Cryo-EM and Molecular Docking Shows Myosin Loop 4 Contacts Actin and Tropomyosin on Thin Filaments

Doran, Matthew; Pavadai, Elumalai; Rynkiewicz, Michael J.; Walklate, Jonathan; Bullitt, Esther; Moore, Jeffrey R.; Regnier, Michael; Geeves, Michael A.; Lehman, William

doi:10.1016/j.bpj.2020.07.006

Cited by 42 publications

(113 citation statements)

References 47 publications

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“…This cryo-EM study of the cardiac thin filament achieved resolution sufficient to create atomic models including most of troponin, in both the presence and absence of Ca 2+ . The structural findings were supported and extended throughout the year by excellent independent reports ( Pavadai et al, 2020b ; Pavadai et al, 2020a ; Oda et al, 2020 ; Doran et al, 2020 ). At the resolution pertinent to the broad themes in our study, these other studies either agree with Yamada et al (2020) or disagree only in the sense of omission (fewer parts of troponin were resolved).…”

Section: Discussionsupporting

confidence: 59%

“…The structure of troponin as bound to the actin filament was derived, in both the presence and absence of Ca 2+ . Although the results may be challenged or revised in time, independent cryo-EM or computational work ( Doran et al, 2020 ; Oda et al, 2020 ; Pavadai et al, 2020a ; Pavadai et al, 2020b ; Burbaum et al, 2020 Preprint ; Wang et al, 2020 Preprint ) is already extending or confirming many aspects, such as the orientation of the troponin core domain. These recent developments are transformative for structural understanding of the primary on-off regulatory switch in cardiac and other striated muscles: reversible Ca 2+ binding to troponin.…”

Section: Introductionmentioning

confidence: 99%

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Cardiomyopathic troponin mutations predominantly occur at its interface with actin and tropomyosin

Tobacman

Cammarato

2021

Journal of General Physiology

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Reversible Ca2+ binding to troponin is the primary on-off switch of the contractile apparatus of striated muscles, including the heart. Dominant missense mutations in human cardiac troponin genes are among the causes of hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy. Structural understanding of troponin action has recently advanced considerably via electron microscopy and molecular dynamics studies of the thin filament. As a result, it is now possible to examine cardiomyopathy-inducing troponin mutations in thin-filament structural context, and from that to seek new insight into pathogenesis and into the troponin regulatory mechanism. We compiled from consortium reports a representative set of troponin mutation sites whose pathogenicity was determined using standardized clinical genetics criteria. Another set of sites, apparently tolerant of amino acid substitutions, was compiled from the gnomAD v2 database. Pathogenic substitutions occurred predominantly in the areas of troponin that contact actin or tropomyosin, including, but not limited to, two regions of newly proposed structure and long-known implication in cardiomyopathy: the C-terminal third of troponin I and a part of the troponin T N terminus. The pathogenic mutations were located in troponin regions that prevent contraction under low Ca2+ concentration conditions. These regions contribute to Ca2+-regulated steric hindrance of myosin by the combined effects of troponin and tropomyosin. Loss-of-function mutations within these parts of troponin result in loss of inhibition, consistent with the hypercontractile phenotype characteristic of HCM. Notably, pathogenic mutations are absent in our dataset from the Ca2+-binding, activation-producing troponin C (TnC) N-lobe, which controls contraction by a multi-faceted mechanism. Apparently benign mutations are also diminished in the TnC N-lobe, suggesting mutations are poorly tolerated in that critical domain.

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Section: Discussionsupporting

confidence: 59%

Section: Introductionmentioning

confidence: 99%

Cardiomyopathic troponin mutations predominantly occur at its interface with actin and tropomyosin

Tobacman

Cammarato

2021

Journal of General Physiology

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“…Although Tpm has a simple coiled-coil structure, it performs multiple functions in the thin filament. In muscle, it is a part of Ca 2+ regulatory mechanism, a stabilizer of the thin filament and, according to the findings, is involved in binding to F-actin, myosin, and troponin [ 34 , 35 , 36 ]. The mechanism of Tpm binding to actin and the detailed mechanism of Tpm shifting upon Ca 2+ activation and/or myosin binding to F-actin is still poorly understood.…”

Section: Discussionmentioning

confidence: 99%

Impact of A134 and E218 Amino Acid Residues of Tropomyosin on Its Flexibility and Function

