Method and composition for crystallizing G protein-coupled receptors

1. A method for analyzing the three dimensional structure of a GPCR on a computer system, comprising:

a) accessing a file containing atomic coordinates of a GPCR using a computer system that comprises a modeling program, wherein said atomic coordinates are produced by subjecting crystals of a GPCR fusion protein to X-ray diffraction analysis, wherein said GPCR fusion protein comprises, from N-terminus to C-terminus:

i. a first portion of a G-protein coupled receptor (GPCR), wherein said first portion comprises TM1, TM2, TM3, TM4 and TM5 regions of said GPCR;

ii. a domain comprising the amino acid sequence of a lysozyme; and

iii. a second portion of said GPCR, wherein said second portion comprises TM6 and TM7 regions of said GPCR;

b) modeling said atomic coordinates on said computer system using said modeling program to produce a model of the three dimensional structure of at least a portion of the GPCR; and

c) displaying the model of said three dimensional structure on the computer system.

2. The method of claim 1 , further comprising identifying a potential modulator of said GPCR.

3. The method of claim 1 , wherein said analyzing comprises identifying a binding site for a modulator of said GPCR.

4. The method of claim 1 , wherein said analyzing comprises identifying a binding site for a ligand of said GPCR.

5. The method of claim 1 , wherein said crystals are made using a bicelle crystallization method or a lipidic cubic phase crystallization method.

6. The method of claim 1 , wherein said domain comprises an amino acid sequence having at least 80% identity to the amino acid sequence of a wild-type lysozyme.

7. The method of claim 1 , wherein said domain comprises an amino acid sequence having at least 95 % identity to the amino acid sequence of a wild-type lysozyme.

8. The method of claim 1 , wherein said domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of T 4 lysozyme.

9. The method of claim 1 , wherein said first and second portions of said GPCR comprise the amino acid sequence of a naturally occurring GPCR.

10. The method of claim 1 , wherein said first and second portions of said GPCR comprise the amino acid sequence of a non-naturally occurring GPCR.

11. The method of claim 1 , wherein the amino acid sequences of said first and second portions of said GPCR are least 80 % identical to amino acid sequences of a naturally occurring GPCR.

12. The method of claim 1 , wherein the GPCR is selected from the group consisting of: a receptor for a biogenic amine, a dopamine receptor, a seratonin receptor, an adrenergic receptor, a β2-adrenergic receptor and chemokine receptor.

13. The method of claim 1 , wherein said crystals comprise a ligand for said GPCR, and the method further comprises identifying the binding site for said ligand in said GPCR using said model.

14. The method of claim 13 , wherein the analyzing comprises identifying amino acids that form polar contacts with said ligand in said binding site, using said model.

15. The method of claim 13 , further comprising determining whether a test compound docks with said binding site using said model.

16. The method of claim 15 , further comprising analyzing the packing of the test compound and amino acids in said binding site, using said model.

17. A method for selecting a modulator of a GPCR, comprising:

a) accessing a file containing atomic coordinates of a GPCR using a computer system that comprises a modeling program, wherein said atomic coordinates are produced by subjecting crystals of a GPCR fusion protein to X-ray diffraction analysis, wherein said GPCR fusion protein comprises, from N-terminus to C-terminus:

i. a first portion of a G-protein coupled receptor (GPCR), wherein said first portion comprises TM 1 , TM 2 , TM 3 , TM 4 and TM 5 regions of said GPCR;

ii. a domain comprising the amino acid sequence of a lysozyme; and iii. a second portion of said GPCR, wherein said second portion comprises TM 6 and TM 7 regions of said GPCR;

b) modeling said atomic coordinates on said computer system using said modeling program to produce a model of the three dimensional structure of at least the ligand binding site of the GPCR;

c) determining, using said computer system, whether a test compound docks with said binding site, wherein a test compound that docks with said binding site is a GPCR modulator; and

d) displaying on the computer system a model of said ligand binding site and said GPCR modulator, docked with said ligand binding site.

18. The method of claim 17 , wherein said domain comprises an amino acid sequence having at least 80% identity to the amino acid sequence of a wild-type lysozyme.

19. The method of claim 17 , wherein said domain comprises an amino acid sequence having at least 95% identity to the amino acid sequence of a wild-type lysozyme.

20. The method of claim 17 , wherein said domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of T 4 lysozyme.

21. The method of claim 17 , wherein said first and second portions of said GPCR comprise the amino acid sequence of a naturally occuring GPCR.

22. The method of claim 17 , wherein said first and second portions of said GPCR comprise the amino acid sequence of a non-naturally occurring GPCR.

23. The method of claim 17 , wherein the amino acid sequences of said first and second portions of said GPCR are at least 80% identical to the amino acid sequence of a naturally occurring GPCR.

24. The method of claim 17 , wherein said method further comprises making said modulator.

25. A method for analyzing the three dimensional structure of a GPCR on a computer system, comprising:

a) accessing a file containing atomic coordinates of a GPCR using a computer system that comprises a modeling program, wherein said atomic coordinates are produced by subjecting crystals of a polypeptide to X-ray diffraction analysis, wherein said polypeptide comprises, from N-terminus to C-terminus:

i. a first portion of a G-protein coupled receptor (GPCR), wherein said first portion comprises the amino acid sequence that is N-terminal to the IC 3 loop of said GPCR;

ii. a domain comprising the amino acid sequence of a lysozyme;

iii. a second portion of said GPCR, wherein said second portion comprises the amino acid sequence that is C-terminal to the IC 3 loop of said GPCR; and

b) modeling said atomic coordinates on said computer system using said modeling program to produce a model of the three dimensional structure of at least a portion of the GPCR; and

c) displaying the model of said three dimensional structure on the computer system.

26. A method for analyzing the three dimensional structure of a GPCR on a computer system, comprising:

a) accessing a file containing atomic coordinates of a GPCR using a computer system that comprises a modeling program, wherein said atomic coordinates are produced by subjecting crystals of a GPCR to X-ray diffraction analysis, wherein said GPCR comprises an IC 3 loop containing a substitution that comprises the amino acid sequence of a lysozyme; and

b) modeling said atomic coordinates on said computer system using said modeling program to produce a model of the three dimensional structure of at least a portion of the GPCR; and

c) displaying the model of said three dimensional structure on the computer.