Predicting Displaceable Water Sites Using Mixed-Solvent Molecular Dynamics

Abstract

Water molecules are an important factor in protein-ligand binding. Upon binding of a ligand with a protein's surface, waters can either be displaced by the ligand or may be conserved and possibly bridge interactions between the protein and ligand. Depending on the specific interactions made by the ligand, displacing waters can yield a gain in binding affinity. The extent to which binding affinity may increase is difficult to predict, as the favorable displacement of a water molecule is dependent on the site-specific interactions made by the water and the potential ligand. Several methods have been developed to predict the location of water sites on a protein's surface, but the majority of methods are not able to take into account both protein dynamics and the interactions made by specific functional groups. Mixed-solvent molecular dynamics (MixMD) is a cosolvent simulation technique that explicitly accounts for the interaction of both water and small molecule probes with a protein's surface, allowing for their direct competition. This method has previously been shown to identify both active and allosteric sites on a protein's surface. Using a test set of eight systems, we have developed a method using MixMD to identify conserved and displaceable water sites. Conserved sites can be determined by an occupancy-based metric to identify sites which are consistently occupied by water even in the presence of probe molecules. Conversely, displaceable water sites can be found by considering the sites which preferentially bind probe molecules. Furthermore, the inclusion of six probe types allows the MixMD method to predict which functional groups are capable of displacing which water sites. The MixMD method consistently identifies sites which are likely to be nondisplaceable and predicts the favorable displacement of water sites that are known to be displaced upon ligand binding.

Figure 1.

ROC plots are given for scoring active-site waters based on occupancies from the MixMD simulations. The x-axis plots the percentage of conserved waters and the y-axis plots the percentage of free (red line) or displaced (blue line) waters. The points of maximum enrichment are marked with circles, and maximum MCC values are marked with triangles. Occupancies (in σ units) are noted next to the points on the graphs.

Figure 2.

Water occupancies are shown as colored mesh from the MixMD simulations (colored by the probe type from each simulation). At high occupancy levels, few water sites are identified. These are sites which are repeatedly occupied by water molecules even in the presence of probe molecules. Water sites that first appear at lower sigma values are less frequently occupied by water when probe molecules are present. Note that these isocontours are for waters, not probes in the simulations.

Figure 3.

Heat Shock Protein 90. Water density is shown at the 23 σ level, colored according to the probe type included in the simulation. Crystallographic waters (PDB: 1AH6) with 3.5 Å of ligands are shown for reference. Site A is a water site found in 100% of homologous structures, predicted to be conserved by MixMD. Site B is a water displaced by the carbonyl of geldanamycin, predicted to be displaced by N-methylacetamide.

Figure 4.

BRD4. Water density is shown at the 23 σ level, colored according to the probe type included in the simulation. Crystallographic waters from the apo structure (PDB:2OSS) that are within 3.5 Å of a ligand are shown for reference. Site A is predicted by MixMD to be displaced, shown with an example ligand (PDB:3UVW, peptide) displacing the site. Site B is a water site found in 97% of comparable structures, predicted by MixMD to be displaceable is shown with an inhibitor displacing this site (PDB:4O7F, 2RQ).

Figure 5.

Dihydrofolate Reductase. Water density is shown at the 23 σ level, colored according to the probe type included in the simulation. Crystallographic waters from the apo structure (PDB:1DG8) that are within 3.5 Å of a ligand are shown for reference. Site A is a water that is found in 100% of comparable crystal structures, predicted to be conserved by MixMD. In site B, this water is seen in structures PDB:4KL9 and 4KLX. It is known to be displaced by nitrogen, and it is predicted by MixMD to be displaced by N-methylacetamide. Site C is a water site known to be displaced by nitrogen, predicted by MixMD to be displaced by N-methylacetamide, acetate/methylammonium, and isopropyl alcohol. (PDB:1DF7(MTX) and 1DG7(WRB))

Figure 6.

β-lactamase. Water density is shown at the 23 σ level, colored according to the probe type included in the simulation. Crystallographic waters from the apo structure (PDB:1ZG4) that are within 3.5 Å of a ligand are shown for reference. Site A is known to be conserved and is well mapped by waters in our simulations. MixMD also correctly predicts many of the waters in the active site of β-lactamase as being displaced, but there is one known discrepancy. Site B is predicted to be conserved when it is actually displaced by a covalent bond between the ligand and protein. This is attributed to the inability to account for potential covalent modifications within an MD simulation. (PDB:1BT5-IM2, 1ERM-BJI)

Figure 7.

Neuraminidase. Crystallographic waters (PDB:4HZV) within 3.5 Å of any ligand are shown, along with water density from the MixMD simulations shown at the 23 σ level, colored according to the probe type included in the simulation. Site A shows a cluster of conserved water sites found in 100% of homologous structures, predicted by MixMD to be conserved. Site B shows water sites displaced by carboxyl of ligand (Zanamivir shown, PDB:4I00, ZMR) and predicted by MixMD to be displaced. The inset figure shows the occupancy of the acetate probe which displaces these sites.

Figure 8.

β-Secretase. Crystallographic waters from the apo structure of BACE (PDB: 1W50) and the bridging water in the ligand bound structure (PDB:4FM7, WAT909) are shown for reference. A) MixMD predicts the displacement of the circled water site by acetate/methylammonium, N-methylacetamide, and pyrimidine probes. The ligand from PDB:4RCD(3LL) is shown for comparison. Water density is shown at the 23 σ level, colored according to the probe type included in the simulation. The inset figure shows the methylammonium density at 150 σ. B) This site may also be conserved and bridge interactions between the ligand and protein, as predicted by the simulations with acetonitrile and isopropyl alcohol. The ligand from PDB:4FM7(OUP) is shown for comparison.

Figure 9.

Thrombin. Crystallographic waters within the active site (PDB:3U69) are shown, along with the water density from the MixMD simulations at the 23 σ level, colored according to the probe type included in the simulation. Site A is a water site that is predicted to be always displaced, shown with a peptide-inhibitor. (PDB:3U8O).

Figure 10.

Penicillopepsin. Crystallographic waters (PDB:3APP) within the active site are shown, along with the water density from the MixMD simulations at the 23 σ level, colored according to the probe type included in the simulation. Site A is a water displaced by phosphonate-containing ligand (PDB:1BXO, PP7⁷³), and it is predicted as displaceable by the MixMD simulations. Site B is an important water site found in 100% of related structures which participates in a network of stabilizing interactions. It is predicted as being conserved.