Molecular Recognition of Corticotropin-releasing Factor by Its G-protein-coupled Receptor CRFR1

Artigo Acesso aberto Revisado por pares

Molecular Recognition of Corticotropin-releasing Factor by Its G-protein-coupled Receptor CRFR1

2008; Elsevier BV; Volume: 283; Issue: 47 Linguagem: Inglês

10.1074/jbc.m805749200

ISSN

1083-351X

Autores

Augen A. Pioszak, Naomi R. Parker, Kelly Suino-Powell, H. Eric Xu,

Tópico(s)

Adrenal Hormones and Disorders

Resumo

The bimolecular interaction between corticotropin-releasing factor (CRF), a neuropeptide, and its type 1 receptor (CRFR1), a class B G-protein-coupled receptor (GPCR), is crucial for activation of the hypothalamic-pituitary-adrenal axis in response to stress, and has been a target of intense drug design for the treatment of anxiety, depression, and related disorders. As a class B GPCR, CRFR1 contains an N-terminal extracellular domain (ECD) that provides the primary ligand binding determinants. Here we present three crystal structures of the human CRFR1 ECD, one in a ligand-free form and two in distinct CRF-bound states. The CRFR1 ECD adopts the α-β-βα fold observed for other class B GPCR ECDs, but the N-terminal α-helix is significantly shorter and does not contact CRF. CRF adopts a continuous α-helix that docks in a hydrophobic surface of the ECD that is distinct from the peptide-binding site of other class B GPCRs, thereby providing a basis for the specificity of ligand recognition between CRFR1 and other class B GPCRs. The binding of CRF is accompanied by clamp-like conformational changes of two loops of the receptor that anchor the CRF C terminus, including the C-terminal amide group. These structural studies provide a molecular framework for understanding peptide binding and specificity by the CRF receptors as well as a template for designing potent and selective CRFR1 antagonists for therapeutic applications. The bimolecular interaction between corticotropin-releasing factor (CRF), a neuropeptide, and its type 1 receptor (CRFR1), a class B G-protein-coupled receptor (GPCR), is crucial for activation of the hypothalamic-pituitary-adrenal axis in response to stress, and has been a target of intense drug design for the treatment of anxiety, depression, and related disorders. As a class B GPCR, CRFR1 contains an N-terminal extracellular domain (ECD) that provides the primary ligand binding determinants. Here we present three crystal structures of the human CRFR1 ECD, one in a ligand-free form and two in distinct CRF-bound states. The CRFR1 ECD adopts the α-β-βα fold observed for other class B GPCR ECDs, but the N-terminal α-helix is significantly shorter and does not contact CRF. CRF adopts a continuous α-helix that docks in a hydrophobic surface of the ECD that is distinct from the peptide-binding site of other class B GPCRs, thereby providing a basis for the specificity of ligand recognition between CRFR1 and other class B GPCRs. The binding of CRF is accompanied by clamp-like conformational changes of two loops of the receptor that anchor the CRF C terminus, including the C-terminal amide group. These structural studies provide a molecular framework for understanding peptide binding and specificity by the CRF receptors as well as a template for designing potent and selective CRFR1 antagonists for therapeutic applications. Corticotropin-releasing factor (CRF) 3The abbreviations used are: CRF, corticotropin releasing factor; CRFR, CRF receptor; Ucn, urocortin; PTH, parathyroid hormone; PTH1R, PTH receptor type 1; GIP, glucose-dependent insulinotropic peptide; GIPR, GIP receptor; GLP1R, glucagon-like peptide 1 receptor; GPCR, G-protein-coupled receptor; PDB, Protein Data Bank; MBP, maltose-binding protein; ECD, extracellular domain; MOPS, 4-morpholinepropanesulfonic acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; SCR, short consensus repeat; MR, molecular replacement; PEG, polyethylene glycol; TLS, translation, libration, screw rotation. is a 41-amino acid, C-terminally amidated neuropeptide originally isolated from sheep hypothalami based on its ability to stimulate secretion of adrenocorticotropin from pituitary cells (1Vale W. Spiess J. Rivier C. Rivier J. Science. 