48Q cTnC variant retains its Ca 2+ -sensitizing effects on thin filament activation when coupled with the D230N-Tm mutation. Thus, we employed a structural model of the cardiac thin filament ( 28 – 31 ) that includes atomically detailed actin, Tm, and the full Tn complex and incorporated into the model the point mutations D230N in Tm and L48Q in cTnC ( Supplemental Figure 3 ). We then quantitatively assessed structural differences between regulatory units (RUs, defined here as Tn-Tm-actin complexes) containing D230N-Tm with and without L48Q cTnC that may influence the Ca 2+ sensitivity of thin filament activation.…”

Section: Resultsmentioning

confidence: 99%

“…The molecular model of the cardiac thin filament was constructed using the previously described initial low-temperature structure ( 28 – 31 ) that includes actin, Tm, and the full Tn complex ( Supplemental Figure 3 ). Point mutations were integrated into each respective protein using the CHARMM42 program ( 53 ) with CHARMM36 parameters, the latest version of the CHARMM force field ( 54 ).…”

Section: Methodsmentioning

confidence: 99%

Modulating the tension-time integral of the cardiac twitch prevents dilated cardiomyopathy in murine hearts

Powers

Kooiker

Mason³

et al. 2020

JCI Insight

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Dilated cardiomyopathy (DCM) is often associated with sarcomere protein mutations that confer reduced myofilament tension–generating capacity. We demonstrated that cardiac twitch tension-time integrals can be targeted and tuned to prevent DCM remodeling in hearts with contractile dysfunction. We employed a transgenic murine model of DCM caused by the D230N-tropomyosin (Tm) mutation and designed a sarcomere-based intervention specifically targeting the twitch tension-time integral of D230N-Tm hearts using multiscale computational models of intramolecular and intermolecular interactions in the thin filament and cell-level contractile simulations. Our models predicted that increasing the calcium sensitivity of thin filament activation using the cardiac troponin C (cTnC) variant L48Q can sufficiently augment twitch tension-time integrals of D230N-Tm hearts. Indeed, cardiac muscle isolated from double-transgenic hearts expressing D230N-Tm and L48Q cTnC had increased calcium sensitivity of tension development and increased twitch tension-time integrals compared with preparations from hearts with D230N-Tm alone. Longitudinal echocardiographic measurements revealed that DTG hearts retained normal cardiac morphology and function, whereas D230N-Tm hearts developed progressive DCM. We present a computational and experimental framework for targeting molecular mechanisms governing the twitch tension of cardiomyopathic hearts to counteract putative mechanical drivers of adverse remodeling and open possibilities for tension-based treatments of genetic cardiomyopathies.

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“…We and others have shown that DSC is a valuable technique for studying thin filament thermodynamics, giving regionally specific information on thermal stability and conformational flexibility of these proteins in complex (9,(29)(30)(31)34). Here we used DSC as a method to biophysically confirm the model-derived changes in Root Mean Square Fluctuation (RMSF), a related standard measure of flexibility that calculates the fluctuation of each alpha carbon in a protein and can then be compared to the WT structure (23). Our data show that D20NTm, a mutation linked to HCM, significantly increases the FWHM of the Tm-Tn array unfolding (Figure 4) consistent with an increase in conformational flexibility of the proteins in the overlap region.…”

Section: Differential Scanning Calorimetrymentioning

confidence: 99%

Computational and biophysical determination of pathogenicity of variants of unknown significance in cardiac thin filament

Mason¹,

Lynn²,

Baldo³

et al. 2021

JCI Insight

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Point mutations within sarcomeric proteins have been associated with altered function and cardiomyopathy development. Difficulties remain, however, in establishing the pathogenic potential of individual mutations, often limiting the use of genotype in management of affected families. To directly address this challenge, we utilized our all-atom computational model of the human full cardiac thin filament (CTF) to predict how sequence substitutions in CTF proteins might affect structure and dynamics on an atomistic level.Utilizing molecular dynamics (MD) calculations, we simulated 21 well-defined genetic pathogenic cardiac troponin T and tropomyosin variants to establish a baseline of pathogenic changes induced in computational observables. Computational results were verified via differential scanning calorimetry on a subset of variants to develop an experimental correlation.Calculations were performed on 9 independent variants of unknown significance (VUS) and results were compared to pathogenic variants to identify high resolution pathogenic signatures.Results for VUS were compared to the baseline set to determine induced structural and dynamic changes and potential variant reclassifications were proposed. This unbiased, highresolution computational methodology can provide unique structural and dynamic information that can be incorporated into existing analyses to facilitate classification both for de novo variants and those where established approaches have provided conflicting information.

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FRET-based analysis of the cardiac troponin T linker region reveals the structural basis of the hypertrophic cardiomyopathy-causing Δ160E mutation

Cited by 11 publications

References 64 publications

Docking Troponin T onto the Tropomyosin Overlapping Domain of Thin Filaments

Docking Troponin T onto the Tropomyosin Overlapping Domain of Thin Filaments

Modulating the tension-time integral of the cardiac twitch prevents dilated cardiomyopathy in murine hearts

Computational and biophysical determination of pathogenicity of variants of unknown significance in cardiac thin filament

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