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. 2015 Feb 3;112(5):E386-91.
doi: 10.1073/pnas.1424461112. Epub 2015 Jan 20.

Kinetics of protein-ligand unbinding: Predicting pathways, rates, and rate-limiting steps

Pratyush Tiwary  1 , Vittorio Limongelli  2 , Matteo Salvalaglio  3 , Michele Parrinello  4
Affiliations

Kinetics of protein-ligand unbinding: Predicting pathways, rates, and rate-limiting steps

Pratyush Tiwary et al. Proc Natl Acad Sci U S A. .

Abstract

The ability to predict the mechanisms and the associated rate constants of protein-ligand unbinding is of great practical importance in drug design. In this work we demonstrate how a recently introduced metadynamics-based approach allows exploration of the unbinding pathways, estimation of the rates, and determination of the rate-limiting steps in the paradigmatic case of the trypsin-benzamidine system. Protein, ligand, and solvent are described with full atomic resolution. Using metadynamics, multiple unbinding trajectories that start with the ligand in the crystallographic binding pose and end with the ligand in the fully solvated state are generated. The unbinding rate k off is computed from the mean residence time of the ligand. Using our previously computed binding affinity we also obtain the binding rate k on. Both rates are in agreement with reported experimental values. We uncover the complex pathways of unbinding trajectories and describe the critical rate-limiting steps with unprecedented detail. Our findings illuminate the role played by the coupling between subtle protein backbone fluctuations and the solvation by water molecules that enter the binding pocket and assist in the breaking of the shielded hydrogen bonds. We expect our approach to be useful in calculating rates for general protein-ligand systems and a valid support for drug design.

Keywords: drug design; enhanced sampling; kinetics; protein–ligand unbinding.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
States relevant for the unbinding of trypsin–benzamidine complex. The specific interactions that stabilize these states are indicated. Also indicated are the water molecules that play defining roles. See text for further details and mean lifetimes.
Fig. 2.
Fig. 2.
Trypsin in its apo state can exist in substates S1 (green) and S2 (red). The key difference between these two states is in the loop L. In S1 the loop is as in the X-ray pose and the protein is available for binding. In S2 the loop has undergone a distortion initiated primarily by glycine–serine residues (S213 and G214) that engage in hydrogen bond interaction with other residues (D216 and Q217). In this state, the protein is temporarily unavailable for binding. See text for further details and mean lifetimes.
Fig. 3.
Fig. 3.
State-to-state transition rates for trypsin–benzamidine unbinding. All rates are in s−1. The respective mean lifetimes for ligand binding states are also shown.
Fig. 4.
Fig. 4.
Typical mechanism of going from state A to P (Top) and A to B (Bottom). For each, typical TSE members as determined by committor analysis are also shown. Relevant residues and water molecules are also indicated. Note that the biological water in state A is removed in the pre-TS state for path 1 (Top) to highlight the role of water molecule coming from solvent. See main text for summary of key interactions, and SI Appendix for more details of the TSE and committor analysis.
See this image and copyright information in PMC

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