Marchenko

Nefedova

Yampolskaya

et al. 2020

IJMS

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Tropomyosin (Tpm) is one of the major actin-binding proteins that play a crucial role in the regulation of muscle contraction. The flexibility of the Tpm molecule is believed to be vital for its functioning, although its role and significance are under discussion. We choose two sites of the Tpm molecule that presumably have high flexibility and stabilized them with the A134L or E218L substitutions. Applying differential scanning calorimetry (DSC), molecular dynamics (MD), co-sedimentation, trypsin digestion, and in vitro motility assay, we characterized the properties of Tpm molecules with these substitutions. The A134L mutation prevented proteolysis of Tpm molecule by trypsin, and both substitutions increased the thermal stability of Tpm and its bending stiffness estimated from MD simulation. None of these mutations affected the primary binding of Tpm to F-actin; still, both of them increased the thermal stability of the actin-Tpm complex and maximal sliding velocity of regulated thin filaments in vitro at a saturating Ca2+ concentration. However, the mutations differently affected the Ca2+ sensitivity of the sliding velocity and pulling force produced by myosin heads. The data suggest that both regions of instability are essential for correct regulation and fine-tuning of Ca2+-dependent interaction of myosin heads with F-actin.

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“…The C-loop is known to participate in energy transduction 42 , actin-activation of myosin ATPase 53 and to modulate myosin in vitro motility velocity in the presence of actin bound tropomyosin 43 . The latter interaction was confirmed in a static structure of a thick filament 54 . It seems that the C-loop maintains resilient links with the MYBPC3 regulatory domain.…”

Section: Discussionmentioning

confidence: 58%

Natural variants with 2D correlation genetics identify domains coordinating sarcomere proteins during contraction

Burghardt

2021

Preprint

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Muscle proteins assemble in a sarcomere then by coordinated action produce contraction force to shorten muscle. In the human heart ventriculum, cardiac myosin motor (βmys) repetitively converts ATP free energy into work. Cardiac myosin binding protein C (MYBPC3) in complex with βmys regulates contraction power generation. Their bimolecular complex βmys/MYBPC3 models the contractile system and is used here to study protein coupling. The database for single nucleotide variants (SNVs) in βmys and MYBPC3 surveys human populations worldwide. It consistently records SNV physical characteristics including substituted residue location in the protein functional domain, the side chain substitution, substitution frequency, and human population group, but inconsistently records SNV implicated phenotype and pathology outcomes. A selected consistent subset of the data trains and validates a feed-forward neural network modeling the contraction mechanism. The full database is completed using the model then interpreted probabilistically with a discrete Bayes network to give the SNV probability for a functional domain location given pathogenicity and human population. Co-domains, intra-protein domains coupling βmys and MYBPC3, are identified by their population correlated SNV probability product for given pathogenicity. Divergent genetics in human populations identify co-domain correlates in this method called 2D correlation genetics. Pathogenic and benign SNV data identify three critical regulatory sites, two in MYBPC3 with links to several domains across the βmys motor, and, one in βmys with links to the known MYBPC3 regulatory domain. Critical sites in MYBPC3 are hinges (one known another proposed) sterically enabling regulatory interactions with βmys. The critical site in βmys is the actin binding C-loop, a contact sensor triggering actin-activated myosin ATPase and contraction velocity modulator coordinating also with actin bound tropomyosin. C-loop and MYBPC3 regulatory domain linkage potentially impacts multiple functions across the contractile system. Identification of co-domains in a binary protein complex implies a capacity to estimate spatial proximity constraints for specific dynamic protein interactions in vivo opening another avenue for protein complex structure/function determination.

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Cryo-EM and Molecular Docking Shows Myosin Loop 4 Contacts Actin and Tropomyosin on Thin Filaments

Abstract: The version in the Kent Academic Repository may differ from the final published version. Users are advised to check http://kar.kent.ac.uk for the status of the paper. Users should always cite the published version of record.

Cited by 42 publications

References 47 publications

Cardiomyopathic troponin mutations predominantly occur at its interface with actin and tropomyosin

Cardiomyopathic troponin mutations predominantly occur at its interface with actin and tropomyosin

Impact of A134 and E218 Amino Acid Residues of Tropomyosin on Its Flexibility and Function

Natural variants with 2D correlation genetics identify domains coordinating sarcomere proteins during contraction

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