Structural analysis of prolyl oligopeptidases using molecular docking and dynamics: insights into conformational changes and ligand binding

Abstract

Prolyl oligopeptidase (POP) is considered as an important pharmaceutical target for the treatment of numerous diseases. Despite enormous studies on various aspects of POPs structure and function still some of the questions are intriguing like conformational dynamics of the protein and interplay between ligand entry/egress. Here, we have used molecular modeling and docking based approaches to unravel questions like differences in ligand binding affinities in three POP species (porcine, human and A. thaliana). Despite high sequence and structural similarity, they possess different affinities for the ligands. Interestingly, human POP was found to be more specific, selective and incapable of binding to a few planar ligands which showed extrapolation of porcine POP in human context is more complicated. Possible routes for substrate entry and product egress were also investigated by detailed analyses of molecular dynamics (MD) simulations for the three proteins. Trajectory analysis of bound and unbound forms of three species showed differences in conformational dynamics, especially variations in β-propeller pore size, which was found to be hidden by five lysine residues present on blades one and seven. During simulation, β-propeller pore size was increased by ∼2 Å in porcine ligand-bound form which might act as a passage for smaller product movement as free energy barrier was reduced, while there were no significant changes in human and A. thaliana POPs. We also suggest that these differences in pore size could lead to fundamental differences in mode of product egress among three species. This analysis also showed some functionally important residues which can be used further for in vitro mutagenesis and inhibitor design. This study can help us in better understanding of the etiology of POPs in several neurodegenerative diseases.

Figure 1. Cavities present in POP.

Bottom arrow indicates β-propeller cavity which continues till active site and form another smaller cavity near active site shown in red color (Tarrago et al 2009), inter-domain cavity is shown in green color.

Figure 2. Model generation.

a) alignment between query (At1g20380) and template (PDB ID: 1E5T, porcine) generated by CLUSTALW, sequence identity was found to be 53%. α/β hydrolase domain is cyan colored and β-propeller is shown in yellow color. Active site residue conservation is shown in red color b) homology model of A. thaliana POP generated using porcine POP as a template. Catalytic triad is shown in magenta color.

Figure 3. Electrostatic charge distribution of POP of three species.

a) down the β-propeller pore view of electrostatic potential. Three species showed differences in electrostatics. A. thaliana POP showed more positive potential; similarly differences were also present in human and porcine POP proteins b) Electrostatic potential of inter-domain cavity and recently reported cavity (Tarrango et al 2009). In A. thaliana later cavity was hidden.

Figure 4. Rigid and flexible docking binding scores.

a) Rigid docking scores of human, porcine and A. thaliana POP. Some of the ligands showed hindrance in binding to human POP as revealed by their positive energy scores. Different ligands showed difference in binding affinities to three species of POP. b) Flexible docking scores of human, porcine and A. thaliana POP. After inducing flexibility in ligands still two of the ligands were incapable of binding to human POP.

Figure 5. Root mean square deviations (rmsd) of backbone Cα of three POP species during simulation.

a) rmsd of bound form of porcine (black), human (red) and A. thaliana (green) POPs b) rmsd of unbound form of porcine, human and A. thaliana POPs. Dotted line indicates rmsd of replicate runs.

Figure 6. Deviations in β-propeller pore size.

During simulation of bound form of porcine, human and A. thaliana POP. In porcine diameter of pore was found to be increasing in while human and A. thaliana it was found to be decreasing (Figure S7 for replicate runs).

Figure 7. 20^th ns bound form structure of porcine, human and A. thaliana POPs.

β-propeller pore size was found to be biggest in porcine, while in human and A. thaliana it was small. Drug (ZPR) is shown in magenta color, active site is colored red.

Figure 8. Conservation of lysine residues across lower to higher organisms.

Lysines were conserved in all mammalian species and also in amphibian Xenopus. Coloring: Archaebacteria (yellow), mammals (dark grey), amphibian (blue), plants (orange), arthropods (blue, below orange), nematodes (light yellow), bacteria (light grey). Presence of lysines are shown using red color, while absence using green color.

Figure 9. Conformational changes in β-propeller pore size in human POP after in silico mutation of lysine residues.

Yellow color indicates change in pore size.

Figure 10. Simulation from blind docking pose where drug was at mouth of β-propeller pore.

a) 0^th ns structure (protein: green, drug: red) and 20^th ns structure (protein: blue, drug: magenta) were superimposed and distance plot of catalytic Ser-554 hydroxyl group and centre of mass of drug was plotted. Distance was found to decrease by more than 2 Å. B) distance plot.

Figure 11. Free energy profile of smaller product movement through β-propeller in porcine POP.

a) 0^th ns structure which showed huge barrier around β-propeller pore. b) 20^th ns structure where free energy barrier was reduced.