GPCR fusion protein containing an N-terminal autonomously folding stable domain, and crystals of the same
View Patent ↗Certain embodiments provide a GPCR fusion protein. In particular embodiments, the GPCR fusion protein comprises: a) a G-protein coupled receptor (GPCR); and b) an autonomously folding stable domain, where the autonomously folding stable domain is N-terminal to the GPCR and is heterologous to the GPCR. The GPCR fusion protein is characterized in that is crystallizable under lipidic cubic phase crystallization conditions. In certain embodiments, the GPCR fusion protein may be crystallizable in a complex with a G-protein or in a complex with an antibody that binds to the IC3 loop of the GPCR.
1. A crystal of a GPCR fusion protein comprising,
a) a G-protein coupled receptor (GPCR); and,
b) an autonomously folding stable domain;
wherein said autonomously folding stable domain is heterologous to said GPCR and fused to said GPCR within 10 amino acids of the TM1 region of the GPCR;
wherein multiple X-ray diffraction patterns of said crystal can be resolved to provide atomic coordinates for at least part of said GPCR; and
wherein (i) the GPCR fusion protein is complexed with one or more of: a ligand for the GPCR, an antibody and a G protein; and/or (ii) the GCR fusion protein comprises one or more stabilizing amino acid substitutions in addition to the autonomously folding stable domain.
2. The crystal of claim 1 , wherein said autonomously folding stable domain comprises the amino acid sequence of lysozyme.
3. The crystal of claim 1 , wherein said GPCR comprises a second autonomously folding stable domain between the TM5 and TM6 regions of said GPCR.
4. The crystal of claim 1 , wherein said GPCR is active.
5. The crystal of claim 1 , wherein said GPCR is naturally occurring.
6. The crystal of claim 1 , wherein said GPCR is non-naturally occurring.
7. The crystal of claim 1 , wherein the crystal comprises a G-protein complexed with said GPCR fusion protein.
8. The crystal of claim 1 , wherein the crystal comprises an antibody that is bound to said GPCR.
9. The crystal of claim 8 , wherein said antibody is a nanobody.
10. The crystal of claim 1 , wherein the GPCR fusion protein is complexed with a ligand for the GPCR.
11. The crystal of claim 10 , wherein said ligand an agonist of said GPCR.
12. The crystal of claim 1 , wherein the GPCR fusion protein comprise a heterologous autonomously folding stable domain between the TM5 and TM6 regions of said GPCR.
13. The crystal of claim 1 , wherein the autonomously folding stable domain is cytochrome 562.
14. The crystal of claim 1 , wherein the autonomously folding stable domain is fused to said GPCR within 5 amino acids of the TM1 region of the GPCR.
15. The crystal of claim 1 , wherein the GPCR is a family A GPCR, a family B GPCR or a family C GCPR.
16. The crystal of claim 1 , wherein the GPCR has an amino acid sequence that is at least 95% identical to a wild type GPCR.
17. The crystal of claim 1 , wherein the GCPR is a biogenic amine receptor, a dopamine receptor, a seratonin receptor, an adrenergic receptor, a β2-adrenergic receptor, a melanocortin receptor, a ghrelin receptor, a metabotropic glutamate receptor or a chemokine receptor.
18. The crystal of claim 1 , wherein the autonomously folding stable domain comprises an amino acid sequence that is at least 90% identical to a wild type protein.
19. The crystal of claim 1 , wherein the GPCR has a truncated C-terminus.
20. The crystal of claim 1 , wherein the crystal comprises a nanobody that is bound to the GPCR and, optionally, a ligand for the GPCR.