Sequences for the NT domains of Calmodulin and Troponin C. Calcium binding residues are in red; α-helices are in green and the EF-hand β-scaffold residues are underlined. The residues at loop position 7 bind using the MC oxygen.
Energy and RMSD measured from the experimentally-derived closed Calmodulin NT structure as a function of accepted ART-event for eight independent simulation started from the experimental apo conformation. (a) and (b) Energy and RMSD results, respectively, for CHARMM19-EEF1; (c) and (d) same for EOPEP. RMSD are computed on Cα, excluding unstable residues 1-3.
Energy and RMSD measured from the experimentally-derived closed CaMnt structure as a function of accepted ART-event for independent simulations started from the experimentally-derived open conformation with Ca ion removed. From a set of 24 trajectories, we present the four simulations with the lowest-energy and RMSD structures, respectively. Panels (a) and (b) show the evolution of the energy and RMSD for CHARMM-EEF1 simulations, respectively; panels (c) and (d) present results for EOPEP simulations.
Folding pathway for simulation eop9h. Each EF-hand pair goes from a rather perpendicular arrangement in the holo state toward a more parallel state in the apo form. (a) 1CLL.pdb model up to residue 76, residues 1-3 added. (b) After a first minimization without Ca2+, we observe an increase in the distance between oxygen carrier residues of the loop. (c) Event 3: consolidation of the β-sheet (magenta) linking the 2 calcium binding loops. A hydrogen bridge is formed between THR29 and ILE61. (d) Event 16: Increase in the number of hydrophobe interactions, the core begin to close, but the presence of PHE19 (cyan) prevent a better packing. (e) Event 19: PHE19 gets out of the hydrophobic core, allowing a better closing of the core. (f) Event 24: The helices pack together near the apo form. The end helices (A and D) are held relatively fixed in these views, in order to show the cooperative motion of helices B and C. For ease of viewing, helices A-B (EF-hand 1) are colored in peach and helices C-D (EF-hand 2) in yellow.
Diagrammatic representation of the Calmodulin NT mechanism. Top: Initial open state. Middle: Consolidation of the β-sheet, widening out the space between helices, freeing PHE19 latch. Bottom: Closing of the helices in parallel configuration, PHE19 getting outside the hydrophobic core.
Evolution of the energy as a function of accepted ART event during eop9h simulation. (a) Evolution of the calcium binding residues. Following calcium removal, the zero temperature minimization already causes a significant energy drop before the first event. (b) Evolution of the six residues participating in the formation of the short β-sheet associated with the closed form. (c) Evolution of the CaMnt 14 hydrophobic residues.
Comparison between open and close models for both (a) CAMnt and (b) TpCnT. Open conformations are shown in blue and closed structure in magenta. The models are aligned on the stable part of their beta sheets. The orange arrows point out the variation of helices B and C relatively to there respective EF-hand partners A and D. Black arrows indicates the most variable location within or nearby the β-scaffold, not the same for both type of molecules. Cyan arrows point the pre-loop 1 phenylalanine, that undergoes an important relocalization relatively to helix B.
Top: Evolution of the angles between various helix pairs as a function of accepted ART event for simulation eop9h. Dashed (dotted) lines indicate the value for the experimentally-derived open (closed) form. Bottom: Evolution of the angles between various pairs of helices as a function of accepted ART event for simulation eop14h. Dashed (dotted) lines indicate the value for the experimentally-derived open (closed) form.
Extension of the 24 EOPEP Calmodulin NT closing simulations, using a 900 K acceptation Metropolis temperature. The vertical continuous black lines represent the beginning of the runs at this temperature following the last 50 events accepted at 300 K. The energy (top) and RMSD from the experimentally-derived apo state (bottom) are shown for the: (a) 5 simulations that had already succeeded PHE19 escape at metropolis 300 K. (b) 6 additional simulations succeeding PHE19 escape; (c) 6 simulations with PHE19 in an intermediate state. (d) 7 simulations with destabilizing trajectory. The dashed vertical lines in (a) and (b) indicate the escape event of PHE19. The line is not shown for simulations 9, 10, 11 and 21 because this event occurred before the beginning of this graph.
Cartoon representation of the PHE19 as it enters and leaves the intermediate pocket. PHE19 of helix A and the tree pocket residues of helix B are represented as spheres: PHE19 in yellow, VAL35 in grey, MET36 in orange and LEU32 in blue. (a) The open Calmodulin NT model. (b) While closing, the LEU-MET-VAL pocket receives PHE19. (c) and (d) Valine 35 swivels and PHE19 finds its way toward (e) exit. (f) Typical unstable structures for simulation at 900 K where the PHE19 is neither trapped in the intermediate state nor ejected outside the hydrophobic core. We see the β-sheet in dark grey at the back of the molecule. This structure appears in (c,d,e) in dark purple.
Energy and RMSD measured from the experimentally-derived closed TpCnt structure as a function of accepted ART-event for independent simulations started from the experimentally-derived open conformation with Ca ion removed. From a set of 24 trajectories, we present the four simulations with the lowest-energy and RMSD structures, respectively. Panels (a) and (b) show the evolution of the energy and RMSD for CHARMM-EEF1 simulations, respectively. RMSD is relative to model 30 of PDB 1TNP; panels (c) and (d) present results for EOPEP simulations, with RMSD relative to model 03 of PDB 1TNP. Simulation eop22h reaches both energy and RMSD criteria.
RMSD statistics for selected trajectories. Averaged displacement measured from the initial minimum to the saddle point and the final minimum for various simulations on both the Calmodulin and Troponin C using OPEP char CHARMM/EEF1 potentials. Statistics are given both for the full sets of events and those accepted only.
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