Conformational Changes of Glutamine 5'-Phosphoribosylpyrophosphate Amidotransferase for Two Substrates Analogue Binding: Insight from Conventional Molecular Dynamics and Accelerated Molecular Dynamics Simulations

Abstract

Glutamine 5'-phosphoribosylpyrophosphate amidotransferase (GPATase) catalyzes the synthesis of phosphoribosylamine, pyrophosphate, and glutamate from phosphoribosylpyrophosphate, as well as glutamine at two sites (i.e., glutaminase and phosphoribosylpyrophosphate sites), through a 20 Å NH₃ channel. In this study, conventional molecular dynamics (cMD) simulations and enhanced sampling accelerated molecular dynamics (aMD) simulations were integrated to characterize the mechanism for coordination catalysis at two separate active sites in the enzyme. Results of cMD simulations illustrated the mechanism by which two substrate analogues, namely, DON and cPRPP, affect the structural stability of GPATase from the perspective of dynamic behavior. aMD simulations obtained several key findings. First, a comparison of protein conformational changes in the complexes of GPATase-DON and GPATase-DON-cPRPP showed that binding cPRPP to the PRTase flexible loop (K326 to L350) substantially effected the formation of the R73-DON salt bridge. Moreover, only the PRTase flexible loop in the GPATase-DON-cPRPP complex could remain closed and had sufficient space for cPRPP binding, indicating that binding of DON to the glutamine loop had an impact on the PRTase flexible loop. Finally, both DON and cPRPP tightly bonded to the two domains, thereby inducing the glutamine loop and the PRTase flexible loop to move close to each other. This movement facilitated the transfer of NH3 via the NH3 channel. These theoretical results are useful to the ongoing research on efficient inhibitors related to GPATase.

Keywords: 5′-phosphoribosylpyrophosphate amidotransferase; accelerated molecular dynamics simulations; conformational changes; molecular dynamics simulations; substrates analogue.

FIGURE 1

The overview of GPATase structure and the ligands. (A) The stereochemical structure of DON and cPRPP; (B) GPATase subunit structure and topological structure of the protein in the glutamine domain and PRTase domain made by Pro-origami.

FIGURE 2

The free energy landscape for Rg and RMSD of (A) GPATase, (B) GPATase–DON, (C) GPATase–cPRPP, and (D) GPATase–DON–cPRPP. (E) Relative frequency of RMSD of the four systems, (F) relative frequency of Rg of the four systems.

FIGURE 3

RMSF values of the backbone atoms of the four systems over time during the simulations. Comparison of the RMSF plots of proteins in (A) GPATase, GPATase–DON, GPATase–DON- cPRPP complexes; (B) GPATase, GPATase–cPRPP, GPATase–DON- cPRPP complexes.

FIGURE 4

Stability analysis of DON binding site and cPRPP binding site for systems during the 200 ns simulations. (A) The RMSD values of DON binding site, (B) RMSD plot of cPRPP binding site of the four systems during 200 cMD simulations. (C) SASA plot for DON binding pocket. (D) SASA plot for cPRPP binding pocket.

FIGURE 5

Analysis of changes in secondary structures. (A) DSSP results of the four systems, the color bar represented different secondary structures. (B) The probability of α-helix (Gln339-Lys349) during 200 cMD simulations. (C) The corresponding cartoon structure of the 0 and 100 ns cMD simulations for the four systems.

FIGURE 6

The residues cross-correlation maps for four systems (A) GPATase, (B) GPATase–DON, (C) GPATase–cPRPP, and (D) GPATase–DON- cPRPP. The positions of glutamine loop and flexible loop were labeled by black boxes.

FIGURE 7

The two-dimensional projection of total cMD conformational space on the first two principal components (PC1 and PC2). FEL maps and PC1 and PC2 structures for (A) GPATase, (B) GPATase–DON, (C) GPATase–cPRPP, and (D) GPATase–DON–cPRPP systems. The conformation of GPATase proteins with one global minimum is marked in red. The depth of the energy landscape indicates the value of the minimum free energy.

FIGURE 8

The subnetwork interaction analysis between ligands and protein. The subnetwork between GPATase and DON in the (A) GPATase–DON complex and (B) GPATase–DON- cPRPP complex. The subnetwork between GPATase and cPRPP in the (C) GPATase–cPRPP complex and (D) GPATase–DON- cPRPP complexes.

FIGURE 9

Comparison of R73–DON salt bridges between the GPATase–DON and GPATase–DON–cPRPP complexes during aMD simulations. The salt bridges are shown as green dashed lines. The glutamine loop is marked in light blue. R73 and DON are presented as sticks. Representative structures of the (A) GPATase–DON and (B) GPATase–DON–cPRPP complexes. (C) Variations in the distance between R73 and DON of the two systems. (D) Relative frequency of distance of the two systems.

FIGURE 10

The conformation change of PRTase flexible loops during 400 ns aMD simulations. Free energy profile maps of flexible loops for the (A) GPATase; (B) GPATase–DON; (C) GPATase–cPRPP; and (D) GPATase–DON–cPRPP complexes as a function of CV1 and CV2 in Å; representative structures of flexible loops for the (E) GPATase; (F) GPATase–DON; (G) GPATase–cPRPP; and (H) GPATase–DON-cPRPP complexes. The PRTase domain active site is shown in cartoon. Cartoons marked in blue and red represent different perspectives.

FIGURE 11

Comparison of the relative position between the glutamine loop and flexible loop of the four systems. Both loops are displayed as cartoons. DON and cPRPP are shown as spheres. Y74 and I335 are depicted as sticks. Representative conformation of the (A) GPATase, (B) GPATase–DON, (C) GPATase–cPRPP, and (D) GPATase–DON–cPRPP complexes during aMD simulations. (E) Distance between Y74 and I335 in the four systems. (F) The average distance between Y74 and I335 in the four systems; standard deviations are labeled in the histogram.