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Journal of Chemical Physics / Volume 136 / Issue 3 / ARTICLES / Biological Molecules, Biopolymers, and Biological Systems
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J. Chem. Phys. 136, 035101 (2012); http://dx.doi.org/10.1063/1.3671986 (13 pages)

Understanding the EF-hand closing pathway using non-biased interatomic potentials

L. Dupuis1 and Normand Mousseau2

1Département de Biochimie, Centre Robert-Cedergren and GEPROM, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, Québec H3C 3J7, Canada
2Département de Physique, Centre Robert-Cedergren and GEPROM, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, Québec H3C 3J7, Canada

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(Received 3 August 2011; accepted 4 December 2011; published online 17 January 2012)

The EF-hand superfamily of proteins is characterized by the presence of calcium binding helix-loop-helix structures. Many of these proteins undergo considerable motion responsible for a wide range of properties upon binding but the exact mechanism at the root of this motion is not fully understood. Here, we use an unbiased accelerated multiscale simulation scheme, coupled with two force fields — CHARMM-EEF1 and the extended OPEP — to explore in details the closing pathway, from the unbound holo state to the closed apo state, of two EF-hand proteins, the Calmodulin and Troponin C N-terminal nodules. Based on a number of closing simulations for these two sequences, we show that the EF-hand β-scaffold, identified as crucial by Grabarek for the EF-hand opening driven by calcium binding, is also important in closing the EF-hand. We also show the crucial importance of the phenylalanine situated at the end of first EF-hand helix, and identify an intermediate state modulating its behavior, providing a detailed picture of the closing mechanism for these two representatives of EF-hand proteins.

© 2012 American Institute of Physics

Article Outline

  1. INTRODUCTION
  2. METHODOLOGY
    1. ART
    2. Holographic multiscale algorithm
    3. Forcefields
    4. Sequences studied
    5. Analysis
  3. RESULTS AND DISCUSSION
    1. Simulations on calmodulin NT
      1. Stability of the native apo conformation
      2. Folding from the unbound holo conformation
      3. Oxygen carrier binding residues
      4. β-sheet scaffold consolidation
      5. Exit of phenylalanine 19
      6. Closure of the hydrophobic core
      7. EF-hand helices angular evolution
      8. Higher temperature simulations: Characterization of the pathways to the apo state from metastable minima
    2. Simulations on Troponin C NT
  4. CONCLUSIONS AND PERSPECTIVES

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KEYWORDS and PACS

Keywords

bonds (chemical), molecular biophysics, molecular configurations, potential energy functions, proteins

PACS

ARTICLE DATA

Digital Object Identifier
http://dx.doi.org/10.1063/1.3671986

PUBLICATION DATA

ISSN

0021-9606 (print)  
1089-7690 (online)

Publisher

$abstract.society.value is a Member of CrossRef American Institute of Physics

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Figures (11) Tables (1)

Figures (click on thumbnails to view enlargements)

FIG.1
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.

FIG.1 Download High Resolution Image (.zip file) | Export Figure to PowerPoint

FIG.2
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.

FIG.2 Download High Resolution Image (.zip file) | Export Figure to PowerPoint

FIG.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.

FIG.3 Download High Resolution Image (.zip file) | Export Figure to PowerPoint

FIG.4
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.

FIG.4 Download High Resolution Image (.zip file) | Export Figure to PowerPoint

FIG.5
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.

FIG.5 Download High Resolution Image (.zip file) | Export Figure to PowerPoint

FIG.6
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.

FIG.6 Download High Resolution Image (.zip file) | Export Figure to PowerPoint

FIG.7
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.

FIG.7 Download High Resolution Image (.zip file) | Export Figure to PowerPoint

FIG.8
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.

FIG.8 Download High Resolution Image (.zip file) | Export Figure to PowerPoint

FIG.9
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.

FIG.9 Download High Resolution Image (.zip file) | Export Figure to PowerPoint

FIG.10
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.

FIG.10 Download High Resolution Image (.zip file) | Export Figure to PowerPoint

FIG.11
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.

FIG.11 Download High Resolution Image (.zip file) | Export Figure to PowerPoint

Tables

Table I. 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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