1981; 213: 1394-1397Crossref PubMed Scopus (4080) Google Scholar). Several other CRF-related peptides have since been identified, including the urocortins (Ucn) I, II, and III in mammals (2Hsu S.Y. Hsueh A.J. Nat. Med. 2001; 7: 605-611Crossref PubMed Scopus (617) Google Scholar, 3Lewis K. Li C. Perrin M.H. Blount A. Kunitake K. Donaldson C. Vaughan J. Reyes T.M. Gulyas J. Fischer W. Bilezikjian L. Rivier J. Sawchenko P.E. Vale W.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7570-7575Crossref PubMed Scopus (843) Google Scholar, 4Reyes T.M. Lewis K. Perrin M.H. Kunitake K.S. Vaughan J. Arias C.A. Hogenesch J.B. Gulyas J. Rivier J. Vale W.W. Sawchenko P.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2843-2848Crossref PubMed Scopus (823) Google Scholar, 5Vaughan J. Donaldson C. Bittencourt J. Perrin M.H. Lewis K. Sutton S. Chan R. Turnbull A.V. Lovejoy D. Rivier C. Rivier J. Sawchenko P.E. Vale W. Nature. 1995; 378: 287-292Crossref PubMed Scopus (1416) Google Scholar). Extensive studies over the last nearly 3 decades have highlighted the critical roles that CRF family peptides play in coordinating endocrine, autonomic, and behavioral responses to stress (reviewed in Refs. 6Bale T.L. Vale W.W. Annu. Rev. Pharmacol. Toxicol. 2004; 44: 525-557Crossref PubMed Scopus (1088) Google Scholar, 7Hauger R.L. Risbrough V. Brauns O. Dautzenberg F.M. CNS Neurol. Disord. Drug Targets. 2006; 5: 453-479Crossref PubMed Scopus (281) Google Scholar). The CRF family of peptides exert their effects through the binding and activation of two paralogous cell surface G-protein-coupled receptors (GPCRs), CRFR1 (8Chen R. Lewis K.A. Perrin M.H. Vale W.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8967-8971Crossref PubMed Scopus (913) Google Scholar) and CRFR2 (9Kishimoto T. Pearse R.V. 2nd, Lin C.R. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1108-1112Crossref PubMed Scopus (377) Google Scholar, 10Lovenberg T.W. Liaw C.W. Grigoriadis D.E. Clevenger W. Chalmers D.T. De Souza E.B. Oltersdorf T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 836-840Crossref PubMed Scopus (839) Google Scholar, 11Perrin M. Donaldson C. Chen R. Blount A. Berggren T. Bilezikjian L. Sawchenko P. Vale W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2969-2973Crossref PubMed Scopus (495) Google Scholar). CRF binds to both receptors but with higher affinity for CRFR1. UcnI binds equally well to both receptors, whereas UcnII and UcnIII are selective for CRFR2. CRF is the primary regulator of central stress responses; its binding to CRFR1 on the surface of pituitary corticotrope cells activates the hypothalamic-pituitary-adrenal axis. Consequently, there has been enormous interest in the therapeutic potential of CRFR1-selective antagonists for the treatment of anxiety, depression, and related disorders (reviewed in Refs. 7Hauger R.L. Risbrough V. Brauns O. Dautzenberg F.M. CNS Neurol. Disord. Drug Targets. 2006; 5: 453-479Crossref PubMed Scopus (281) Google Scholar, 12Grammatopoulos D.K. Chrousos G.P. Trends Endocrinol. Metab. 2002; 13: 436-444Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). The CRF receptors belong to the class B/Secretin family of GPCRs (13Fredriksson R. Lagerstrom M.C. Lundin L.G. Schioth H.B. Mol. Pharmacol. 2003; 63: 1256-1272Crossref PubMed Scopus (2257) Google Scholar), whose members include receptors for parathyroid hormone, calcitonin, glucagon, glucagon-like peptides, and other therapeutically important peptides. In addition to a 7-transmembrane helical domain common to all GPCRs, class B receptors have an N-terminal extracellular domain (ECD) of roughly 100-160 amino acids that contains three conserved disulfide bonds. Ligand binding and activation of the receptors are thought to occur by a two-domain model (reviewed in Ref. 14Hoare S.R. Drug Discov. Today. 