Binding domains directed against GPCR:G protein complexes and uses derived thereof
The present invention relates to the field of G protein coupled receptor (GPCR) structural biology and signaling. In particular, the present invention 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 invention 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 nanobody comprising:
four framework regions (FR1 to FR4) and three complementary determining regions (CDR1 to CDR3), according to Formula (I)
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (I)
wherein the nanobody specifically binds to a complex comprising a GPCR and a G protein;
wherein the nanobody specifically binds to the G protein and not to the GPCR; and
wherein
CDR1 is SEQ ID NO: 13, CDR2 is SEQ ID NO: 25, and CDR3 is SEQ ID NO: 37; or
CDR1 is SEQ ID NO: 14, CDR2 is SEQ ID NO: 26, and CDR3 is SEQ ID NO: 38; or
CDR1 is SEQ ID NO: 15, CDR2 is SEQ ID NO: 27, and CDR3 is SEQ ID NO: 39; or
CDR1 is SEQ ID NO: 16, CDR2 is SEQ ID NO: 28, and CDR3 is SEQ ID NO: 40; or
CDR1 is SEQ ID NO: 17, CDR2 is SEQ ID NO: 29, and CDR3 is SEQ ID NO: 41; or
CDR1 is SEQ ID NO: 18, CDR2 is SEQ ID NO: 30, and CDR3 is SEQ ID NO: 42.
2. The nanobody of claim 1 , wherein the nanobody binds with higher affinity to the complex as compared to binding with the heterotrimeric G protein alone.
3. The nanobody of claim 1 , wherein the complex further comprises a receptor ligand.
4. The nanobody of claim 3 , wherein the complex consists of a GPCR, a G protein, and a receptor ligand.
5. The nanobody of claim 3 , wherein the receptor ligand is an agonist.
6. The nanobody of claim 1 , wherein the G protein is in a nucleotide free form.
7. The nanobody of claim 1 , wherein the nanobody specifically binds a conformational epitope at the interface between the alpha and beta subunit of the G protein.
8. The nanobody of claim 1 , wherein the nanobody
a) prevents or inhibits the dissociation of the complex in the presence of nucleotides, or
b) prevents or inhibits the binding of nucleotides to the G protein, or
c) displaces nucleotides from the G protein.
9. The nanobody of claim 6 , wherein the nucleotides are guanine nucleotides or analogs thereof.
10. The nanobody of claim 1 , wherein the GPCR is in an active conformation.
11. The nanobody of claim 1 , wherein the G protein is Gs.
12. The nanobody of claim 1 , wherein the GPCR is a Gs coupled receptor.
13. The nanobody of claim 1 , wherein the GPCR is a human protein.
14. The nanobody of claim 1 , wherein the nanobody comprises SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6.
15. The nanobody of claim 1 , wherein the nanobody is comprised in a polypeptide.
16. The nanobody of claim 1 , wherein the nanobody is immobilized on a solid support.
17. A complex comprising the nanobody of claim 1 .
18. The complex of claim 17 , wherein the complex further comprises a GPCR, and a G protein.
19. The complex of claim 17 , wherein the complex is crystalline.