A novel coarse-grained molecular dynamics method for the accurate prediction of helix-helix interactions in GPCRs

This thesis describes a novel computational method developed to identify and characterise points of protein-protein interaction between two G protein-coupled receptors (GPCRs). An ensemble-based coarse-grained molecular dynamics (eCG-MD) approach was applied to GPCR oligomers with experimentally-determined contact interfaces (adenosine A2A receptor, rhodopsin, CXCR4 and β1AR). Error analysis was used to determine 1) the number of replicas in an ensemble and 2) the simulation time for each replica that were needed to obtain convergence with experimental results. Error analysis also enabled identification of non-interacting regions. This novel method yielded calculations of distance between rhodopsin, CXCR4 and β1AR transmembrane domains reported to form contact points in homodimers that correlated well with the corresponding measurements obtained from the structural data, demonstrating an ability to predict contact interfaces computationally. The method gave distance measurements between residues shown to be involved in oligomerisation of the fifth transmembrane domain from the adenosine A2A receptor that were in very good agreement with the existing biophysical data. Further, the method provided information about the nature of the contact interface that could not be determined experimentally. This CG-MD method was then used as a high-throughput screen to identify novel sites of interaction in the adenosine A2A receptor, informing the design of future experimental work. Experimental methods to investigate interactions are also described in this thesis. These were less successful in identifying contact points, however, the present computational method will enable novel interaction points between GPCRs to be predicted and tested experimentally using assays of ligand binding and receptor signaling. In conclusion, this work provides an accurate, reproducible and reliable method for determining the specific points of interaction between GPCR dimers. The eCG-MD method discriminates between residues in TM helices that form specific interactions and residues that are in close proximity but do not interact