Figure 1.
Cartoon representations of DHDPS crystallographic structures.
(A) Wild-type E. coli; (B) E. coli L197Y mutant dimer; (C) wild-type MRSA dimer. The arrows indicate locations of the active sites (1 per monomer) and tight-dimer interfaces; (D) Active site alignments of tetramer and dimers. Wild-type E. coli tetramer (dark blue), E. coli L197 mutant dimer (light blue), MRSA wild-type dimer (green).
Figure 2.
Overall simulations results for E. coli DHDPS tetramer and dimer.
(A) Cα RMSDs over the course of the simulations, for dimers from tet-1 & tet-2 (shades of grey), dim-A (blue), dim-B (light blue); (B) Cartoon representation of monomer-monomer reorientation during simulation of dimers. The relative rotation of monomers is represented by dotted lines and an arrow. Cartoons are shown for extreme conformations taken from dim-B (light-blue at 70 ns, blue at 433 ns), and mrsa-1 (green at 430 ns). Cα RMSD between extreme conformations are: 4.0 Å for the E.coli and 3.8 Å for the MRSA dimers. (C) Cα RMSD values for monomers from tet-1 & tet-2 (shades of grey), dim-A & dim-B (shades of blue); (D) Angles of rotation corresponding to monomer rearrangement. Only tet1-A (black), dim-A (blue) and dim-B (light blue) are represented for clarity, the thick lines represent the spline fit of the values.
Figure 3.
Flexibility and stereochemistry of active sites in E. coli DHDPS tetramer and dimer simulations.
(A) RMSDs of active site residues for E. coli tetramers and dimers: tet-1 & tet-2 (grey shades, 8 curves overlayed for 2×4 active sites), dim-A (light blue, 2 curves), dim-B (dark blue, 2 curves). (B) Individual RMSFs of active site residues, averaged over all simulations, with error bars: tet-1 and tet-2 (grey, 2×4 active sites), dim-A and dim-B (blue, 2×2 active sites), mrsa-1 and mrsa-2 (green, 2×2 active sites; E. coli numbering). (C) and (D) Ramachandran plots of the Y107 backbone dihedral angles in the E. coli tetramer and dimer simulations, respectively. Red crosses indicate the crystallographic geometries. The orange contour map (or “favoured” region) accounts for 98% of the phi-psi angles analysed by Lovell et al [25]. Pale orange contour maps account for 99.95% (“allowed”). Percentages represent the time spent in the 3 regions of the plot.
Figure 4.
Snapshots of active site residues taken from: (A) E. coli tetramer (tet-1), (B) E. coli mutant dimer (dim-A), and (C) MRSA simulations (mrsa-1).
Y107 (E. coli)/109 (MRSA) is highlighted in purple (A), blue (B) or pale green (C). Snaphots are taken every 100 ns from each trajectory.
Figure 5.
A detailed view of the tight-dimer interface in E. coli and MRSA DHDPS.
Surfaces of both enzymes with the residues involved in the tight-dimer interface represented in light orange. Residues involved in hydrogen bonds are shown in red and in salt-bridges in yellow, as calculated by the PISA server (A) Dimer from E. coli wild-type tetramer (PDB ID: 1YXC); (B) MRSA wild-type dimer (PDB ID: 3DAQ).
Figure 6.
(A) Cα RMSDs over the course of the MRSA simulations, for dimers from mrsa-1 (green) and mrsa-2 (turquoise); (B) RMSDs of active site residues over the course of the MRSA simulations (4 active sites); (C) Ramachandran plot showing backbone dihedral angles of residue Y109 during the MRSA simulations.
The red crosses indicate the crystallographic conformations. Percentages represent the time spent in the 3 regions of the plot.
Figure 7.
Network of essential active site interactions over the course of the simulations.
(A) Electrostatic interactions and residues considered: Wild-type E. coli tetramer (dark blue), E. coli L197 mutant dimer (light blue), MRSA wild-type dimer (green). Only Wild-type E. Coli interactions (dashed lines) are shown for clarity. (B) Distance between T44-Oγ and Y133-Oç. (C) Distance between T44-Oγ and Y107-Oç. (D) Distance between K161-Nζ and Y133-Oç. Equivalent MRSA residues are T46, Y135, Y109 and K163.
Figure 8.
Changes at the tight-dimer interface during simulations.
(A). Interfacial surface area buried for both monomers; (B) Number of interfacial hydrogen-bonds (tet-1:black; dim-A: blue; mrsa-1: green). Spline fits (thick lines) of the values (thin lines) are represented for clarity.
Figure 9.
Cavities in DHDPS active sites.
(A) Wild-type E. coli DHDPS; (B) L197Y E. coli engineered dimer; (C) Wild-type MRSA DHDPS. Active site cavities are represented as mesh surfaces (yellow) for the last 100 ns of dim-A, tet-1 and mrsa-1.