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Directed evolution of nanobody chaperones that stabilize GPCR:G‐protein complexes
Author(s) -
Manglik Aashish,
Kruse Andrew C.,
Koehl Antoine,
Kobilka Brian K.,
Ring Aaron M.
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.278.1
Subject(s) - heterotrimeric g protein , g protein coupled receptor , g protein , chemistry , biochemistry , microbiology and biotechnology , receptor , biology , biophysics
G protein‐coupled receptors (GPCRs) sense external signals through a stereotyped mechanism that couples ligand‐induced conformational changes to nucleotide exchange in associated heterotrimeric G proteins. The conformational states in both GPCRs and G proteins that mediate this process are transient and thus challenging to study. Single‐domain camelid antibody fragments (“nanobodies”) have proven invaluable tools to capture GPCRs in particular conformations for crystallography, spectroscopy, and in vivo studies. Nb35 is a conformationally‐selective nanobody that binds to Gs to stabilize the nucleotide‐free state and its complex with activated GPCRs, including the Beta‐2 adrenergic receptor. Similar chaperones for other heterotrimeric G proteins such as Gi and Gq have not yet been described. To obtain these valuable probes, we used structure‐based directed evolution of Nb35 with yeast surface display to alter its specificity for Gi and Gq. After selection, we obtained two variants, NbD7 and NbD12 that respectively bind Gi and Gq in a fashion analogous to Nb35 for Gs. Biochemically, NbD7 and NbD12 protect GPCR:G‐protein complexes from GTP‐induced disassociation. Furthermore, NbD7 enables crystallization of a complex of heterotrimeric Gi and a GPCR. Together with Nb35, NbD7 and NbD12 allow for conformational control of the vast majority of GPCR:G‐protein complexes and provide a facile template to prepare additional nanobody chaperones against other G protein families.