Chemokines are small 70-120 amino acid proteins that serve as traffic signals for cell migration in development, routine immune surveillance, and inflammation. They function by binding to seven transmembrane G Protein-Coupled Receptors (GPCRs) on leukocytes, where they induce conformational changes that trigger intracellular signaling pathways involved in adhesion, cell movement and ultimately, defense-related immune responses. Although the chemokine system evolved for protection of the host, inappropriate regulation of chemokine-mediated processes contributes to the pathology of many diseases, including inflammatory diseases (e.g. asthma, rheumatoid arthritis, multiple sclerosis) and HIV. As such, the receptors have become important drug targets in the pharmaceutical industry where structural information is highly desired to aid the drug discovery process. As allosteric membrane proteins, chemokine receptors like other GPCRs are intrinsically unstable and dynamic by nature, which has made them difficult structural targets. Nevertheless, since 2007 there has been a virtual explosion in the field of GPCR structural biology with the determination of many new structures including one chemokine receptor, CXCR4, in complex with a small molecule and cyclic peptide antagonist. Despite these breakthroughs, GPCR structural biology remains non- trivial, particularly with respect to crystallization of complexes with other proteins and in relevant functional states. For example, determining structures of chemokine receptors in complex with their natural protein ligands has proven more difficult than small molecule complexes. The challenges include (i) the sensitivity of the complexes to crystallization conditions because of the importance of polar interactions in complex formation, and (ii) the fact that although chemokines bind receptors as monomers, many chemokines oligomerize. Thus at the concentrations used for crystallization, chemokine oligomers may be present along with chemokine:receptor complexes unless the gap between the stability of the chemokine oligomers and the chemokine:receptor complexes can be widened by stabilization of the receptor complexes. To this end, the use of Fab (fragment antigen binding) and other scaffolds has emerged as an important approach for crystallization of difficult targets. Thus the goal of this proposal is to use phage display to generate Fabs that stabilize discrete chemokine receptor complexes with their natural ligands while also acting as crystallization handles. The proposed approach capitalizes on our ability to express chemokine receptors, incorporate them into stable membrane mimetics called nanodiscs, immobilize the nanodisc particles through site specific biotinylation, and to make fluorescent chemokines for use in a robust screen to generate Fabs against any chemokine:receptor complex of interest. Ultimately, these methods can also be used to generate Fabs to stabilize receptors with other ligands and downstream signaling molecules, and to generate valuable reagents for cell biology and pharmacology to probe chemokine:receptor signaling and function in normal physiology and disease.
Chemokines and their receptors are involved in a broad spectrum of diseases. To aid drug discovery efforts, the goal of this proposal is to engineer antibody (Fab) fragments that will stabilize chemokine:receptor complexes for crystallographic studies. However, the same methods will also be applicable for the generation of Fab fragments for a wide variety of applications--for example interrogation of signal transduction pathways and applications involving conformation-dependent Fabs.
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