Method and composition for crystallizing G protein-coupled receptors
View Patent ↗Certain embodiments provide a method for crystallizing a GPCR. The method may employ a fusion protein comprising: a) a first portion of a G-protein coupled receptor (GPCR), where the first portion comprises the TM1, TM2, TM3, TM4 and TM5 regions of the GPCR; b) a stable, folded protein insertion; and c) a second portion of the GPCR, where the second portion comprises the TM6 and TM7 regions of the GPCR.
1. A fusion protein comprising, from N-terminus to C-terminus:
a) 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;
b) a heterologous domain comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of a soluble, autonomously folding, wild-type protein that aids in the formation of lattice contacts during crystallization of the fusion protein;
c) a second portion of said GPCR, wherein said second portion comprises TM6 and TM7 regions of said GPCR.
2. The fusion protein of claim 1 , wherein said GPCR is active.
3. The fusion protein of claim 1 , wherein said GPCR is naturally occurring.
4. The fusion protein of claim 1 , wherein said GPCR is non-naturally occurring.
5. The fusion protein of claim 4 , wherein said GPCR has an amino acid sequence that is at least 80% to a naturally occurring GPCR.
6. The fusion protein of claim 5 , wherein the domain of b) is in the range of from 100 to 200 amino acids in length.
7. The fusion protein of claim 1 , wherein said stable, folded protein insertion comprises the amino acid sequence of a lysozyme.
8. A method of determining a crystal structure, comprising:
receiving a fusion protein of claim 1 ,
crystallizing said fusion protein to produce a crystal; and
obtaining atomic coordinates of said fusion protein from said crystal.
9. A method of determining a crystal structure, comprising:
forwarding a fusion protein of claim 1 to a remote location,
receiving atomic coordinates of said fusion protein.
10. The fusion protein of claim 1 , wherein the domain of b) spaces the C-terminal end of the TM 5 region and the N-terminal end of the TM6 region of said GPCR such that the closest alpha carbon atoms at said C-terminal end and said N-terminal end are spaced by a distance in the range of from 6 Å to 16 Å.
11. A nucleic acid encoding a fusion protein comprising, from N-terminus to C-terminus:
a) 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;
b) a heterologous domain comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of a soluble, autonomously folding, wild-type protein that aids in the formation of lattice contacts during crystallization of the fusion protein;
c) a second portion of said GPCR, wherein said second portion comprises TM6 and TM7 regions of said GPCR.
12. A cell containing a nucleic acid encoding a fusion protein comprising, from N-terminus to C-terminus:
a) 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;
b) a heterologous domain comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of a soluble, autonomously folding, wild-type protein that aids in the formation of lattice contacts during crystallization of the fusion protein;
c) a second portion of said GPCR, wherein said second portion comprises TM6 and TM7 regions of said GPCR.
13. The cell of claim 12 , wherein said fusion protein is expressed and disposed on the plasma membrane of said cell.
14. A crystal comprising a fusion protein comprising, from N-terminus to C-terminus:
a) 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;
b) a heterologous domain comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of a soluble, autonomously folding, wild-type protein;
c) a second portion of said GPCR, wherein said second portion comprises TM6 and TM7 regions of said GPCR.
15. The crystal of claim 14 , wherein the GPCR fusion protein is co-crystallized with a ligand for the GPCR.
16. A method comprising:
culturing a cell comprising a nucleic acid encoding a fusion protein comprising, from N-terminus to C-terminus:
a) 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;
b) a heterologous domain comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of a soluble, autonomously folding, wild-type protein that aids in the formation of lattice contacts during crystallization of the fusion protein;
c) a second portion of said GPCR, wherein said second portion comprises TM6 and TM7 regions of said GPCR;
to produce said fusion protein; and
isolating said fusion protein from said cell.
17. The method of claim 16 , further comprising:
crystallizing said fusion protein to make crystals.
18. The method of claim 17 , wherein said method comprises combining said fusion protein with lipid prior to crystallization.
19. The method of claim 18 , wherein said fusion protein is crystallized using a bicelle crystallization method or a lipidic cubic phase crystallization method.
20. The method of claim 17 , further comprising:
obtaining atomic coordinates of said fusion protein from said crystal.