2005; 10: 417-427Crossref PubMed Scopus (312) Google Scholar). The C-terminal portion of the peptide ligand provides the primary receptor binding determinants and interacts with the ECD to bring the N-terminal portion of the peptide in proximity to the 7-transmembrane helical domain where it activates the receptor. Many studies support the two-domain model for CRFR1. Chimeric receptor studies indicated that the CRFR1 ECD provides the primary ligand binding determinants (15Dautzenberg F.M. Wille S. Lohmann R. Spiess J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4941-4946Crossref PubMed Scopus (57) Google Scholar, 16Perrin M.H. Sutton S. Bain D.L. Berggren W.T. Vale W.W. Endocrinology. 1998; 139: 566-570Crossref PubMed Scopus (95) Google Scholar, 17Wille S. Sydow S. Palchaudhuri M.R. Spiess J. Dautzenberg F.M. J. Neurochem. 1999; 72: 388-395Crossref PubMed Scopus (79) Google Scholar). Moreover, recombinant expression and purification of the isolated ECD confirmed its ability to bind ligands (18Klose J. Fechner K. Beyermann M. Krause E. Wendt N. Bienert M. Rudolph R. Rothemund S. Biochemistry. 2005; 44: 1614-1623Crossref PubMed Scopus (21) Google Scholar, 19Perrin M.H. Fischer W.H. Kunitake K.S. Craig A.G. Koerber S.C. Cervini L.A. Rivier J.E. Groppe J.C. Greenwald J. Moller Nielsen S. Vale W.W. J. Biol. Chem. 2001; 276: 31528-31534Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). A chimeric receptor in which the ECD of CRFR1 was replaced with the 16 N-terminal residues of CRF exhibited constitutive activation (20Nielsen S.M. Nielsen L.Z. Hjorth S.A. Perrin M.H. Vale W.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10277-10281Crossref PubMed Scopus (70) Google Scholar), and the isolated 7-transmembrane helical domain was activated by agonist peptides, albeit with reduced potency (21Hoare S.R. Sullivan S.K. Schwarz D.A. Ling N. Vale W.W. Crowe P.D. Grigoriadis D.E. Biochemistry. 2004; 43: 3996-4011Crossref PubMed Scopus (75) Google Scholar). It has long been recognized that the C-terminal amide moiety of CRF and a helical conformation of the peptide are critical for high affinity binding to the receptor (1Vale W. Spiess J. Rivier C. Rivier J. Science. 1981; 213: 1394-1397Crossref PubMed Scopus (4080) Google Scholar, 22Rivier J. Rivier C. Vale W. Science. 1984; 224: 889-891Crossref PubMed Scopus (453) Google Scholar). Truncation of N-terminal residues of CRF creates competitive antagonists (22Rivier J. Rivier C. Vale W. Science. 1984; 224: 889-891Crossref PubMed Scopus (453) Google Scholar). Numerous such CRF analogs have been synthesized that display varying potency and selectivity for CRFR1 and CRFR2 (23Hernandez J.F. Kornreich W. Rivier C. Miranda A. Yamamoto G. Andrews J. Tache Y. Vale W. Rivier J. J. Med. Chem. 1993; 36: 2860-2867Crossref PubMed Scopus (86) Google Scholar, 24Miranda A. Koerber S.C. Gulyas J. Lahrichi S.L. Craig A.G. Corrigan A. Hagler A. Rivier C. Vale W. Rivier J. J. Med. Chem. 1994; 37: 1450-1459Crossref PubMed Scopus (59) Google Scholar, 25Ruhmann A. Bonk I. Lin C.R. Rosenfeld M.G. Spiess J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15264-15269Crossref PubMed Scopus (169) Google Scholar). Astressin, a high affinity antagonist that binds both receptors, is a modified version of CRF-(12-41)-NH2 constrained by a helix-stabilizing lactam bridge (26Gulyas J. Rivier C. Perrin M. Koerber S.C. Sutton S. Corrigan A. Lahrichi S.L. Craig A.G. Vale W. Rivier J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10575-10579Crossref PubMed Scopus (229) Google Scholar). Potent astressin-like antagonists as short as fragments 27-41 or 30-41 have also been reported (27Rijkers D.T. Kruijtzer J.A. van Oostenbrugge M. Ronken E. den Hartog J.A. Liskamp R.M. ChemBioChem. 