Binding domains directed against GPCR:G protein complexes and uses derived thereof
The present disclosure relates to the field of G protein coupled receptor (GPCR) structural biology and signaling. In particular, the present disclosure relates to binding domains directed against and/or specifically binding to GPCR:G protein complexes. Also provided are nucleic acid sequences encoding such binding domains and cells expressing or capable of expressing such binding domains. The binding domains of the present disclosure can be used as universal tools for the structural and functional characterization of G-protein coupled receptors in complex with downstream heterotrimeric G proteins and bound to various natural or synthetic ligands, for investigating the dynamic features of G protein activation, as well as for screening and drug discovery efforts that make use of GPCR:G protein complexes.
1. A method for identifying a test compound that binds to a GPCR/G-protein complex, the method comprising:
determining that a protein binding domain binds to the G-protein in the GPCR/G-protein complex and stabilizes the GPCR/G-protein complex, wherein the protein binding domain is a nanobody;
forming a complex of the GPCR, the G-protein, and the protein binding domain;
contacting the complex with the test compound; and
determining binding between the test compound and the complex.
2. The method according to claim 1 , further comprising:
determining that the test compound binds to the GPCR in the complex.
3. The method according to claim 1 , further comprising:
determining that the test compound binds to the G-protein in the complex.
4. The method according to claim 1 , wherein the test compound is selected from the group consisting of a polypeptide, a peptide, a small molecule, a natural product, a peptidomimetic, a nucleic acid, a lipid, a lipopeptide, a carbohydrate, an antibody or an antigen-binding fragment thereof, a heavy chain antibody (hcAb), a single domain antibody (sdAb), a minibody, a variable domain derived from a camelid heavy chain antibody, a variable domain of an immunoglobulin new antigen receptor (V NAR ), and a protein scaffold.
5. The method according to claim 4 , wherein the antigen-binding fragment thereof is selected from the group consisting of a Fab, a Fab′, a F(ab′)2, an Fd, a single-chain Fvs (scFv), a single-chain antibody, a disulfide-linked Fv (dsFv), a fragment comprising a VL domain and a fragment comprising a VH domain.
6. The method according to claim 4 , wherein the protein scaffold is selected from the group consisting of an alphabody, a protein A, a protein G, a designed ankyrin-repeat domain (DARPin), a fibronectin-type III repeat, an anticalin, a knottin, and an engineered CH2 domain.
7. The method according to claim 1 , wherein the protein binding domain binds to an intracellular domain of the GPCR/G-protein complex.
8. The method according to claim 1 , wherein the protein binding domain binds to an intracellular domain of the G-protein.
9. The method according to claim 8 , wherein the protein binding domain binds to a conformational epitope at the interface between the alpha and beta subunits of the G-protein.
10. The method according to claim 1 , wherein the protein binding domain (a) is derived from an immunoglobulin, (b) comprises an amino acid sequence comprising 4 framework regions and 3 complementary determining regions or any suitable fragment thereof, (c) is derived from a camelid antibody, and/or (d) comprises a nanobody sequence or any suitable fragment thereof.
11. The method according to claim 1 , wherein the test compound has higher affinity that for the GPRC/G-protein/protein binding domain complex than the same complex in the absence of the protein binding domain.