Fig 1.
A schematic catalytic cycle for CYPs, showing all the key species, and the uncoupling process.
Fig 2.
a,b) Cartoon representation of the CYP-2B4 crystal structure (PDB ID 1SUO) with specific domains coloured and labeled according to Scott et al.;[12] the heme group, the peroxide anion and Cys426 are shown as ball and stick. The enzyme is shown looking from the distal (a) or proximal (b) side of the heme group. To be coherent with the literature, the same color map reported in the original experimental work has been used. (c,d) Detail of the active site.
The protein backbone is shown as a transparent cyan ribbon, while Cpd0 is shown in and stick style, together with the various rotamers of residue 429. Cys 436, residue 429 and the A and D propionate groups are labeled.
Table 1.
For a given nC (first row) the Dunn index (DI), the Davies-Bouldin index (DBI) and the size of the various clusters are shown. The column showing the best nC value is typed in italics. Frame set #1 was used in the analysis.
Fig 3.
Clustering of CYP2-2B4 conformations.
Distribution of structures from 17500 frames artificial trajectory (frame set #2) in clusters (y axis) and simulations (x axis) using GROMOS. Snapshots originally sampled in the 15 trajectories are represented as black circles (WT), blue squares (F429A), magenta triangles (F429E), orange squares (F429H) and green down pointing triangles (F429L).
Fig 4.
Projection of the trajectories of Cα atoms from the larger artificial trajectory in the volume spanned by the first 3 eigenvectors obtained by the principal component analysis, from 2 different points of view. Frames have been represented as dots coloured in black (WT), blue (F429A), magenta (F429E), orange (F429H) and green (F429L).
Fig 5.
Root Mean Square Fluctuations in clusters and specific systems.
a) Cα atom RMSF in the 3 clusters obtained by clustering. Cyan dashed line: cluster #1 (i.e. frames from the F429A, F429E and F429H simulations); full black line: cluster #2 (WT); magenta dot-dashed line: cluster #3 (F429L). b) difference of RMSF calculated from the trajectories of 4 mutants with respect to the WT trajectory. Full blue line: F429A; magenta dashed line: F429E; orange dot-dashed line: F429H; green dotted line: F429L.
Fig 6.
Per residue averaged root mean square fluctuation.
Average structures of the 3 clusters, depicted using ribbon colours and thickness proportional to the RMSF. a) First cluster (F429A, F429E, F429H). b) Second cluster (Wild Type enzyme). c) Third cluster (F429L).
Fig 7.
Radial distribution functions of water molecules (center of mass) around the Sγ atom in C436 calculated from the WT (full black line), F429A (blue dashed line), F429E (magenta dot-dashed line), F429H (orange dashed line) and F429L (green dotted line).
Fig 8.
The heme group is shown in purple and the protein backbone as a cyan transparent ribbon. Cys436 is always shown in ball and stick. a) Tunnels in the centroid of clusters #1. The bottleneck residues E429 and L437 are represented in licorice. b) Tunnel of the F429H simulation; residues G438 and L437 are also shown.
Table 2.
Pathways towards the Sγ atom in C436.
The table shows the length, bottleneck radius and throughput. The last column summarizes the simulation(s) in which a given tunnel was observable.
Fig 9.
Radial distribution function of water molecules (center of mass) around the OOH- anion calculated for the WT, F429A, F429E, F429H and F429L, respectively.
Table 3.
Pathways properties for frame set #2.
The table shows the total number of frames in which a specific tunnel cluster was present, the average length and bottleneck radius and the throughput, i.e. the average of e-cost across all frames using the cheapest tunnel in each frame. Pathways are shown from highest to lowest average throughput; average values refers to total number of frames in which a pathways was existent. The last column summarizes the simulation(s) in which a given tunnel cluster was observable; systems for which the pathway was less important are shown in brackets.
Fig 10.
Bottleneck radii of pathways frame set #2.
a) bottleneck radius of pathways #1 (blue), #5 (orange) and #6 (magenta). b) bottleneck radius of pathway #2 (black), #3 (red) and #4 (green). Any given configuration may be easily related to the simulation it belongs to dividing the graph in windows 150 ns wide (see also the graph top).
Fig 11.
The heme group, OOH- and Cys436 are shown in magenta. The enzyme and tunnel coordinates shown are those of the corresponding centroid as in Fig 8. a) Pathways #1 (red), #2 (green) have been represented; coordinates from the F429H centroid were used. Bottleneck lining residues for pathway #1 and #2 have been represented in ball stick or licorice, respectively. b) Pathway #3 (F429L simulation) and corresponding bottleneck residues. c) Pathways #4 (WT simulation) and corresponding bottleneck residues.