2004; 5: 340-348Crossref PubMed Scopus (18) Google Scholar, 28Yamada Y. Mizutani K. Mizusawa Y. Hantani Y. Tanaka M. Tanaka Y. Tomimoto M. Sugawara M. Imai N. Yamada H. Okajima N. Haruta J. J. Med. Chem. 2004; 47: 1075-1078Crossref PubMed Scopus (23) Google Scholar). Recently, considerable insight into the ligand binding mechanisms of class B GPCRs has been gained from several reports of ECD·peptide complex structures determined by NMR or x-ray crystallographic methods. The NMR solution structure of astressin bound to the mouse CRFR2β ECD showed that the ECD consists of two antiparallel β-sheets, each with two β-strands that are held together by the conserved disulfide bonds. The arrangement of the CRFR2β ECD resembles the short consensus repeat (SCR) fold that is also present in the Ig family of proteins (29Grace C.R. Perrin M.H. Gulyas J. Digruccio M.R. Cantle J.P. Rivier J.E. Vale W.W. Riek R. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 4858-4863Crossref PubMed Scopus (123) Google Scholar). The astressin 27-41-amino acid fragment forms an amphipathic α-helix that interacts with a hydrophobic surface of the ECD at the interface of three loop regions. Subsequent reports described the structures of pituitary adenylate cyclase-activating polypeptide, glucose-dependent insulinotropic peptide (GIP), exendin-4, and parathyroid hormone (PTH) in complex with their cognate receptor ECDs (30Parthier C. Kleinschmidt M. Neumann P. Rudolph R. Manhart S. Schlenzig D. Fanghanel J. Rahfeld J.U. Demuth H.U. Stubbs M.T. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 13942-13947Crossref PubMed Scopus (208) Google Scholar, 31Pioszak A.A. Xu H.E. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5034-5039Crossref PubMed Scopus (226) Google Scholar, 32Runge S. Thogersen H. Madsen K. Lau J. Rudolph R. J. Biol. Chem. 2008; 283: 11340-11347Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 33Sun C. Song D. Davis-Taber R.A. Barrett L.W. Scott V.E. Richardson P.L. Pereda-Lopez A. Uchic M.E. Solomon L.R. Lake M.R. Walter K.A. Hajduk P.J. Olejniczak E.T. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7875-7880Crossref PubMed Scopus (127) Google Scholar). No structure of the CRFR1 ECD has been reported to date, although the conformation of a short astressin-like antagonist when bound to the CRFR1 ECD was determined by NMR methods (34Mesleh M.F. Shirley W.A. Heise C.E. Ling N. Maki R.A. Laura R.P. J. Biol. Chem. 2007; 282: 6338-6346Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). A high resolution structure of the CRF·CRFR1 ECD complex is required to understand how the endogenous ligand binds the receptor and will provide insight into ligand selectivity and aid rational drug design targeting CRFR1. We previously reported a general methodology for the expression, purification, and crystallization of the N-terminal ECD of class B GPCRs and demonstrated its applicability for the PTH1R ECD (31Pioszak A.A. Xu H.E. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5034-5039Crossref PubMed Scopus (226) Google Scholar). The PTH1R ECD was expressed as a fusion to bacterial maltose-binding protein (MBP) in the oxidizing cytoplasm of an Escherichia coli trxB gor host to facilitate disulfide bond formation, and the fusion protein was purified and subjected to in vitro disulfide shuffling in a redox buffer to maximize the yield of properly folded protein. The MBP tag facilitated crystallization of the PTH1R ECD by providing a large surface area for crystal contacts. Here we show that the methodology is applicable to the ECD of human CRFR1, and we describe the crystal structures of the CRFR1 ECD in the ligand-free and CRF-bound states, discuss conformational changes associated with CRF binding, and compare the CRFR1 ECD structures to those of the mouse CRFR2β ECD and other class B GPCR ECDs. Molecular Biology Methods—The plasmid for expression of the human CRFR1 ECD as a fusion to bacterial maltose-binding protein (MBP) was constructed as described previously for the PTH1R ECD (31Pioszak A.A. Xu H.E. