Development and crystallization of a minimal thermostabilised G protein-coupled receptor

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Abstract

Structure determination of G protein-coupled receptors is still in its infancy and many factors affect whether crystals are obtained and whether the diffraction is of sufficient quality for structure determination. We recently solved the structure of a thermostabilised turkey β1-adrenergic receptor by crystallization in the presence of the detergent octylthioglucoside. Three factors were essential for this success. Firstly, truncations were required at the N-terminus to give optimal expression. Secondly, 6 thermostabilising point mutations were incorporated to make the receptor sufficiently stable in short-chain detergents to allow crystallization. Thirdly, truncations at the C-terminus and within cytoplasmic loop 3, in combination with the removal of the palmitoylation site, were required to obtain well-diffracting crystals in octylthioglucoside. Here, we describe the strategy employed and the utility of thermostability assays in assessing how point mutations, truncations, detergents and ligands combine to develop a construct that forms diffraction-grade crystals.

Section snippets

Removal of flexible regions in β1AR

It is generally considered that flexible regions in a protein might prevent the growth of well-ordered crystals and that it might be beneficial to remove them, either by site-directed mutagenesis or by limited proteolysis of the purified protein. An alignment of the β-adrenergic receptors (not shown) clearly indicated three areas that were non-conserved and thus potentially represented regions that might be flexible and redundant for ligand binding, namely the N-terminus, the C-terminus and

Discussion

No three dimensional crystals of the full-length, non-stabilised β1AR6 construct were ever been obtained from sitting or hanging drop crystallization trials, despite the high expression level, the purity of the preparation, the steps taken to reduce heterogeneity due to post-translational modifications and the implementation of large-scale robotic crystallization screening. We ascribe this to three major problems, namely receptor flexibility, heterogeneity of signalling states and poor

Materials

Sf9 and Tni (High 5) cells were obtained from Invitrogen, the baculovirus transfer vector pBacPAK8 was from BD Clontech, and linearized baculovirus DNA (Baculogold) was from Pharmingen. All detergents were from Anatrace, with the exception of LDAO that was from Fluka and C8E4 that was from Bachem. Alprenolol sepharose CL-4B was synthesized as described previously [29]. All other chromatographic equipment, columns and materials were from GE Healthcare, PEGs were from Fluka, (−)-alprenolol and (

Acknowledgments

This work was supported by a joint grant from Pfizer Global Research and Development and from the MRCT Development Gap Fund, in addition to core funding from MRC. G.F.X.S. was financially supported by a Human Frontier Science Project (HFSP) programme grant (RG/0052), a European Commission FP6 specific targeted research project (LSH-2003-1.1.0-1) and an ESRF long-term proposal. We also thank C. Riekel and M. Burghammer at ID13, D. Flot and S. McSweeney at ID 23.2, which are both microfocus

References (35)

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    They were not authenticated further. The parent β1AR construct for in vitro translation was created by inserting the coding region of residues 20-424 of turkey β1AR-B6m23 (Warne et al., 2009) into an SP64 based vector containing an HA affinity tag at the N terminus and the unstructured cytosolic domain of Sec61β (residues 2-69, with the single Cysteine and predicted Glycosylation acceptor sequence mutated to Serine and Glutamine, respectively) followed by a 6-Histidine tag at the C terminus. A glycosylation acceptor site (NGT) was introduced at residues 22-24 within the β1AR sequence.

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