Stable proteins

Abstract

The invention provides a fusion protein comprising, from N-terminus to C-terminus: a) a first portion of a Family B G-protein coupled receptor (GPCR) that comprises transmembrane helix (TM)-1, TM2 and TM3 of the GPCR; b) a stable protein domain; and c) a second portion of the GPCR comprising TM4, TM5, TM6 and TM7 of the GPCR. The invention also provides a method of crystallising a GPCR comprising providing the fusion protein of the invention and crystallising it to obtain crystals.

Claims

1. A fusion protein comprising, from N-terminus to C-terminus: a. a first portion of a Family B G-protein coupled receptor (GPCR) that comprises transmembrane helix (TM)-1, TM2 and TM3 of the GPCR, wherein the TM1 and the TM2 are joined by intracellular loop 1 (ICL1), and the TM2 and TM3 are joined by extracellular loop 1 (ECL1); b. a stable protein domain; and c. a second portion of the GPCR comprising TM4, TM5, TM6 and TM7 of the GPCR wherein the TM4 and the TM5 are joined by extracellular loop 2 (ECL2), the TM5 and the TM6 are joined by intracellular loop 3 (ICL3), and the TM6 and the TM7 are joined by extracellular loop 3 (ECL3); wherein the stable protein domain comprises a soluble, well-folded polypeptide that provides N- and C-termini, the distance between which approximates the distance between helices 3 and 4 in the Family B GPCR, and that provides a hydrophilic surface for crystal lattice contacts, thereby facilitating crystallisation, wherein the stable protein domain is inserted into the intracellular loop 2 (ICL2) region of the GPCR which loop joins the TM3 in the first portion of the GPCR and the TM4 in the second portion of the GPCR, and wherein the fusion protein displays reduced aggregation in the presence of a detergent solution, as compared to a GPCR without insertion of the stable protein domain into the ICL2 region of the GPCR.

2. The fusion protein according to claim 1, wherein the stable protein domain is inserted into the ICL2 region of the GPCR at a position between amino acid residues that correspond to amino acids Phe 257 and Ser 261 of human GLP1R, wherein Phe 257 corresponds to the fifth amino acid in SEQ ID NO: 30 and Ser 261 corresponds to the ninth amino acid in SEQ ID NO: 30.

3. The fusion protein according to claim 2, wherein the stable protein domain is inserted into the ICL2 region of the GPCR after an amino acid corresponding to amino acid Phe 257 of human GLP1R and before an amino acid corresponding to amino acid Ser 261 or Phe 260 or Val 259, wherein Phe 257 corresponds to the fifth amino acid in SEQ ID NO: 30, Ser 261 corresponds to the ninth amino acid in SEQ ID NO: 30, Phe 260 corresponds to the eighth amino acid in SEQ ID NO: 30, and Val 259 corresponds to the seventh amino acid in SEQ ID NO: 30.

4. The fusion protein according to claim 2, wherein the stable protein domain is inserted into the ICL2 region of the GPCR after an amino acid corresponding to amino acid Ser 258 of human GLP1R and before an amino acid corresponding to amino acid Ser 261 or Phe 260 or Val 259 of human GLP1R, wherein Ser 258 corresponds to the sixth amino acid in SEQ ID NO: 30, Ser 261 corresponds to the ninth amino acid in SEQ ID NO: 30, Phe 260 corresponds to the eighth amino acid in SEQ ID NO: 30, and Val 259 corresponds to the seventh amino acid in SEQ ID NO: 30.

5. The fusion protein according to claim 1, wherein the stable protein domain comprises a lysozyme and wherein the amino acid sequence of the lysozyme is at least 90% identical to SEQ ID NO: 24.

6. The fusion protein according to claim 1, further comprising a detectable moiety.

7. The fusion protein according to claim 6, wherein the detectable moiety is EGFP.

8. A crystal comprising the fusion protein of claim 1.

9. The fusion protein according to claim 1, which is in a solubilised form or which is substantially free of other proteins or which is immobilised to a solid support.

10. The fusion protein according to claim 1, wherein the stable protein domain reduces the inherent flexibility of the GPCR fusion protein.

11. The fusion protein according to claim 1, wherein the stable protein domain: a. has an N-terminus that is within 5-17 of its C-terminus; b. is resistant to thermal and chemical denaturation, as well as proteolytic denaturation; and c. is highly crystallisable in a variety of space groups and crystal packing arrangements.

