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Molecular dynamics simulations of the cardiac troponin complex performed with FRET distances as restraints.

Jayasundar JJ, Xing J, Robinson JM, Cheung HC, Dong WJ - PLoS ONE (2014)

Bottom Line: In the presence of saturating Ca(2+) the above said phenomenon were absent.We postulate that the secondary structure perturbations experienced by the cTnI regulatory region held within the cTnC N-domain hydrophobic pocket, coupled with the rotation of the cTnC N-domain would control the cTnI mobile domain interaction with actin.Concomitantly the rotation of the cTnC N-domain and perturbation of the D/E linker rigidity would control the cTnI inhibitory region interaction with actin to effect muscle relaxation.

View Article: PubMed Central - PubMed

Affiliation: Voiland School of Chemical Engineering and Bioengineering and The Department of Integrated Physiology and Neuroscience, Washington State University, Pullman, Washington, United States of America.

ABSTRACT
Cardiac troponin (cTn) is the Ca(2+)-sensitive molecular switch that controls cardiac muscle activation and relaxation. However, the molecular detail of the switching mechanism and how the Ca(2+) signal received at cardiac troponin C (cTnC) is communicated to cardiac troponin I (cTnI) are still elusive. To unravel the structural details of troponin switching, we performed ensemble Förster resonance energy transfer (FRET) measurements and molecular dynamic (MD) simulations of the cardiac troponin core domain complex. The distance distributions of forty five inter-residue pairs were obtained under Ca(2+)-free and saturating Ca(2+) conditions from time-resolved FRET measurements. These distances were incorporated as restraints during the MD simulations of the cardiac troponin core domain. Compared to the Ca(2+)-saturated structure, the absence of regulatory Ca(2+) perturbed the cTnC N-domain hydrophobic pocket which assumed a closed conformation. This event partially unfolded the cTnI regulatory region/switch. The absence of Ca(2+), induced flexibility to the D/E linker and the cTnI inhibitory region, and rotated the cTnC N-domain with respect to rest of the troponin core domain. In the presence of saturating Ca(2+) the above said phenomenon were absent. We postulate that the secondary structure perturbations experienced by the cTnI regulatory region held within the cTnC N-domain hydrophobic pocket, coupled with the rotation of the cTnC N-domain would control the cTnI mobile domain interaction with actin. Concomitantly the rotation of the cTnC N-domain and perturbation of the D/E linker rigidity would control the cTnI inhibitory region interaction with actin to effect muscle relaxation.

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The starting structure for MD simulations.The crystal structure (1J1E.pdb [10]) of the cTn complex at saturating Ca2+ is depicted with the N-terminal extension of cTnI (cTnI-Nxt) docked over the cTnC N-domain. The N-terminal extension of cTnI was docked to the cTn complex using Hex, a protein-protein docking program. The cTnT and cTnI are colored cyan and blue respectively. The cTnC helices N, A, B, C, D in the N-domain are colored, red, mustard, orange, yellow and lime green. The bound Ca2+ ions at sites 2, 3, and 4 are rendered as spheres and colored cyan.
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pone-0087135-g002: The starting structure for MD simulations.The crystal structure (1J1E.pdb [10]) of the cTn complex at saturating Ca2+ is depicted with the N-terminal extension of cTnI (cTnI-Nxt) docked over the cTnC N-domain. The N-terminal extension of cTnI was docked to the cTn complex using Hex, a protein-protein docking program. The cTnT and cTnI are colored cyan and blue respectively. The cTnC helices N, A, B, C, D in the N-domain are colored, red, mustard, orange, yellow and lime green. The bound Ca2+ ions at sites 2, 3, and 4 are rendered as spheres and colored cyan.

Mentions: The distances and half-widths generated from the FRET experiments were used to model the cardiac troponin complex. The crystal structure of the cardiac troponin core domain that was resolved in the Ca2+-saturated state [10] did not have structural information pertaining to the physiologically important N-terminal and inhibitory regions of cTnI. To acquire this information and to shed light on the structure and structural dynamics of the cardiac troponin in the Ca2+-free and Ca2+-saturated states, we carried out MD simulations. The cardiac troponin core domain was simulated in three different conditions. The first was restrained MD simulations. During the restrained MD simulations the experimentally determined FRET distances and half-widths were applied as distance restraints and bond energies. This approach helped us select the starting structure shown in Fig. 2. The details of the validation process are detailed in the next paragraph. The second MD simulation was carried out in such a way that at the end of the first FRET distance restrained MD simulations, the restraints were removed and the simulation was allowed to continue for 250 ns. The results of this simulation give insight into the dynamics of the cTn complex. This helped us understand whether the cardiac troponin complex when in one biochemical state is always locked in that state, or if there are fluctuations in between states. The data is presented in figures S1, S2, S3. The third MD simulation of the cardiac troponin complex displayed in Fig. 2 was performed for 150 ns without any restraints. Since there was unfolding of cTnC helices in first simulation we wanted to show that the unfolding was due to the presence of restraints and was not because the force field was set up incorrectly. The results of the MD simulation of the troponin complex from the third set of simulations are given in the File S1. The data is presented in figures S4, S5, S6,S7.


