HomolWat: a web server tool to incorporate 'homologous' water molecules into GPCR structures

Abstract

Internal water molecules play an essential role in the structure and function of membrane proteins including G protein-coupled receptors (GPCRs). However, technical limitations severely influence the number and certainty of observed water molecules in 3D structures. This may compromise the accuracy of further structural studies such as docking calculations or molecular dynamics simulations. Here we present HomolWat, a web application for incorporating water molecules into GPCR structures by using template-based modelling of homologous water molecules obtained from high-resolution structures. While there are various tools available to predict the positions of internal waters using energy-based methods, the approach of borrowing lacking water molecules from homologous GPCR structures makes HomolWat unique. The tool can incorporate water molecules into a protein structure in about a minute with around 85% of water recovery. The web server is freely available at http://lmc.uab.es/homolwat.

Figure 1.

Schematic representation of the HomolWat reference database (A) and overall pipeline (B). In (B), the user provides an input 3D structure file of a GPCR—that could contain ligands or water molecules—in PDB format and obtains an output PDB file of the same structure with additional homologous internal water molecules inserted. As a first step, HomolWat extracts the protein sequence from the PDB file and runs Blast+ on all receptor sequences in HomolWatDB. This provides a list of receptors sorted by Blast score from high to low sequence homology. Subsequently, HomolWat uses this list to assess if a water molecule fits in the receptor, considering previously inserted water molecules. This assessment is performed for every water molecule across all receptors within chosen Blast+ score, and across every structure for each receptor.

Figure 2.

HomolWat test case: the crystal structure of the inactive serotonin 5-HT_2A receptor (PDB id 6A94) without internal water molecules. (A) Screenshot of the results for the Blast+ alignment to receptors in HomolWatDB. (B) Screenshot of the output PDB structure with the introduced internal water molecules displayed in NGL viewer. (C-E) Details of the clusters of water molecules associated to functionally important residues at different levels ranging from the orthosteric site to the G-protein binding crevice upon incorporation to its receptor core using HomolWat. The receptor is shown in light blue cartoons and water molecules are displayed as red spheres.

Figure 3.

Validation of HomolWat and comparison to Dowser+. Number of water molecules recovered using Homolwat (red squares) and Dowser+ (blue dots) upon removal of the originally resolved water molecules (black star) in each PDB structure (mapped to their Uniprot accession code).