12. The fusion protein according to claim 1, wherein the stable protein domain has an N-terminus that is within 6-16 , or within 7-15 , or within 7-10 , or within 10-13 , or within 12-15 of its C-terminus.

13. The fusion protein according to claim 1, wherein the fusion protein is characterised in that it is crystallisable.

14. A fusion protein according to claim 1, wherein the stable protein domain comprises a polypeptide selected from a cytochrome.sub.b562, a flavodoxin, a -lactamase and a 70 kDa heat shock ATPase domain.

15. The fusion protein according to claim 1, wherein the Family B GPCR is a glucagon-like peptide 1 receptor (GLP1R), glucagon-like peptide 2 receptor (GLP2R), or glucagon receptor.

16. The fusion protein according to claim 1, wherein the Family B GPCR is a calcitonin receptor (CT).

17. The fusion protein according to claim 1, wherein the Family B GPCR is an amylin/CGRP receptor (AMY.sub.1), amylin receptor (AMY.sub.2), amylin/CGRP receptor (AMY.sub.3), CGRP/adrenomedullin receptor (CGRP.sub.1), adrenomedullin/CGRP receptor (AM.sub.1), or adrenomedullin/CGRP receptor (AM.sub.2 receptor).

18. The fusion protein according to claim 1, wherein the Family B GPCR is a corticotropin releasing factor receptor (CRF.sub.1), or urocortins receptor (CRF.sub.2).

19. The fusion protein according to claim 1, wherein the Family B GPCR is a growth hormone releasing hormone receptor (GHRH).

20. The fusion protein according to claim 1, wherein the Family B GPCR is a gastric inhibitory polypeptide receptor (GIP).

21. The fusion protein according to claim 1, wherein the Family B GPCR is a secretin receptor.

22. The fusion protein according to claim 1, wherein the Family B GPCR is a TIP-39 receptor (PTH2), or parathyroid hormone receptor (PTH1).

23. The fusion protein according to claim 1, wherein the Family B GPCR is a VIP/PACAP receptor (VPAC.sub.1), PACAP receptor (PAC.sub.2), or VIP/PACAP receptor (VPAC.sub.2).

24. The fusion protein according to claim 5, wherein the lysozyme is T4 lysozyme.

25. The fusion protein according to claim 6, wherein the detectable moiety is a fluorescent label, a radiolabel, or an enzymatic label.

Description

(1) The invention will now be described with the aid of the following figures and examples.

(2) FIGS. 1A-1B. Design of T4L fusion constructs with GLP1R. T4L was inserted after the indicated residues in ICL2 (left) and ICL3 (right). Construct 1 a means that T4L was inserted between L255 and 8261. The model of TM domains and loops are based on reference [4].

(3) FIG. 2. Total EGFP signal of GLP1R-T4L fusion constructs compared to the wild-type (WT) and the mock transfected (U) samples. Each measurement was done on 50 ug of total cellular material in duplicate. Error bars represent standard deviation from mean.

(4) FIG. 3. Typical fSEC elution profile of wild-type GLP1R.

(5) FIGS. 4A-4C. fSEC elution profiles DOM solubilised GLP1 R-T4L fusion constructs in the ICL2 overlaid the wild-type profile. In each case, the wild-type profile is shown in red the fusion constructs in blue.

(6) FIGS. 5A-5E. fSEC elution profiles DOM solubilised GLP1R-T4L fusion constructs in the ICL3 overlaid the wild-type profile. In each case, the wild-type profile is shown in red the fusion constructs in blue.

(7) FIG. 6. Amino acid sequences of Family B GPCRs showing position of TM3, ICL2 and TM4 (SEQ ID Nos: 1-22). The portion of ICL2 that was replaced with T4L in GLP1R fusion construct 1c is highlighted in other Family B receptors from mouse, rat and human.

(8) FIG. 7. Amino acid sequences of T4 phage lysozyme: (A) Sequence inserted into ICL3 of Family A receptors [2], [3] (SEQ ID No: 23); (B) Sequence inserted into ICL2 of Family B receptors (SEQ ID No: 24) (see Examples). Differences are indicated in boxes.

EXAMPLE 1

Insertion of T4L into ICL2 Improves Biochemical Properties of GLP1R

(9) Summary

(10) We have tested the effect of inserting T4L in the internal loops of Family B receptors. Our data indicate that Family B receptors cannot tolerate T4L fusion in ICL3, however, adding T4L to ICL2 improves the biochemical properties of the receptor.

