Applying molecular dynamics simulations to identify rarely sampled ligand-bound conformational states of undecaprenyl pyrophosphate synthase, an antibacterial target

Abstract

Undecaprenyl pyrophosphate synthase is a cis-prenyltransferase enzyme, which is required for cell wall biosynthesis in bacteria. Undecaprenyl pyrophosphate synthase is an attractive target for antimicrobial therapy. We performed long molecular dynamics simulations and docking studies on undecaprenyl pyrophosphate synthase to investigate its dynamic behavior and the influence of protein flexibility on the design of undecaprenyl pyrophosphate synthase inhibitors. We also describe the first X-ray crystallographic structure of Escherichia coli apo-undecaprenyl pyrophosphate synthase. The molecular dynamics simulations indicate that undecaprenyl pyrophosphate synthase is a highly flexible protein, with mobile binding pockets in the active site. By carrying out docking studies with experimentally validated undecaprenyl pyrophosphate synthase inhibitors using high- and low-populated conformational states extracted from the molecular dynamics simulations, we show that structurally dissimilar compounds can bind preferentially to different and rarely sampled conformational states. By performing structural analyses on the newly obtained apo-undecaprenyl pyrophosphate synthase and other crystal structures previously published, we show that the changes observed during the molecular dynamics simulation are very similar to those seen in the crystal structures obtained in the presence or absence of ligands. We believe that this is the first time that a rare 'expanded pocket' state, key to drug design and verified by crystallography, has been extracted from a molecular dynamics simulation.

Figure 1

Grid points used to calculate pocket volumes. (A) Points spaced 1 Å apart were positioned along a grid that encompassed the UPPS IPP-binding pocket. (B) Those grid points near protein atoms were removed, leaving only points within the active site, from which the volume was calculated.

Figure 2

(A) Superposition of the docked (colored by atom type) and co-crystallized poses (green) of BPH-629 bound to the 2E98 crystal structure. (B) Docked poses of 29 BPH inhibitors into the 2E98 structure. (C) Docked poses of 1i, 1j, 4a, 4g, 4j, 4l, and 4m into the 2E98 structure. Ligand poses showed a very poor alignment with the substrate, farnesyl pyrophosphate (FPP) (shown in green). (D) Docked poses of 1i, 1j, 4a, 4g, 4j, 4l, and 4m into the fourth most populated MD-derived structure. All ligand poses showed good alignment with the substrate, FPP (shown in green).

Figure 3

Volume distribution of the HIP43 undecaprenyl pyrophosphate synthase binding pocket 2E98 crystal structure and the apo crystal structure. (A) Volume of the binding pocket along the MD trajectory. The black line shows data taken every 10 ps, the overlayed gray line is the average over every 100ps. (B) Frequency at which different volumes of the pocket are sampled. The size of the bisphosphonate-bound crystal structure (2E98), the newly described apo crystal structure (Apo), and cluster 4 that docked tetramic acids well, are represented by labeled dashed lines in both graphs.

Figure 4

(A) The apo crystal structure in green and the bisphosphonate-bound crystal structure in blue. (B) The apo crystal structure with 1 Å spheres filling the active site pocket. (C) The bisphosphonate-bound crystal structure with 1 Å spheres filling the active site pocket. Note the significantly larger pocket size in the bisphosphonate-bound structure when compared to the apo crystal structure.

Figure 5

Principal component analyses (PCA). (A) Principal component analysis (PCA) calculated from the HID43 trajectory. (B) The extreme conformations of the flexible loop (residues 72–82) in HID43 are shown in red and blue. (C) PCA calculated from the HIP43 trajectory. The green circle indicates the largest conformation, and the blue circle indicates cluster 4. (D) The extreme conformations of the flexible loop (residues 72–82) in HIP43 are shown in red and blue.

Scheme 1

Condensation reaction of IPP and farnesyl pyrophosphate by undecaprenyl pyrophosphate synthase.

Chart 1

Undecaprenyl pyrophosphate synthase inhibitors. IC₅₀ values shown in parentheses. ^aPeukert 2008, (S. pneumoniae UPPS tested). ^bGuo 2007, (E. coli UPPS tested).