Molecular dynamics simulations of the cardiac troponin complex performed with FRET distances as restraints.

Jayasundar JJ, Xing J, Robinson JM, Cheung HC, Dong WJ - PLoS ONE (2014)

The starting structure for MD simulations.The crystal structure (1J1E.pdb [10]) of the cTn complex at saturating Ca2+ is depicted with the N-terminal extension of cTnI (cTnI-Nxt) docked over the cTnC N-domain. The N-terminal extension of cTnI was docked to the cTn complex using Hex, a protein-protein docking program. The cTnT and cTnI are colored cyan and blue respectively. The cTnC helices N, A, B, C, D in the N-domain are colored, red, mustard, orange, yellow and lime green. The bound Ca2+ ions at sites 2, 3, and 4 are rendered as spheres and colored cyan.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928104&req=5

pone-0087135-g002: The starting structure for MD simulations.The crystal structure (1J1E.pdb [10]) of the cTn complex at saturating Ca2+ is depicted with the N-terminal extension of cTnI (cTnI-Nxt) docked over the cTnC N-domain. The N-terminal extension of cTnI was docked to the cTn complex using Hex, a protein-protein docking program. The cTnT and cTnI are colored cyan and blue respectively. The cTnC helices N, A, B, C, D in the N-domain are colored, red, mustard, orange, yellow and lime green. The bound Ca2+ ions at sites 2, 3, and 4 are rendered as spheres and colored cyan.
Mentions: The distances and half-widths generated from the FRET experiments were used to model the cardiac troponin complex. The crystal structure of the cardiac troponin core domain that was resolved in the Ca2+-saturated state [10] did not have structural information pertaining to the physiologically important N-terminal and inhibitory regions of cTnI. To acquire this information and to shed light on the structure and structural dynamics of the cardiac troponin in the Ca2+-free and Ca2+-saturated states, we carried out MD simulations. The cardiac troponin core domain was simulated in three different conditions. The first was restrained MD simulations. During the restrained MD simulations the experimentally determined FRET distances and half-widths were applied as distance restraints and bond energies. This approach helped us select the starting structure shown in Fig. 2. The details of the validation process are detailed in the next paragraph. The second MD simulation was carried out in such a way that at the end of the first FRET distance restrained MD simulations, the restraints were removed and the simulation was allowed to continue for 250 ns. The results of this simulation give insight into the dynamics of the cTn complex. This helped us understand whether the cardiac troponin complex when in one biochemical state is always locked in that state, or if there are fluctuations in between states. The data is presented in figures S1, S2, S3. The third MD simulation of the cardiac troponin complex displayed in Fig. 2 was performed for 150 ns without any restraints. Since there was unfolding of cTnC helices in first simulation we wanted to show that the unfolding was due to the presence of restraints and was not because the force field was set up incorrectly. The results of the MD simulation of the troponin complex from the third set of simulations are given in the File S1. The data is presented in figures S4, S5, S6,S7.

Bottom Line: In the presence of saturating Ca(2+) the above said phenomenon were absent.We postulate that the secondary structure perturbations experienced by the cTnI regulatory region held within the cTnC N-domain hydrophobic pocket, coupled with the rotation of the cTnC N-domain would control the cTnI mobile domain interaction with actin.Concomitantly the rotation of the cTnC N-domain and perturbation of the D/E linker rigidity would control the cTnI inhibitory region interaction with actin to effect muscle relaxation.

View Article: PubMed Central - PubMed

Affiliation: Voiland School of Chemical Engineering and Bioengineering and The Department of Integrated Physiology and Neuroscience, Washington State University, Pullman, Washington, United States of America.

ABSTRACT
Cardiac troponin (cTn) is the Ca(2+)-sensitive molecular switch that controls cardiac muscle activation and relaxation. However, the molecular detail of the switching mechanism and how the Ca(2+) signal received at cardiac troponin C (cTnC) is communicated to cardiac troponin I (cTnI) are still elusive. To unravel the structural details of troponin switching, we performed ensemble Förster resonance energy transfer (FRET) measurements and molecular dynamic (MD) simulations of the cardiac troponin core domain complex. The distance distributions of forty five inter-residue pairs were obtained under Ca(2+)-free and saturating Ca(2+) conditions from time-resolved FRET measurements. These distances were incorporated as restraints during the MD simulations of the cardiac troponin core domain. Compared to the Ca(2+)-saturated structure, the absence of regulatory Ca(2+) perturbed the cTnC N-domain hydrophobic pocket which assumed a closed conformation. This event partially unfolded the cTnI regulatory region/switch. The absence of Ca(2+), induced flexibility to the D/E linker and the cTnI inhibitory region, and rotated the cTnC N-domain with respect to rest of the troponin core domain. In the presence of saturating Ca(2+) the above said phenomenon were absent. We postulate that the secondary structure perturbations experienced by the cTnI regulatory region held within the cTnC N-domain hydrophobic pocket, coupled with the rotation of the cTnC N-domain would control the cTnI mobile domain interaction with actin. Concomitantly the rotation of the cTnC N-domain and perturbation of the D/E linker rigidity would control the cTnI inhibitory region interaction with actin to effect muscle relaxation.

Show MeSH
Related in: MedlinePlus