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5034-5039Crossref PubMed Scopus (226) Google Scholar). Briefly, a DNA fragment corresponding to residues 24-119 of human CRFR1 (excluding the native signal peptide residues 1-23) was PCR-amplified with a C-terminal six histidine residue tag from a CRFR1 cDNA clone obtained from the UMR cDNA resource center. After digestion with EcoRI and NotI restriction endonucleases, the fragment was ligated into an isopropyl 1-thio-β-d-galactopyranoside-inducible, T7 promoter-driven, bacterial expression vector that permits co-expression of the MBP-CRFR1-ECD-H6 protein with the bacterial disulfide isomerase/chaperone DsbC as described previously (31Pioszak A.A. Xu H.E. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5034-5039Crossref PubMed Scopus (226) Google Scholar). Single amino acid substitutions in MBP were introduced by site-directed mutagenesis of the expression vector using the Stratagene Quikchange kit according to the manufacturer's directions. All plasmid constructs were verified by DNA sequencing. Protein Expression and Purification—The CRFR1 ECD was expressed as a fusion protein with maltose-binding protein (MBP) at its N terminus and a His6 tag at its C terminus in the E. coli strain Origami B (DE3) (Novagen) as described previously (31Pioszak A.A. Xu H.E. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5034-5039Crossref PubMed Scopus (226) Google Scholar). The purification protocol was as described previously for the MBP-PTH1R ECD fusion protein (31Pioszak A.A. Xu H.E. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5034-5039Crossref PubMed Scopus (226) Google Scholar), with the exceptions noted below. First, the fusion protein was purified by affinity chromatography via the His6 and MBP tags on nickel-chelating Sepharose resin (GE Healthcare) followed by amylose resin (New England Biolabs). Second, in vitro disulfide shuffling in a 1 mm GSH, 1 mm GSSG redox buffer was performed to increase the yield of properly folded protein. Third, Superdex 200 gel filtration (GE Healthcare) chromatography was used to separate the properly folded and misfolded protein. Finally, the protein was subjected to QFF anion exchange (GE Healthcare) chromatography. The disulfide shuffling reaction mixture was incubated at 13 °C overnight and did not require the addition of purified DsbC, thus permitting application of the shuffling reaction mixture to the gel filtration column without the need to first remove DsbC. Proteins with site-specific amino acid substitutions in MBP were purified in the same manner as wild type, with the exception of the MBP(A326E)-CRFR1 ECD protein for which the amylose step was omitted. (The numbering of MBP residues is based on our synthetic construct.) Protein concentrations were determined by the method of Bradford (35Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (223660) Google Scholar) with bovine serum albumin as the standard. Native gel electrophoresis was performed as described (31Pioszak A.A. Xu H.E. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5034-5039Crossref PubMed Scopus (226) Google Scholar). Peptide Synthesis—Peptides were custom-synthesized and high pressure liquid chromatography-purified by SynBioSci (Livermore, CA). The concentrations of stock solutions were determined based on the theoretical peptide content reported by SynBioSci. Peptide integrity was internally verified by analysis of aliquots by Tris-Tricine SDS-PAGE and mass spectrometry. All peptides contain a C-terminal amide group unless indicated otherwise. Peptide Binding Assay—Association of CRF with MBP-CRFR1-ECD was determined by an AlphaScreen™ luminescent proximity assay (PerkinElmer Life Sciences) using a histidine detection kit similar to a previously described assay (31Pioszak A.A. Xu H.E. