Fig 1.
Crystal structure and cis-1,4-polyisoprene cleavage reaction catalyzed by LcpK30.
(a) Oxidative cleavage of cis-1,4-polyisoprene catalyzed by LcpK30. (b) Crystal structure of LcpK30 (PDB ID: 5O1L) with a close view of heme in the active site..Coordination of an imidazole molecule to the distal site of heme fixes the protein structure in its open conformation (PDB: 5O1L), while the coordination by the sidechain of Lys167 forces the closed state of the enzyme (PDB: 5O1M).
Fig 2.
Static tunnel mapping and pocket identification in the LcpK30 open structure without substrate.
(a) Two dominant tunnels identified by CAVER-Pymol plugin 3.0.3 (tunnel 1 in blue and tunnel 2 in green); (b) five detected pockets or cavities closest to heme in the LcpK30 static X-ray structure, identified by automated geometry-based fpocket tool. The pockets are pocket 1 (olive), pocket 3 (cyan), pocket 4 (orange), pocket 9 (magenta) and pocket 12 (yellow).
Fig 3.
Principal component analysis (PCA) of MD simulations of open and closed-like states of LcpK30 without substrate.
PCA was performed considering cartesian coordinates of protein backbone atoms (N, Cα, C and O). (a) MD snapshots projected on the first two principal components. Blue and red circles represent MD snapshots belonging to open and closed-like states, respectively. (b) Normal mode displacement vectors associated with the first two principal components showing only motions longer than 2 Å as porcupine in both directions. The backbone is shown as cartoon representation and colored by the mobility where lower and higher flexibility is depicted in blue and red, respectively.
Fig 4.
Dynamical tunnel mapping and pocket identification in the LcpK30 open structure without substrate.
(a) Dynamical tunnel calculations with CAVER Analyst 2 using MD simulations of LcpK30 in open conformation. First 5 dominant tunnel clusters were calculated on 1500 snapshots from MD simulations of the open state. Two superclusters are formed, colored in green (comprised of clusters 1 and 3) and blue (comprised of clusters 2 and 5). Clustering displays the center lines for all tunnels computed for all snapshots at once. Center lines are colored according to their related clusters. (b) Pockets detected closest to heme calculated on 1500 snapshots from MD simulations of LcpK30 in the open state.
Table 1.
Features of first 5 dominant dynamical tunnel clusters in LcpK30 open and closed-like states.
Fig 5.
Substrate model with 10 repeating isoprene units (C50H82) used in this work.
Table 2.
Structural features of the best docking poses obtained from induced fit docking within GOLD.
Docking solutions are ranked based on the fitness score from highest to lowest.
Fig 6.
RMSD and RMSF profiles for LcpK30 in the open state with cis-1,4-polyisoprene (C50H82) substrate model bound.
C-alpha RMSD (left) and RMSF (right) were calculated during 100 ns MD simulations, starting from 10 docking poses obtained with (a) ChemPLP and (b) ChemScore. All values are calculated from the reference X-ray structure of the enzyme. Residues 29–49 from N-terminus were omitted from the analysis to minimize the noise.
Fig 7.
Dynamical tunnel mapping and pocket identification in the LcpK30 open structure with bound substrate.
First 10 dominant tunnel clusters were calculated on 1000 snapshots from MD simulations of the open state LcpK30 with cis-1,4-polyisoprene substrate bound, using CAVER Analyst 2.
Table 3.
Features of first 10 dominant tunnel clusters in LcpK30 bound to substrate.
Fig 8.
Ensemble of 50 superimposed snapshots from 100 ns MD simulations of cis-1,4-polyisoprene near heme, obtained starting from the 20 top ranked docking poses.
a) Snapshots obtained from ChemPLP fitness function; b) Snapshots obtained from ChemScore fitness function. Trajectory smoothing window size was 5. Protein and hydrogens were omitted for the sake of clarity.
Fig 9.
Residues that frequently interact with the substrate during MD simulations.
(a) Close contacts between frequently occurring bound conformations of the substrate (extended and folded) and LcpK30, calculated from 100 ns MD simulations of 20 docking poses obtained with ChemPLP and ChemScore fitness function. The profile was constructed by averaging total fractions obtained for similar conformations. Conformations were clustered based on the contacts and the features from the visual inspection; (b) Representative snapshots extracted from MD simulations showing the main extended (ChemPLP pose 10 and ChemScore pose 5 shown as dark green and light green licorice, respectively) and folded (ChemScore pose 1 shown as magenta licorice) conformations and protein residues (shown as lines) that interact with the cis-1,4-polyisoprene substrate model bound to LcpK30 near heme (sphere representation). The three tunnels and the hydrophobic cavity are shown as blue, green, orange and yellow surface, respectively. The rest of the LcpK30 and hydrogen atoms are omitted for clarity.
Fig 10.
Linear interaction energy analysis between the ligand and LcpK30.
Calculations were performed on 100 ns MD simulations of 10 docking poses obtained with (a) ChemPLP and (b) ChemScore fitness function. The Eele is the electrostatic energy (orange circles) and EvdW is van der Waals energy (blue circles). The total energy Etot (green circles) is calculated as a sum of Eele and EvdW. The binding energy ΔEbind (yellow circles) was calculated on 50 snapshots from MD simulations with the MM/GBSA method.
Fig 11.
Cleavage frequency profile constructed from distance histograms indicating a total count of each of the ten C = C double bonds (coloured differently) from the cis-1,4-polyisoprene substrate that are found in the near vicinity (below 4 Å) of the distal oxygen atom in the heme-bound O2 molecule. The profile is calculated from 100 ns MD simulations of LcpK30 in the presence of the substrate for all 20 docking poses obtained with ChemPLP and ChemScore fitness functions.
Fig 12.
Proposed gateway for the substrate entry into LcpK30.
The gateway residues were proposed based on the results from static and dynamic modelling of LcpK30 with cis-1,4-polyisoprene ligand. The representative snapshots of (a) extended and (b) folded conformation of cis-1,4-polyisoprene in complex with LcpK30 highlighting their interaction with the putative tunnels and the hydrophobic protein cavity. The O2 bound on heme was omitted for the sake of clarity.