Molecular dynamics provides new insights into the mechanism of calcium signal transduction and interdomain interactions in cardiac troponin

Fig. 1. Cardiac troponin molecule TnC subunit Ca2+ binding site II residues and sequence conservation. (A) The thin filament Ca2+ sensor cardiac troponin molecule depicted as NewCartoon representation. TnI, TnC and TnT subunits are shown in blue, red and yellow. The Ca2+ binding site II is surrounded by a gray rectangle. In the expanded view, the Ca2+ binding site II is shown as a tube representation, and Ca2+ binding site residues are shown in the CPK representation; each residue number is shown per its position within the site, and the Ca2+ ion is shown in an ice‐blue vdW representation. (B) The amino acid sequence in the Ca2+ binding site II, including noncoordinating residues, exhibits high conservation in both cardiac and skeletal isoforms. The sequence is identical among several vertebrate TnC molecules. One notable exception is quail cardiac TnC, in which Gln76 replaces Glu. bov., bovine; c, cardiac; hum., human; por., porcine; ss, slow skeletal.

Fig. 2. Ca2+‐saturated and ‐depleted state behavior of TnC site II N2‐OE12/N9‐OE12 interaction. Dynamic behavior of TnC site II N2‐OE12/N9‐OE12 interaction during 80 ns of Ca2+‐saturated state equilibrium MD simulation, followed by extraction of the Ca2+ ion from site II by SMD, followed by >40 ns of Ca2+‐depleted state equilibrium MD simulation. The distances between 12/Glu76OE:2/Glu66HN shown in green and 12/Glu76OE:9/Asp73HN shown in yellow. Distance of Ca2+ to atom Thr71O is shown in black to highlight the Ca2+ state of site II. Using SMD, after t = 80 ns, the Ca2+ is extracted and pulled away to 40‐Å distance from site II. The N2‐OE12/ N9‐OE12 interaction is well maintained in the Ca2+ bound state but is immediately released and does not re‐form in the Ca2+‐depleted state.

Fig. 3. Binding of Ca2+ into the TnC N‐lobe regulatory site II. (a) Capture: Glu66, Asp67, Asp73, Asp75 capture the floating Ca2+ in a cooperative motion (orange arrow), but the Ca2+ ion also may be captured directly by ASP67 in bidentate mode (green arrow). If Asp75 captures the Ca2+ ion, it may get trapped between residues Asp75 and Asp73 and was not transposed into the binding site (gray arrow) during the duration of the simulation. (b) Toward Ca2+ coordination: Asp67 (evolving from bidentate to monodentate coordination) cooperatively with Glu76 (evolving from monodentate to bidentate coordination) transpose the ion inward; the binding site starts closing around the Ca2+, while Phe74 serves as an anchor; Asp66 and Glu75 serve as gatekeepers so the Ca2+ does not escape back into the solvent. Asp65 and Thr71O also are now close to Ca2+. A water molecule present inside the site hinders those residues from joining the coordination set. (c) Expulsion of the water molecule: Ca2+ is positioned away from the outside solvent environment when the water molecule is expelled from the site into the outside cavity by switching places with the Ca2+ ion; Asp65 and Thr71O also start coordinating the…

Fig. 4. Key distances within the Ca2+ binding site in holo‐state and apo‐state. (A) Coordination distance between the Ca2+ ion and site II residues. Evolution from holo‐state to apo‐state and back to holo‐state. Distances of the Ca2+ ion to the coordinating ligands are depicted. Starting from t = 80 ns, the Ca2+ ion is extracted via SMD from the binding site but binds back after force application is stopped. Initially, it is captured by Glu67 (yellow line) as evidenced by the reduction in separating distance, and Glu67 starts in bidentate coordination followed by cooperation with Glu76 (purple line). Glu67 switches to monodentate (jump in yellow line, drop in purple line); also, at ~ 97 ns, the two ligands briefly switch coordination mode. The final big decline in distance to Thr71O (to ~ 2.5 A) and Asp65 is when the Ca2+ ion and the water molecule switch places and the coordination plane around the ion is finally closed. (B) N2‐OE12/N9‐OE12 H‐bond interaction distances. Evolution from holo‐state to apo‐state and back to holo‐state. In the Ca2+‐saturated state (until t = 80 ns), the N2‐OE12/N9‐OE12 distances are well maintained. Removal of Ca2+ causes the N2‐OE12/N9‐OE12 interactions to…

Fig. 5. Key residues involved in TnC–TnI interdomain interaction. (A, B) Focus on TnI: hydrophobic amino acids Val147 and Ile149 and the positively charged amino acid Arg145 participating in interdomain interaction with TnC residues. The TnI chain is shown in blue and NewCartoon representation, and the TnC chain is shown in red and NewCartoon representation. Alpha‐C atoms of TnC residues Glu15 and Leu48 of the TnC N‐lobe hydrophobic pocket opening are shown as gray vdW spheres. (B) Ninety‐degree rotation of (A). (C, D) Focus on TnC: hydrophobic residues Val44, Met45, Leu48, Pro52, Glu56, Leu57 and Met60 participating in interdomain interaction with TnI residues. The TnI chain is shown in blue and NewCartoon representation, and the TnC chain is shown in red and NewCartoon representation. Alpha‐C atoms of TnC residues Glu15 and Leu48 of the TnC N‐lobe hydrophobic pocket opening are shown as gray vdW spheres. (C) Ninety‐degree rotation of (D).

Fig. 6. Interdomain interaction between TnC and TnI. (A) Hydrophobic interaction i1 between TnI residue Ile149 (shown in white) and TnC residue Val44 (shown in green), TnC residue Met45 (shown in purple) and TnC residue Leu48 (shown in black) (TnI residue Ile149 is surrounded by nonpolar residues). (B) Hydrophobic interaction i2 between TnI residue Val147 (shown in white) and TnC residue Met45 (shown in purple), TnC residue Pro52 (shown in green), TnC residue Leu57 (shown in blue) and TnC residue Met60 (shown in pink). TnI residue Val147 is surrounded by nonpolar residues. (C) H‐bond interaction i3 between TnI residue Arg145 (shown in atom colors) and TnC residue Glu56 (shown in atom colors).

Fig. 7. Starting conformation for TnI switch region SMD extraction, the two distinct switch extraction conformations used for post‐extraction equilibrium MD sim1 and sim2 simulations, and evolution of the TnC N‐lobe hydrophobic pocket opening distance in sim1 and sim2. Inset: starting conformation TnI switch extraction by SMD. The TnI switch is embedded in the TnC N‐lobe hydrophobic pocket. Residues involved in TnI–TnC interdomain interaction are shown as vdW spheres; force application is donated by F. (A) Starting conformation for post‐TnI switch extraction equilibrium MD simulation sim1 after translation of ~ 45 Å of the pulling atom from the starting conformation is achieved by SMD. The TnI switch is pulled from the TnC N‐lobe hydrophobic pocket; however, the three TnC–TnI interactions (i1, i2 and i3) are maintained. (B) Starting conformation for post‐TnI switch extraction equilibrium MD simulation sim2 after translation of ~ 60 Å of the pulling atom from the starting conformation is achieved by SMD. The TnI switch is pulled from the TnC N‐lobe hydrophobic pocket, TnI residues Ile149 and Val147 are extracted from the hydrophobic niches in TnC (interactions i1 and i2), while the H‐bond…