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5034-5039Crossref PubMed Scopus (226) Google Scholar). The reaction mixtures contained 5 μg/ml each of streptavidin-coated donor beads and nickel-chelate-coated acceptor beads, and biotin-Gly-Gly-Gly-CRF-(12-41)-NH2 and MBP-CRFR1-ECD-H6 as indicated in a buffer of 50 mm MOPS, pH 7.4, 100 mm NaCl, and 0.1 mg/ml bovine serum albumin. Equilibrium was achieved after incubation at 22 °C for 4.5 h, at which point signal recording was performed in a 384-well microplate with an Envision 2104 plate reader (PerkinElmer Life Sciences). For competition experiments, unlabeled competitor peptides were added at time 0, and the reactions were allowed to reach equilibrium before signal recording. Nonlinear regression as implemented in Prism 5.0 (GraphPad Software, San Diego) was used to fit the data to a variable slope dose-response inhibition equation for determination of IC50 values. Control experiments to ensure that inhibition of the signal by unlabeled peptides was specific were carried out using a biotin-Gly6-His6 peptide (25 nm) in place of the biotinylated CRF-(12-41) and MBP-CRFR1-ECD-H6. Crystallization and Data Collection—Crystal growth was carried out at 20 °C. For ligand-free MBP-CRFR1-ECD-H6, a protein sample in 10 mm Tris-HCl, pH 7.5, 1 mm EDTA, and 1 mm maltose was concentrated to ∼14 mg/ml using an Amicon ultracentrifugal filter device (Millipore) with a molecular mass cutoff of 3 kDa. For the receptor·peptide complexes, a protein sample in 10 mm Tris-HCl, pH 7.5, 50 mm NaCl, 1 mm EDTA, and 1 mm maltose was complexed with a synthetic CRF fragment at a molar ratio of 1:1.2 (protein:peptide) and incubated on ice for 30 min, after which the mixture was concentrated to ∼18 mg/ml as above. Initial crystal screening utilized kits from Hampton Research and an Art Robbins Instruments Phoenix robot. Optimizations of the initial hits were performed manually using the hanging drop vapor diffusion method with drops containing equal volumes of protein and reservoir solution. Large, bipyramidal crystals of the ligand-free protein (crystal form I) were grown over a reservoir solution of 0.1 m sodium acetate, pH 4.7, 1.8 m NaCl, and 30% (w/v) sucrose. For crystallization of the receptor·ligand complexes, MBP-CRFR1 ECD fusion proteins containing the site-specific alterations F94E or A326E in MBP were used to prevent an unfavorable crystal packing interaction with the CRFR1 ECD that prevented crystallization of the wild-type fusion protein in complex with CRF. Plate-shaped crystals of the CRF-(22-41)-bound receptor (crystal form II) were grown with the A326E-altered fusion protein over a reservoir of 0.1 m BisTris, pH 6.75, 0.1 m CaCl2, 22% (v/v) polyethylene glycol (PEG) monomethyl ether 550, and 3% (v/v) tert-butyl alcohol. Microseeding was used to obtain single plate crystals for crystal form II. Bipyramidal crystals of the CRF-(12-41)-bound receptor (crystal form III) were grown with the F94E-altered fusion protein over a reservoir of 0.1 m BisTris, pH 6.25, 0.2 m Li2SO4, and 20% (v/v) PEG 3350. All crystals appeared and completed growth within a few days. The crystals were flash-cooled in cryoprotectant solution by plunging into liquid nitrogen. Crystal form I was suitably cryoprotected in its mother liquor. For crystal form II, the PEG monomethyl ether 550 concentration was raised to 31% by vapor diffusion overnight. For crystal form III, the PEG 3350 concentration was raised to 28% by serial transfer of the crystal into solutions of increasing PEG concentration. Native diffraction data sets were collected from single crystals, with the data for form I and form III crystals collected at beamline 21-ID-D of the Advanced Photon Source (Argonne, IL), and data for crystal form II collected at beamline 21-ID-F. The datasets were processed and scaled with the HKL2000 package (36Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38782) Google Scholar). The data collection statistics are summarized in Table 1.TABLE 1Data collection and refinement statisticsCrystal formI, ligand-freeII, CRF-(22-41)-NH2-boundIII, CRF-(27-41)-NH2-boundData collectionBeamlineAPS 21-ID-DAPS 21-ID-FAPS 21-ID-DSpace groupP41212P1P41212a, b, c112.02, 112.02, 145.79 Å49.20, 63.48, 85.88 Å112.92, 112.92, 158.17 Åα, β, γ90.0, 90.0, 90.0°99.75, 106.28, 101.67°90.0, 90.0, 90.0°Resolution range50.00-2.75 Å (2.85-2.75 Å)aValues in parentheses are for the highest resolution shell.50.00-1.96 Å (2.03-1.96 Å)50.00-3.40 