Technical Highlight - October 2012
Short description: A method to identify fusion partners for GPCRs can lead to new structures.
Despite their importance and ubiquity in key cellular processes, relatively few crystal structures of G protein-coupled receptors (GPCRs) are available. Like other transmembrane receptors, the structural characterization of GPCRs has been slowed by difficulties in expressing, purifying and crystallizing these proteins. As a result, researchers have had to employ alternative methods to make these proteins more amenable to structural analyses. One such workaround consists of replacing the unstructured third intracellular loop (ICL3) with a soluble protein or domain—most commonly the T4 lysozyme (T4L).
While insertion of T4L into ICL3 has proven successful in crystallizing and solving the structures of several GPCRs, many others have remained intractable to this technique. To expand the number of tools available for improving the crystallization of GPCRs, Hanson, Stevens and colleagues (PSI GPCR Network) have identified and characterized new GPCR fusion proteins to provide alternatives to T4L chimeras.
To find suitable fusion partners, the authors screened the PDB for proteins and domains based on size, charge and other criteria. Eventually, five proteins were chosen and inserted into the ICL3 of the GPCRs A2AAR and β2AR. After optimizing the region flanking the insertion site and analyzing the thermostability of the fusion proteins, one A2AAR and one β2AR fusion construct were crystallized, each partnered with thermostabilized cytochrome b562 (A2AAR-BRIL and β2AR-BRIL), The crystal structure of A2AAR-BRIL (PDB 4EIY) showed a higher similarity to a thermostabilized version of A2AAR (PDB 3PWH), in which ICL3 is intact, than did A2AAR-T4L (PDB 3EML), in which the insertion of T4L caused distortions in the helices of A2AAR. These types of structural analyses and comparisons may lead to the rational design of GPCR-fusion domain junctions to improve protein stability and solubility.
This work not only expands the repertoire of GPCRs and possibly other proteins that are amenable to structural analyses, but also has the potential to provide a general framework in screening for improved fusion domains for a variety of purposes.
E. Chun et al. Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors.
Structure. 20, 967-976 (2012). doi:10.1016/j.str.2012.04.010