Uncovering water effects in protein-ligand recognition: importance in the second hydration shell and binding kinetics

Abstract

Developing a ligand with high affinity for a specific protein target is essential for drug design, and water molecules are well known to play a key role in protein-drug recognition. However, predicting the role of particularly ordered water molecules in drug binding remains challenging. Furthermore, hydration free energy contributed from the water network, including the second shell of water molecules, is far from being well studied. In this research we focused on these aspects to accurately and efficiently evaluate water effects in protein-ligand binding affinity. We developed a new strategy using a free-energy calculation method, VM2. We successfully predicted the stable ordered water molecules in a number of protein systems: PDE 10a, HSP90, tryptophan synthase (TRPS), CDK2 and Factor Xa. In some of these, the second shell of water molecules appeared to be critical in protein-ligand binding. We also applied the strategy to largely improve binding free energy calculation using the MM/PBSA method. When applying MM/PBSA alone for two systems, CDK2 and Factor Xa, the computed binding free energy resulted in poor to moderate R² values with experimental data. However, including water free energy correction greatly improved the free energy calculation. Furthermore, our work helped to explain how xk263 is a 1000 times faster binder to HIVp than ritonavir, a potentially useful tool for investigating binding kinetics. Our studies reveal the importance of fully considering water effects in therapeutic developments in pharmaceutical and biotechnology industries and for fundamental research in protein-ligand recognition.

Figure 1.

(A) Molecular structure of compound 9; (B) molecular structure of compound 18; (C) interactions in the complex of PDE 10a and Compound 9; (D) interactions in the complex of PDE 10a and Compound 18. In (C) and (D), the protein residues involved in hydrogen bonds are in ball-and-stick, and ligands and water molecules are in licorice.

Figure 1.

(A) Molecular structure of compound 9; (B) molecular structure of compound 18; (C) interactions in the complex of PDE 10a and Compound 9; (D) interactions in the complex of PDE 10a and Compound 18. In (C) and (D), the protein residues involved in hydrogen bonds are in ball-and-stick, and ligands and water molecules are in licorice.

Figure 2.

The hydrogen bonding network among HSP90, PU1 and three water molecules. Molecule representations are the same as in Figure 1.

Figure 3.

Intermediate 2-aminophenol quinonoid in the catalytic site of TRPS β-subunit, together with two water molecules nearby.

Figure 4.

Correlation between the experimental free energies and MM/PBSA results. (A) MM/PBSA alone for CDK2 inhibitors; (B) MM/PBSA with water energy correction for CDK2 inhibitors; (C) MM/PBSA alone for factor Xa inhibitors; (D) MM/PBSA with water energy correction for factor Xa inhibitors.

Figure 4.

Correlation between the experimental free energies and MM/PBSA results. (A) MM/PBSA alone for CDK2 inhibitors; (B) MM/PBSA with water energy correction for CDK2 inhibitors; (C) MM/PBSA alone for factor Xa inhibitors; (D) MM/PBSA with water energy correction for factor Xa inhibitors.

Figure 5.

Free energy needed to remove a specific water molecule during ritonavir dissociation. (A) A bridging water molecule was found between ARG8 of HIVP and ritonavir; (B) computed water removal free energy for this transient bridging water molecule with > 100-ps dwell time. An average removal free energy is ~5 kcal/mol.

Scheme 1:

Illustration of water removal, where red, green, white with two blue lines, white and blue background represent the protein, the ligand, a water molecule, a cavity and the continuum aqueous solution, respectively. A bound water molecule is removed from a protein-ligand complex (1) into bulk solution (3, 4), leaving just an empty cavity with the same shape as that of the water (2).

Scheme 2:

The free energy of a system of a protein and two water molecules, W1 and W2, can be decomposed into several energy components as shown in the matrix below. As an example, the shaded area has the components from the water molecule itself G_W2, along with the water–protein interaction G_Protein–W2, and the water–water interaction G_W1–W2, and all together, they contribute to the water removal energy for W2.