title: Conformational thermostabilisation of corticotropin releasing factor receptor 1.
abstract: Recent technical advances have greatly facilitated G-protein coupled receptors crystallography as evidenced by the number of successful x-ray structures that have been reported recently. These technical advances include novel detergents, specialised crystallography techniques as well as protein engineering solutions such as fusions and conformational thermostabilisation. Using conformational thermostabilisation, it is possible to generate variants of GPCRs that exhibit significantly increased stability in detergent micelles whilst preferentially occupying a single conformation. In this paper we describe for the first time the application of this technique to a member of a class B GPCR, the corticotropin releasing factor receptor 1 (CRF1R). Mutational screening in the presence of the inverse agonist, CP-376395, resulted in the identification of a construct with twelve point mutations that exhibited significantly increased thermal stability in a range of detergents. We further describe the subsequent construct engineering steps that eventually yielded a crystallisation-ready construct which recently led to the solution of the first x-ray structure of a class B receptor. Finally, we have used molecular dynamic simulation to provide structural insight into CRF1R instability as well as the stabilising effects of the mutants, which may be extended to other class B receptors considering the high degree of structural conservation.
authors.affiliation: Heptares Therapeutics Ltd, BioPark, Broadwater Road, Welwyn Garden City, AL7 3AX, UK.
authors.forename: James
authors.initials: J
authors.lastname: Kean
authors.affiliation: Heptares Therapeutics Ltd, BioPark, Broadwater Road, Welwyn Garden City, AL7 3AX, UK.
authors.forename: Andrea
authors.initials: A
authors.lastname: Bortolato
authors.affiliation: Heptares Therapeutics Ltd, BioPark, Broadwater Road, Welwyn Garden City, AL7 3AX, UK.
authors.forename: Kaspar
authors.initials: K
authors.lastname: Hollenstein
authors.affiliation: Heptares Therapeutics Ltd, BioPark, Broadwater Road, Welwyn Garden City, AL7 3AX, UK.
authors.forename: Fiona H
authors.initials: FH
authors.lastname: Marshall
authors.affiliation: Heptares Therapeutics Ltd, BioPark, Broadwater Road, Welwyn Garden City, AL7 3AX, UK.
authors.forename: Ali
authors.initials: A
authors.lastname: Jazayeri
mesh_terms: D000631:Aminopyridines / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D054314:Drug Inverse Agonism; D057809:HEK293 Cells; D006207:Half-Life; D006801:Humans; D056004:Molecular Dynamics Simulation; D016296:Mutagenesis; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D018019:Receptors, Corticotropin-Releasing Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000099052:CRF Receptor, Type 1
chemical_list: C529296:(3,6-dimethyl-2-(2,4,6-trimethylphenoxy)pyridin-4-yl)(1-ethylpropyl)amine; D000631:Aminopyridines; D018019:Receptors, Corticotropin-Releasing Hormone; D000099052:CRF Receptor, Type 1