(11) Results

(12) The loop regions of GLP1R were determined according to the model of GLP1R and the DNA encoding T4L was inserted in different locations within ICL2 and ICL3 (FIG. 1). The GLP1R construct was C-terminally tagged with the EGFP in order to monitor total expression as well as monodispersity using fluorescent-detection size exclusion chromatography.

(13) Following sequence confirmation, these constructs were expressed in HEK293T transiently. As an initial analysis, the EGFP signal in whole cells was measured to assess the total levels of expression. Interestingly, the constructs in ICL3 failed to produce any EGFP signal, indicating that T4L fusion in this region of GLP1R is incompatible with the overall architecture of this receptor. However, fusions in the ICL2 resulted in the robust expression of GLP1R (FIG. 2).

(14) In order to analyse the biochemical properties of GLP1R-T4L fusions, cells expressing these constructs were solubilised in dodecyl maltoside (DDM) and applied to fluorescence-detection size-exclusion chromatography (fSEC). fSEC has been used widely to provide data regarding the monodispersity and the aggregation status of the proteins, particularly in pre-crystallisation screens [5]. In general, the more favourable conditions will result in more monodispersity and reduced aggregation. The fSEC elution profile of DDM-solubilised wild-type GLP1R shows the presence of the main monodispersed peak with an aggregation shoulder as well as free EGFP species that are the result of proteolytic degredation (FIG. 3).

(15) Consistent with the EGFP signal data showed in FIG. 2, the elution profiles of ICL2 T4L fusion constructs 1a, 1b, 2a, 2b, 3a and 3b indicates that cells failed to express these fusions (FIG. 4). This is most likely due to the proximity of the N-terminus of T4L to TM III, leading to the disruption of the overall structure. In contrast, the elution profiles of constructs 1c, 1d, 2c, 2d, 3c and 3d revealed that productive fusion receptors were expressed and more significantly it appears that T4L fusion results in the reduction of the aggregation peak and concomitant improvement of the monodispersed peak, which together indicate that T4L insertion in this region of the receptor has beneficial effect on the biochemical properties of solubilised receptor (FIG. 4). This effect is most pronounced in the constructs 1c and 2c.

(16) The same analysis was carried out for the ICL3 fusion constructs and in agreement with the EGFP signal data showed in FIG. 2, none of the T4L fusions in the ICL3 resulted in the expression of any productive fusion receptor (FIG. 5).

(17) Taken together, these data indicate that T4L fusion in the third cytoplasmic loop of GLP1R is not tolerated, however, insertion of T4L in certain positions in the second cytoplasmic loop not only is tolerated, it also improves the biochemical properties of the solubilised receptor. Given the high sequence homology amongst members of the Family B GPCRs, we suggest that these observations can be extended to other members of this Family. The portion of the ICL2 that was replaced in the best construct (1c) is highlighted in other Family B members as shown in FIG. 6.

(18) Methods and material

(19) T4 lysozyme was inserted in the second and third cytoplasmic loops of human GLP1R using standard molecular biology techniques. These constructs were transiently expressed from a modified pcDNA3.1 in HEK293T cells, generating receptors fused to EEGFP at their C-termini. Transfections were carried out using GeneJuice (Merck Biosciences) according to the manufacturer's guideline. Typically, 6 ug of DNA was used to transfect 310.sup.6 adherent cells in 10 cm plates. Cells were harvested about 40 hours post transfection and re-suspended in 50 mM HEPES pH 7.5/150 mM NaCl/0.5 mM EDTA complemented with Complete EDTA-free protease inhibitor cocktail (Roche). Typically 650 ug of each sample was solubilised with 1% DDM in total volume of 200 uL for 1 hour at 4 C. followed by centrifugation at 50000 rpm for 30 minutes. 50 uL of the supernantant was loaded onto BioSEep-SEC-S3000 column (Phenomenex), pre-equilibrated with SEC buffer (50 mM HEPES pH 7.5/150 mM NaCl/0.5 mM EDTA/0.03% DDM) and run at the flow rate of 1 mL/minute for 15 minutes. The eluent was passed through an on-line fluorometer with the following settings: excitation 490 nm, emission 513 nm and gain of 13.

REFERENCES

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