GPCRmd - A web platform for collection, visualization and analysis of molecular dynamics data for G protein-coupled receptors : Bridging the gap between dynamics and receptor functionality

In this thesis, we present GPCRmd, an online repository with a submission system and visualization platform specifically designed for storing and providing easy access to molecular dynamics (MD) data of G protein-coupled receptor (GPCR). This database stores MD trajectories and necessary metadata (e.g. force-field, simulation software, integration time-step) for posterior analysis, ensuring data reproducibility and integrity. Importantly, we demonstrate the usefulness of implemented analysis tools in two case studies related to GPCR signaling bias and membrane-induced GPCR modulation. These tools enabled us to detect important structural rearrangement in the initial phase of β-arrestin signaling in the δ-opioid receptor. In addition, we captured relevant molecular mechanisms which are responsible for cholesterol-induced modulation of the 5-HT2A receptor.Aquí presentem el GPCRmd, un repositori en línia amb un sistema de dipòsit i una plataforma de visualització dissenyats específicament per oferir l’emmagatzematge i un fàcil accés a dades dinàmica molecular (MD) de receptors acoblats a proteïnes G (GPCRs). Aquesta base de dades emmagatzema trajectòries de MD i les metadades (per exemple, el programari de simulació, el camp de força o el temps d'integració) necessàries per a una anàlisi posterior, garantint la reproductibilitat i la integritat de les dades. És important destacar la utilitat de les eines d'anàlisi implementades en dos estudis de cas relacionats amb el biaix de senyalització de GPCRs i la modulació de GPCRs induïda per membrana. Aquestes eines ens han permès detectar una important reordenació estructural en la fase inicial de senyalització per β-arrestina en el receptor δ-opioide. A més, hem capturat el mecanismes moleculars rellevants responsables de la modulació induïda per colesterol del receptor 5-HT2A

Performance of virtual screening against GPCR homology models: Impact of template selection and treatment of binding site plasticity

Rational drug design for G protein-coupled receptors (GPCRs) is limited by the small number of available atomic resolution structures. We assessed the use of homology modeling to predict the structures of two therapeutically relevant GPCRs and strategies to improve the performance of virtual screening against modeled binding sites. Homology models of the D2 dopamine (D2R) and serotonin 5-HT2A receptors (5-HT2AR) were generated based on crystal structures of 16 different GPCRs. Comparison of the homology models to D2R and 5-HT2AR crystal structures showed that accurate predictions could be obtained, but not necessarily using the most closely related template. Assessment of virtual screening performance was based on molecular docking of ligands and decoys. The results demonstrated that several templates and multiple models based on each of these must be evaluated to identify the optimal binding site structure. Models based on aminergic GPCRs showed substantial ligand enrichment and there was a trend toward improved virtual screening performance with increasing binding site accuracy. The best models even yielded ligand enrichment comparable to or better than that of the D2R and 5-HT2AR crystal structures. Methods to consider binding site plasticity were explored to further improve predictions. Molecular docking to ensembles of structures did not outperform the best individual binding site models, but could increase the diversity of hits from virtual screens and be advantageous for GPCR targets with few known ligands. Molecular dynamics refinement resulted in moderate improvements of structural accuracy and the virtual screening performance of snapshots was either comparable to or worse than that of the raw homology models. These results provide guidelines for successful application of structure-based ligand discovery using GPCR homology models

Electron paramagnetic resonance gives evidence for the presence of type 1 gonadotropin-releasing hormone receptor (GnRH-R) in subdomains of lipid rafts

This study investigated the effect of type 1 gonadotropin releasing hormone receptor (GnRH-R) localization within lipid rafts on the properties of plasma membrane (PM) nanodomain structure. Confocal microscopy revealed colocalization of PM-localized GnRH-R with GM1-enriched raft-like PM subdomains. Electron paramagnetic resonance spectroscopy (EPR) of a membrane-partitioned spin probe was then used to study PM fluidity of immortalized pituitary gonadotrope cell line αT3-1 and HEK-293 cells stably expressing GnRH-R and compared it with their corresponding controls (αT4 and HEK-293 cells). Computer-assisted interpretation of EPR spectra revealed three modes of spin probe movement reflecting the properties of three types of PM nanodomains. Domains with an intermediate order parameter (domain 2) were the most affected by the presence of the GnRH-Rs, which increased PM ordering (order parameter (S)) and rotational mobility of PM lipids (decreased rotational correlation time (τc)). Depletion of cholesterol by methyl-β-cyclodextrin (methyl-β-CD) inhibited agonist-induced GnRH-R internalization and intracellular Ca2+ activity and resulted in an overall reduction in PM order; an observation further supported by molecular dynamics (MD) simulations of model membrane systems. This study provides evidence that GnRH-R PM localization may be related to a subdomain of lipid rafts that has lower PM ordering, suggesting lateral heterogeneity within lipid raft domains