Building a Transition Path between Open and Closed Forms of TcPRAC and Virtual Screening

The structure of hemi-bounded TcPRAC (1W62 hemi complex PDB) is asymmetric and shows one closed subunit binding PYC (chain A), and one unbound opened subunit (chain B) [18]. By swapping the chain labels and reorientation on the original structure we built an inverted structure where chain A is opened and chain B is closed (see Figure 2A). Internal coordinates and Cartesian coordinates of the two extreme structures were compared to remove unnecessary symmetric side chain flips between the two structures. Then, we built a transition path connecting the two structures, hence modeling opening of chain A and closing of chain B when moving from the original structure to the inverted one (see schematic view in Figure 2B). For that, we used the “Path Optimization and Exploration” approach (POE) described in ref. [25]. This approach iteratively uses the Conjugated Peak Refinement (CPR) [26] method of the CHARMM program [27] to refine the path, explore possible shorter local paths and reassembles them to reconstruct the whole path. The transition path was built on all atomic degrees of freedom (here 19488), as a curved trajectory formed by a series of N (here 49 at the end of the procedure) ordered and low energy intermediate states (X i , i in [1,.,N]) avoiding energetic barriers. Following CPR construct, it ensures the absence of “hidden” barriers by probing the energy of structures along a straight line between consecutive intermediates of the path ([X i ,X i+1 ], with i in [1,.,N−1]), and checks that it is lower than a given (low) threshold (E[(1–λ)X i +λX i+1 ]< E max , λ in [0,1]). To facilitate the first iteration, the procedure was initiated with a Molecular Dynamics (MD) trajectory starting from the original structure and drawn towards the inverted structure by a linear constraint of 0.5 kcal/mol/Å/atom. MD was 200,000 steps long recorded every 100 steps with a Langevin thermostat at 300 K and a friction coefficient of 100 ps−1 to remove excess heat. The solvent is modeled implicitly for CPR is an optimization method and, thus requires instantaneous solvent equilibration at room temperature while the structure energy is being minimized, which corresponds to a low temperature. The analytical continuum electrostatics (ACE2) potential [28] was used with default dielectric constants (1 inside the protein and 80 outside) to avoid formation of artifactual cavities. POE includes various procedures above simple CPR to reduce the number of intermediates for the same level of optimization (all intermediate have energies lower than a given threshold, here −34000 kcal/mol). (i) Intermediate structures are systematically removed if any linear interpolations between remaining consecutive structures are still below an energy threshold (-34000 kcal/mol) in a procedure implemented as “backward reduction” [25]. (ii) Sub-paths are recalculated with CPR between intermediate X i and X j , j−i>n min , at distance d ij <d max , when the latter are separated by a sequence intermediate X i+1 ,…X j−1 covering a length (l i,j = d i,i+1 +…+d j−1,j ) that is K time larger than d ij . K, d max and n min are adjusted according to path refinement according to computer resources available (e.g. maintain the list of i,j pairs lower than 1000). Distance between X i and X j , d i,j is the RMS difference between respective coordinates expressed in Å. In early phases the path is rugged and K has to be set at high level, say 5, d max low, say 1.5 Å, and n min high, say 10 or 20. At late phases the path is smoother, the number of pairs fulfilling the early criteria decreases and K can be set to lower values, say 1.5, d max higher, say 2–3 Å, and n min lower say 5. A Recalculated sub-path replaces the original one when its maximum energy is lower or equal to that of the replaced sub-path and the number of intermediates it contains is lower.

PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 2. PYC-induced structural rearrangement of TcPRAC prompted a virtual screening strategy. (A) In the crystal structure (1W62.PDB), the substrate-binding cavity of the “closed” protomer, shown as red ribbons and cylinders, is completely buried, as illustrated by the small green volume surrounding PYC whose nitrogen, oxygen and carbon appear as blue, red and cyan small spheres, respectively. In the absence of PYC, the TcPRAC protomer has a more relaxed, open structure, shown as blue ribbons and cylinders, with the void volume of the active site accessible from the bulk solvent shown in green. (B) Schematic overview of the virtual screening strategy. The 3D structure of the hemi saturated complex shown in A is represented by circles of the same color on the left. It was used to build the symmetric form of the complex as depicted by the circles on the right. Molecular mechanics and molecular dynamics were used to model TcPRAC motion (horizontal arrow) triggered by inhibitor binding. Known (weak) inhibitors derived from PYC were used to select intermediate conformations on the basis of their docking scores. https://doi.org/10.1371/journal.pone.0060955.g002

The volume of the cavity was calculated with a cavity search program developed in the laboratory and described in [29], but without outer sphere limit. Within the cavity, the points accessible to the center of the probe sphere are labeled (i.e. points excluded from the protein atoms when their canonical exclusion radii had been incremented by the solvent probe sphere radius). The cavity extension is calculated as the maximum distance between the latter points. Xmgrace was used to prepare the graphics of Figures 3A, 3B and 3C and Figure 4A [http://plasma-gate.weizmann.ac.il/Grace/]. Ligand sizes, l max , were measured on canonical conformers in the computer graphic program VMD, which was also used to prepare Figure 2A and Figure 4B [30].

PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 3. Transition path characteristics. (A) Energy profile and metric quantities for the TcPRAC transition path. The energy profile (full line) shows that the intermediate states have low energy and do not present any energy barriers. Dotted and dashed lines show the distance from the first structure (d 1,i ) and the cumulative distance covered from the first structure (l 1,i ), respectively (RMS in Å, see Material and Methods). Little swerving was necessary to avoid the energy barriers. The points corresponding to the intermediate structures used in the screening are marked by crosses. (B) Scores of known ligands, synthesized analogues, and new inhibitors when docked in the selected binding site models. Br-OxoPA could not be docked in the crystallographic structure and its score in the fourth conformation is circled. The score threshold that was chosen in the subsequent virtual screening phase for ligand selection is indicated by a dashed line and the exclusion region is striped. Transition path characteristics. (A) Energy profile and metric quantities for the TcPRAC transition path. The energy profile (full line) shows that the intermediate states have low energy and do not present any energy barriers. Dotted and dashed lines show the distance from the first structure (d 1,i ) and the cumulative distance covered from the first structure (l 1,i ), respectively (RMS in Å, see Material and Methods). Little swerving was necessary to avoid the energy barriers. The points corresponding to the intermediate structures used in the screening are marked by crosses. (B) Scores of known ligands, synthesized analogues, and new inhibitors when docked in the selected binding site models. Br-OxoPA could not be docked in the crystallographic structure and its score in the fourth conformation is circled. The score threshold that was chosen in the subsequent virtual screening phase for ligand selection is indicated by a dashed line and the exclusion region is striped. (C) Cavity volume and extension in transition path intermediates, and docked molecules properties. Volume and extension are calculated as explained in Material and Methods. The volume is displayed by the thin line curve. Cavity extension is displayed by the thick curve and crosses mark intermediates that were used for virtual screening. The extension of PYC is shown by the horizontal line, that of OxoPA in all-trans conformation is shown by the horizontal dashed line. The extension of BrOxoPA in all-trans conformation is given in dotted line for two extreme rotamers on the C4–C5 bond. The average molecular weight of the library compounds successfully docked in conformers 1, 4 and 10 is displayed by filled circles. For clarity, the average mass has been divided by 2 to fit the same scale as the cavity volume. https://doi.org/10.1371/journal.pone.0060955.g003

PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 4. Selection of active site conformations for virtual screening. (A) Four of the 49 conformations defining the path were selected for virtual screening. Protein secondary structures are shown schematically as in Figure 2. Transparent green spheres show the enclosed void volume of the pocket, with the ligand inside the first three structures (opaque green). (B) Structure of the two identified novel inhibitors of TcPRAC. https://doi.org/10.1371/journal.pone.0060955.g004