Chimeric polypeptides comprising G protein-coupled receptors and VHH antibodies
Described are polypeptides and their use for screening and drug discovery. More specifically, the disclosure provides chimeric polypeptides comprising a membrane protein, in particular a GPCR, fused to a binding domain, wherein the binding domain is directed against and/or specifically binds to the membrane protein. In particular, the chimeric polypeptides are single proteins wherein, in an intramolecular reaction, the binding domain stabilizes the membrane protein in a conformation of interest. Also provided are nucleic acid sequences encoding such chimeric polypeptides, cells capable of expressing such chimeric polypeptides as well as cellular compositions derived thereof. Also screening methods for compounds using the chimeric polypeptides.
1. A chimeric polypeptide comprising:
a G protein-coupled receptor (GPCR) fused to a conformation-selective binding domain,
wherein the binding domain is a VHH antibody directed against and/or able to specifically bind to the GPCR;
wherein the GPCR is stabilized in an active or inactive conformation upon binding of the binding domain to the GPCR; and
wherein the stabilized GPCR has increased affinity for a conformation-selective ligand as compared to GPCR when not fused to the binding domain.
2. The chimeric polypeptide of claim 1 , wherein the binding domain is fused to the GPCR either directly or through a linker.
3. The chimeric polypeptide of claim 1 , wherein the GPCR is stabilized in an active conformation upon binding of the binding domain.
4. The chimeric polypeptide of claim 3 , wherein the active conformation is an agonist conformation, a partial agonist conformation, or a biased agonist conformation.
5. The chimeric polypeptide of claim 1 , wherein the GPCR is stabilized in an inactive conformation upon binding of the binding domain.
6. The chimeric polypeptide of claim 5 , wherein the inactive conformation is an inverse agonist conformation.
7. The chimeric polypeptide of claim 1 , wherein the binding domain binds to an intracellular epitope of the GPCR.
8. The chimeric polypeptide of claim 7 , wherein said intracellular epitope is comprised in a binding site for a downstream signaling protein.
9. The chimeric polypeptide of claim 7 , wherein said intracellular epitope is comprised in the G protein binding site.
10. The chimeric polypeptide of claim 1 , wherein the GPCR is a naturally occurring GPCR.
11. The chimeric polypeptide of claim 1 , wherein the GPCR is a variant of a naturally occurring GPCR or a truncated form of a naturally occurring GPCR.
12. The chimeric polypeptide of claim 1 , wherein the GPCR is selected from the group consisting of a GPCR of the Glutamate family of GPCRs, a GPCR of the Rhodopsin family of GPCRs, a GPCR of the Adhesion family of GPCRs, a GPCR of the Frizzled/Taste2 family of GPCRs, and a GPCR of the Secretin family of GPCRs.
13. A complex comprising:
the chimeric polypeptide of claim 1 , and
a receptor ligand.
14. The chimeric polypeptide of claim 1 , which is crystalline.
15. A nucleic acid molecule comprising:
a polynucleotide encoding the chimeric polypeptide of claim 1 .
16. A host cell comprising the nucleic acid molecule of claim 15 .
17. The host cell according to claim 16 , which is a bacterial cell, a yeast cell, a mammalian cell, or an insect cell.
18. A method to produce a chimeric polypeptide comprising a G protein-coupled receptor (GPCR) fused to a binding domain, the method comprising:
culturing the host cell of claim 16 under suitable conditions.
19. A method to display a G protein-coupled receptor (GPCR) in an active or inactive conformation at the cellular surface or in a particular cellular membrane fraction of a host cell, the method comprising:
culturing the host cell of claim 16 under suitable conditions to express the chimeric polypeptide.
20. A membrane composition comprising the chimeric polypeptide of claim 1 .
21. The method according to claim 18 , further comprising:
isolating the chimeric polypeptide.
22. A kit comprising the chimeric polypeptide of claim 1 .