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Direct interaction between troponin and myosin enhances the ATPase activity of heavy meromyosin

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Abstract

Contractility of the heart muscle is a result of sliding movements between thick and thin filaments, produced by interactions between actin and myosin during the cross-bridge cycle. Activation of the myofilament is triggered by Ca2+ binding to cardiac troponin C and is regulated through an “on/off” switching process occurring in the thin filament. Beside Ca2+ regulation, strongly bound cross-bridges exert a positive feedback on myofilament regulation. Despite the importance of this positive feedback mechanism, its full molecular basis has so far remained elusive. Ca2+-regulated interactions between thick and thin filaments are widely regarded as an allosteric system, which means that multiple protein-protein interactions at their interface may exert alternative feedback effects on myofilament activation. To advance knowledge about these regulatory feedback mechanisms, we investigated a previously unstudied, hypothetical interaction between cardiac troponin and myosin, and how this interaction affects the function of myosin. Our results strongly suggest that myosin does indeed interact with the N-terminus of cardiac troponin I and the C-terminus of cardiac troponin T, suggesting a possible direct interaction between myosin and the IT-arm of troponin. We also conducted an in vitro heavy meromyosin (HMM) ATPase assay, and found that troponin significantly enhanced the actin-activated ATPase activity of HMM, both in the absence of tropomyosin and at the activated state of thin filament.

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Abbreviations

cTnC:

cardiac troponin C

cTnI:

cardiac troponin I

cTnT:

cardiac troponin T

DTT:

dithiothreitol

EGTA:

ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid

HMM:

heavy meromyosin

IVM:

in vitro motility assay

MOPS:

3-(N-morpholino)propanesulfonic acid

N-cTnC:

N-terminus of cardiac troponin C

Pi:

inorganic phosphate

SL:

sarcomere length

TBS/T:

Tris-buffered saline solution containing 1% tween-20

TF:

thin filament

Tm:

tropomyosin

Tn:

troponin

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Authors and Affiliations

  1. The Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA, 99164, USA

    Nazanin Bohlooli Ghashghaee, King-Lun Li & Wen-Ji Dong

  2. The Department of Integrative Physiology and Neuroscience, Washington State University, Pullman, WA, 99164, USA

    Wen-Ji Dong

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  1. Nazanin Bohlooli Ghashghaee
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  2. King-Lun Li
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  3. Wen-Ji Dong
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Correspondence to Wen-Ji Dong.

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Ghashghaee, N.B., Li, KL. & Dong, WJ. Direct interaction between troponin and myosin enhances the ATPase activity of heavy meromyosin. Biologia 72, 702–708 (2017). https://doi.org/10.1515/biolog-2017-0079

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  • DOI: https://doi.org/10.1515/biolog-2017-0079

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