Fig 1.
Sampling space for oseltamivir in the simulations.
(a) The diffusion region of oseltamivir in the free-binding MD simulation; oseltamivir was restricted in a space centered on the beta carbon of N346 with a radius of 27 Å. (b) and (c) Side and top views of the sampling region (cuboid box with a size of 46 Å×38 Å×32 Å) for the meta-eABF simulation.
Fig 2.
Typical RMSDs of oseltamivir in the trajectories produced binding events.
The RMSD of heavy atoms of oseltamivir was calculated by referring those of oseltamivir of the crystal structure.
Fig 3.
Interaction of oseltamivir with NA in metastable S0-S9.
The position of the residue that participated in H-bond interactions with oseltamivir is marked with black dots. The red spheres in S8 and S9 represent the oxygen atoms of water molecules (the hydrogen atoms of water molecules are not shown), which participated in the formation of water-mediated H-bonds. The 150 loop, 370 loop and 430 loop regions are highlighted in pink, ice blue and green, respectively.
Fig 4.
Comparison of the conformations of sialic acid and oseltamivir at the secondary binding site.
The position outlined by the black dotted square in the left panel is the secondary binding site of NA. Emerald green in the right panel represents oseltamivir and NA in the simulation, and magenta represents sialic acid and NA in the crystal structure (PDB ID: 1MWE). Hydrogen bonds between sialic acid and partial residues in the secondary binding site are shown as dashed red lines.
Fig 5.
Transition modes for the metastable states in the main binding pathways.
The circles refer to oseltamivir in the metastable states, and the arrows indicate transitions between the states. S1 and S9 are the start and end states of the binding process, respectively.
Fig 6.
Free-energy landscape and the binding pathways of oseltamivir and NA.
(a) Free-energy landscape of oseltamivir along the NA surface. Collective variables x and y represent the projection of the distance vector between the centroids of oseltamivir and NA to the x-axis and y-axis of the system, respectively. The occurrence of the region with very low free energy in the upper right corner of the free-energy landscape will be explained in S1 Appendix, Section 2. (b) Top view of the sampling space of NA in the meta-eABF simulation. (c) Fusion image of NA conformation and free-energy landscape superimposed on Fig 6A and 6B. The metastable states are represented by circles and the pathways connecting the metastable states are shown as dashed lines. (d) Free-energy profiles as a function of the position (s) along the pathways. Pathway 1’: S7→S9, pathway 2’: S2→S7→S8→S9, pathway 3’: S6→S3→S9, pathway 4’: S0A→S2→S7→S9, pathway 5’: S0B→S2→S7→S9. S = 0.0 in pathways 1, 2, 3, 4 and 5 represents S7, S2, S6, S0A and S0B, respectively, and S = 1.0 in pathways 1, 2, 3, 4 and 5 represents S9.
Table 1.
The free energy of metastable states.
Fig 7.
Comparison of the oseltamivir-NA interactions in the crystal structure and simulation.
The H-bond is represented as an orange dotted line. (a and b) Oseltamivir-NA interaction in the crystal structure (PDB ID: 2QWK) in which oseltamivir forms a direct H-bond with R118. (c and d) Oseltamivir-NA interactions in the simulation (S9) in which oseltamivir forms a water-mediated H-bond with R118. (e) Per-residue decomposition energies for selected amino acids in the crystal structure and simulation.