Novel Technology to Study Chemokine Receptor Signaling Complexes

Abstract

C-C chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) involved in immune responses and it is the primary co-receptor required for HIV-1 cellular entry. To obtain functional heterologous over-expression of engineered GPCRs is one of the major hurdles in GPCR research aimed at elucidating structure-activity relationships. It can be especially difficult to obtain structural information about GPCRs in signaling complexes with other cellular proteins that form a so-called “signalosome.” We have developed two new technologies for investigating the structure and function of GPCRs, which we have now applied to CCR5. First, we have established a membrane nanoparticle system called NABBs (nanoscale apolipoprotein bound bilayers), which are self-assembling discs that maintain receptors in a native-like membrane environment outside of the cell. We have incorporated functional CCR5 into NABBs and plan to use this platform to reconstitute the ternary complex with chemokine ligand and G protein from purified components. In addition, we have adapted unnatural amino acid mutagenesis for use with GPCRs. This is a method to incorporate amino acids with unique side chains at specific sites in the receptor. We introduced p-benzoyl-L-phenylalanine, into CCR5 at various positions on both the extracellular and intracellular surface of the protein using the amber suppression technology. Since the benzoyl moiety is a photo-activatable crosslinker, these mutants can now be used to map the specific sites of interaction between ligand, receptor, and G protein as predicted from molecular modeling and molecular dynamics simulations. Our methods will form the basis of a new experimental paradigm in the structural biology of signaling complexes on a mesoscopic level. Ultimately, these methods will be useful for developing a chemically-precise model for how an extracellular ligand stimulates a GPCR to activate a cytosolic G protein.