Thermodynamics of Water in an Enzyme Active Site: Grid-Based Hydration Analysis of Coagulation Factor Xa

Abstract

Water molecules in the active site of an enzyme occupy a complex, heterogeneous environment, and the thermodynamic properties of active-site water are functions of position. As a consequence, it is thought that an enzyme inhibitor can gain affinity by extending into a region occupied by unfavorable water or lose affinity by displacing water from a region where it was relatively stable. Recent advances in the characterization of binding-site water, based on the analysis of molecular simulations with explicit water molecules, have focused largely on simplified representations of water as occupying well-defined hydration sites. Our grid-based treatment of hydration, GIST, offers a more complete picture of the complex distributions of water properties, but it has not yet been applied to proteins. This first application of GIST to protein-ligand modeling, for the case of Coagulation Factor Xa, shows that ligand scoring functions based on GIST perform at least as well as scoring functions based on a hydration-site approach (HSA), when applied to exactly the same simulation data. Interestingly, the displacement of energetically unfavorable water emerges as the dominant factor in the fitted scoring functions, for both GIST and HSA methods, while water entropy plays a secondary role, at least in the present context.

Figure 1

Comparison of the GIST and HSA representations of water density in the Factor Xa binding pocket. HSA sites (blue spheres) are the same in all three panels. From left to right, the GIST contour levels are at g = 6, 4, and 2. The GIST water densities are based on the occupancy of grid voxels by water oxygens, and the boundaries of the grid box may be discerned in the right-hand panel. A smoothed protein surface is shown in order to highlight the water data.

Figure 2

GIST contours in binding site of FXa, in molecular surface representation. Left: Energy scoring region, which meets both density and energy cutoff criteria for the combined energy–entropy scoring function. Middle: Entropy scoring region, which meets both density and entropy cutoff criteria for the combined energy–entropy scoring function. Right: Normalized total water energy, contoured at −2.6 kcal/mol/water. White arrows: entropy scoring subregions at the polar surface. Magenta arrows: entropy scoring subregions at hydrophobic surface. Red: oxygen. Blue: nitrogen. Pale blue: carbon. Graphic generated with VMD.

Figure 3

Convergence of R² values for GIST scoring functions, as a function of simulation duration. Top row: combined energy/entropy scoring function. Bottom row: energy-only scoring function. Left: 100 ns simulation, frames saved at 1 ps intervals. Middle: The first 20 ns of the same 100 ns simulation, frames saved every 0.05 ps. Right: 20 ns simulation initiated from the last frame of the 100 ns simulation, frames saved every 0.05 ps.

Figure 4

Comparison of GIST and HSA scoring locations, in the context of the FXa binding site (gray). Left: Scoring sites from the capped HSA energy-only scoring function (blue spheres) and energy scoring regions from the GIST energy-only scoring function (orange contours). GIST results are present only within the limits of the GIST grid (orange lines). Right: a representative ligand, PDB HET ID 4QC, in van der Waals representation (pink).