Figure 1.
Mechanism of tetrapyrrole chain elongation catalyzed by PBGD.
The scheme shows the deamination of porphobilinogen (PBG) to methylene pyrrolenine (MePy), the nucleophilic attack by the B ring of dipyrromethane (DPM) on MePy forming an intermediate that undergoes deprotonation to form a tripyrrole moiety (P3M). Subsequent additions of PBG elongates the chain to form tetrapyrrole (P4M), pentapyrrole (P5M) and hexapyrrole (P6M) moieties. In the last step, the tetrapyrrole product, 1-hydroxymethylbilane (HMB) is hydrolyzed from DPM. The rings of the elongating pyrrole chain are labeled as A, B, C, D, E and F starting from the pyrrole ring covalently attached to C242. The acetate and propionate side-groups of the pyrroles are denoted by -Ac and -Pr, respectively.
Figure 2.
Loop modeled structure of E.coli PBGD and its conserved residues in the active site.
A. The 3D coordinates of the E.coli PBGD were obtained from PDB (ID: 2YPN) and the missing loop region (42–60) was modeled using Modeller9v8. The same domain color scheme is followed throughout. B. Stereo view of the active site residues R11, D84, R131, R132, R149, R155, R176 and R232 along with conserved residues K55 and K59 (in green) and the DPM cofactor (in pink) shown as sticks.
Figure 3.
Root mean square deviation of the protein during pyrrole chain elongation.
RMSD of the protein backbone, with respect to the 2YPN reference structure, at each stage of chain elongation.
Figure 4.
Protein dynamics during pyrrole chain elongation.
A. RMSF plot of the protein from DPM to P6M stages. The color bar at the bottom corresponds to the domain demarcation (domain 1 – blue, domain 2 – red and domain 3 – green, active site loop – cyan, hinge regions – black). B. HeatMap showing the residue-wise contribution to RMSD of the protein through the different stages of simulation indicated by a color bar at the right. The simulation stages are denoted by a color bar along the abscissa. C. Solvent Accessible Surface Area (SASA) and Radius of gyration (Rgyr) values show the loss of compactness of the protein on addition of each PBG molecule through the stages of simulation from DPM to P6M. The error bars shown in the figure represent the standard deviation of the data from the mean.
Figure 5.
Loop dynamics during pyrrole chain elongation.
A. Plot of distance between centers of mass of the loop residues (42–60) and the active site residues (11, 19, 84, 131, 132, 155, 176, 242) to track the loop movement in the different stages of the simulation. B. Interaction of K55 with E88 (open loop conformation denoted in blue color) and with V306, E305 and Q243 (closed loop conformation denoted in green color) regulate the loop movement in DPM stage. C. Distance graphs depicting the interaction of D50 with R149 during DPM stage to regulate loop movement (along with K55 interactions); interaction of D50 with R149 (black), D50 with G150 (red) and K55 with E88 (green), involved in loop movement during the P3M stage; Interaction of K55 with E239 (black) and G60 with E88 (red) involved in loop movement during the P4M stage.
Figure 6.
Structural changes in PBGD during DPM to P6M stages.
Structural changes observed in the protein in DPM and P6M stages show the length of beta sheets and helices in domain 1 shortens (red arrow) in P6M stage, a shorter helix is observed in domain 3 (green arrow) and the hinge region between domain 1 and domain 2 uncoils (pink arrow) in the P6M stage.
Figure 7.
Combined essential dynamics and DCCM of the concatenated trajectory.
A. Principal component 1 (Video S1) and B. Principal component 2 (Video S2) of the combined trajectory analysis from DPM to P6M stages. C. RMSF corresponding to the principal component 1 and 2 with domain demarcation along the abscissa. D. Dynamic Cross Correlation Map on the concatenated trajectory of all stages of simulation showing the correlation between the domain movements with the color bar at right indicating the intensity of correlation. Domain demarcations and the secondary structure elements (helix and sheet) are also shown along the axes.
Figure 8.
Polypyrrole chain accommodation.
A. Volume of the active site and cumulative SASA of the active site residues (as reported in Table 1) show an increase from DPM to P6M stage with the addition of each PBG molecule. B. Graph showing the increase in domain separation between domain 1 and 2 during the catalytic stages of PBGD. C. Polypyrrole accommodation within the active site cleft as a result of major domain movements during chain elongation; snapshots of only DPM & P6M stages are shown. D. Graphs of the interaction between W18 and R176 with B ring of pyrrole chain in P3M stage and with C ring of pyrrole chain in P4M stage showing the shift in interactions to accommodate the polypyrrole chain.
Table 1.
Amino acids in PBGD interacting with the polypyrrole chain during each stage of chain elongation.
Figure 9.
Interactions of R11, F62, D84 and R176.
A. A closer view of the stacking interaction of R11 with F62 present at the base of the active site loop measured by the distance between the CZ atom of R11 and center of mass of the phenyl ring of F62 in the DPM stage. B. In the P4M stage D84 interacts with R11, disrupting the stacking of R11 with F62. C. Stacking interaction of R11 with F62 keeps the active site loop in a position facilitating its movement during DPM and P3M stages, shown as a distance graph between the CZ atom of R11 and the center of mass of the phenyl ring of F62 in DPM, P3M and P4M stages. D. Distance graph depicting the interaction of R11 with D84 in the P4M stage which causes the stacking between R11 and F62 to break. E. Distance graphs depicting the interaction of R176 with the B (in black) and C (in red) rings of the polypyrrole chain during the stages of chain elongation.
Figure 10.
Conformation of 1-hydroxymethylbilane during the HMB stage simulation.
With reference to initial conformation of HMB (green) the C and F rings are displaced by a distance of 3.5 Å and 5.3 Å respectively in the structure at the end of simulation (cyan) during the HMB stage.
Figure 11.
Exit mechanism of HMB from PBGD.
A. Structure of PBGD showing probable exit directions, either from C or F ring of the HMB unit, that are considered for SMD simulations: C1 (Direction from the center of mass of the C ring in HMB towards the interface of domain 1 and domain 2), F1 (Direction from the center of mass of the F ring in HMB towards the active site loop), F2 (Direction from the center of mass of the F ring in HMB towards the interface between the active site loop and domain 1). B. Surface representation of the structure of PBGD showing the most probable path predicted for the exit of HMB through the space between domain 1, domain 2 and the active site loop (Video S3). C. Force as a function of time during the SMD runs in 3 different exit paths: C1, F1 and F2. D. Graphs showing the interactions of R11, Q19 and R176 with HMB during the SMD runs through C1, F1, and F2 path, indicating the possible role of these catalytically important residues in the exit of the product.
Table 2.
Residue-wise interactions with HMB during its exit in the SMD runs.
Figure 12.
Protein relaxation after product exit.
A. Probability distribution graph of radius of gyration of the protein in DPM, P6M and no-HMB stages showing that in the no-HMB stage, the Rgyr falls back close to the DPM stage. DCCM plots of B. no-HMB; C. DPM; and D. P6M stages, showing the differences and similarities in correlation to the DPM stage as the protein relaxes from no-HMB stage. The marked regions in the no-HMB stage resemble more to DPM stage during protein relaxation.
Figure 13.
Comparison of average structures of DPM and no-HMB stages.
Superposed average structures of the DPM and no-HMB stages. The encircled regions show secondary structures that are yet to be regained after the protein relaxation for 150 ns.