Intriguing role of water in protein-ligand binding studied by neutron crystallography on trypsin complexes

Johannes Schiebel,Gerhard Klebe,Andreas Ostermann,Roberto Gaspari,Khang Ngo,Tobias Wulsdorf,Andreas Heine,Tobias E Schrader,Andrea Cavalli,Christian Sohn

doi:10.1038/s41467-018-05769-2

Abstract

Hydrogen bonds are key interactions determining protein-ligand binding affinity and therefore fundamental to any biological process. Unfortunately, explicit structural information about hydrogen positions and thus H-bonds in protein-ligand complexes is extremely rare and similarly the important role of water during binding remains poorly understood. Here, we report on neutron structures of trypsin determined at very high resolutions ≤1.5 Å in uncomplexed and inhibited state complemented by X-ray and thermodynamic data and computer simulations. Our structures show the precise geometry of H-bonds between protein and the inhibitors N-amidinopiperidine and benzamidine along with the dynamics of the residual solvation pattern. Prior to binding, the ligand-free binding pocket is occupied by water molecules characterized by a paucity of H-bonds and high mobility resulting in an imperfect hydration of the critical residue Asp189. This phenomenon likely constitutes a key factor fueling ligand binding via water displacement and helps improving our current view on water influencing protein–ligand recognition.

Highlights

Hydrogen bonds are key interactions determining protein-ligand binding affinity and fundamental to any biological process
This modulation depends on the shape and physicochemical properties of the solute. Proteins and their ligands are surrounded by water molecules that need to be shed, entrapped, rearranged, and modulated in their dynamic properties when the water-exposed surfaces of the proteins or ligands change for instance upon formation of assemblies such as enzyme-substrate or protein-inhibitor complexes[2]
Individual water molecules located in the deep S1 pocket of the trypsin-like serine proteases are known to drastically alter the ligand’s free enthalpy of binding depending on changes induced upon complex formation[6]

Summary

Introduction

Hydrogen bonds are key interactions determining protein-ligand binding affinity and fundamental to any biological process. The role of water molecules during ligand recognition is believed to be multifactorial and highly complex explaining why binding affinity is often difficult to predict even when structural information is available[2,3] Based on their unique properties, water molecules fulfill multiple functions in protein–ligand complexes, e.g. by mediating hydrogen bonds between interaction partners due to their dual ability to act either as donor or acceptor[3]. The importance of water molecules during ligand binding is generally accepted and some key concepts have been proposed and computationally studied, experimental and in particular structural data deciphering how water molecules act exactly during protein-ligand complex formation is rare[7,8,9,10] In part, this lack of knowledge arises from the fact that macromolecular X-ray crystallography can hardly detect hydrogen atoms reliably unless ultra-high resolution data had been collected, which may allow detection of the structurally most defined H-atoms. The experimental studies were complemented by an in-depth computational characterization of both ligand-binding events using metadynamics and QM-based approaches, which provide a detailed structural and dynamic view of protein-ligand complex formation

Methods

Results

Conclusion