Viral GPCR recognition of chemokines and engineered ligands

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Abstract: Despite a great deal of progress in understanding the structure-function properties of G protein-coupled receptors (GPCR) that serve as receptors for small molecules, there remains a dearth of knowledge about GPCRs that engage protein ligands. There are several classes of GPCR that respond to protein ligands and it is unclear how the mechanisms of recognition and receptor activation relate to those of small molecule ligands. This is an important issue to clarify given that drug discovery efforts are focused almost exclusively on small molecule GPCR, yet many therapeutic opportunities exist for proteins that signal through GPCR. The largest class of proteins that use GPCR as signaling receptors are chemokines, which bind to and signal through a large family of chemokine GPCR. Chemokines are potent immune-modulators, play important roles in inflammation, during infection, and their activities have been implicated in many human diseases. Some viruses have hijacked and `repurposed' chemokine GPCR for immune evasion and to enhance virulence. Viral GPCR can mediate oncogenic transformation and contribute to an array of clinical problems. The P.I.'s laboratory recently made an advance in our understanding of chemokine GPCR structure, by determining the crystal structure of a virally encoded (Human cytomegalovirus - HCMV) GPCR, US28, bound to the human chemokine CX3CL1 (Fractalkine). US28 possesses many interesting functional properties that make it an excellent system to probe mechanisms and structure-function properties of chemokine GPCRs as a whole. For example, US28 is constitutively active, which is important for HCMV pathogenesis, and is the only chemokine GPCR to engage both CXC and CC classes of chemokines. US28 also serves as an entry co-receptor for human immunodeficiency virus type 1 (HIV-1) gp120, which typically uses the human chemokine GPCR CXCR4 and CCR5 as entry receptors. US28 has particularly favorable biochemical properties compared to other chemokine receptors that make it an outstanding system for structure- function analysis. We have robust biochemical and structural access to US28, having developed a high level expression protocol, isolated Alpaca nanobodies, and have the ability to crystallize different complexes of US28 with chemokines and gp120. Here we propose to merge structural efforts on US28- chemokine, US28-gp120, and US28 G-protein complexes, with combinatorial biology to engineer novel chemokine and protein-based surrogate ligands for US28. These studies will collectively probe the structural basis US28-chemokine recognition and signaling properties, elucidate how gp120 HIV engages chemokine GPCR as co-receptors, and explore new therapeutic opportunities for engineering chemokine GPCR ligands with biased signaling properties.