Å (3.52-3.40 Å)Wavelength0.97872 Å0.97872 Å0.99999 ÅNo. of observations335,781217,545100,651Unique reflections24,25364,91914,697Completeness98.4% (90.2%)94.7% (75.2%)99.4% (97.2%)Redundancy13.83.46.8I/σ33.26 (2.99)13.49 (2.32)20.81 (1.88)RmergebRmerge = ∑|I - 〈I〉| > |/∑|I|, where I is the intensity measurement for a given reflection, and is the average intensity for multiple measurements of the reflection.5.9% (57.3%)11.3% (33.7%)8.5% (73.9%)Mosaicity0.441°0.909°0.623°RefinementResolution range50.00-2.7639.50-1.9650.00-3.40No. of reflections (total/test)22,995/123861,357/325413,897/739RcrystcRcryst = ∑(|Fo| - K|Fc|)/∑|Fo|./RfreedRfree was calculated using a randomly selected 5% test set of the total reflections that was omitted from the refinement.20.7%/24.0%20.9%/25.6%21.8%/25.2%MBP-ECD molecules/ASU121No. of TLS groups261Mean B value111.60 Å232.13 Å2156.49 Å2No. of protein atoms353970443586No. of water atoms64500No. of heterogen atoms23 (1 maltose molecule)90 (2 maltose, 2 calcium, 2 Bis Tris, and 2 PEG molecules)23 (1 maltose molecule)r.m.s. bond length deviation0.011 Å0.012 Å0.006 År.m.s. bond angle deviation1.233°1.285°0.997°Ramachandran plot, % residues ineData were as defined in Procheck (42).Most favored91.992.289.7Additional allowed7.67.69.5Generously allowed0.50.30.8Disallowed000a Values in parentheses are for the highest resolution shell.b Rmerge = ∑|I - 〈I〉| > |/∑|I|, where I is the intensity measurement for a given reflection, and is the average intensity for multiple measurements of the reflection.c Rcryst = ∑(|Fo| - K|Fc|)/∑|Fo|.d Rfree was calculated using a randomly selected 5% test set of the total reflections that was omitted from the refinement.e Data were as defined in Procheck (42Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Open table in a new tab Structure Solution and Refinement—The CCP4 suite was used to convert the Scalepack intensities to structure factor amplitudes and flag 5% of the reflections for cross-validation (37Collaborative Computational Project, Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19879) Google Scholar). All three structures were solved by the molecular replacement (MR) method using Phaser (38McCoy A.J. Grosse-Kunstleve R.W. Adams P.D. Winn M.D. Storoni L.C. Read R.J. J. Appl. Crystallogr. 2007; 40: 658-674Crossref PubMed Scopus (15286) Google Scholar). The ligand-free structure (crystal form I) was solved using separate search models for MBP and a model of the PTH1R ECD with the N-terminal α-helix removed. The coordinates used were from our previously reported structure of MBP-PTH1R-ECD, PDB code 3C4M (31Pioszak A.A. Xu H.E. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5034-5039Crossref PubMed Scopus (226) Google Scholar). The ligand-bound structures (crystal forms II and III) were solved using separate search models for MBP and the CRFR1 ECD from the ligand-free structure (this work). The MR solutions were subjected to restrained refinement with Refmac5 (39Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (14027) Google Scholar). The 2Fo - Fc and Fo - Fc electron density maps from the refined MR solutions were all sufficiently clear as to obviate the need for density modification. The MR solutions were verified by clear electron density for the maltose molecule, which was not included in the MR search models, as well as clear density for the peptide ligand for crystal forms II and III. Iterative cycles of manual rebuilding in O (40Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. 1991; 47: 110-119Crossref PubMed Scopus (13055) Google Scholar) and restrained refinement with Refmac5 were used to finish the models. Noncrystallographic symmetry restraints were applied for crystal form II in the initial stages and gradually released as the model improved. TLS refinement was included for all three structures (41Winn M.D. Isupov M.N. Murshudov G.N. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 122-133Crossref PubMed Scopus (1660) Google Scholar). Two TLS groups corresponding to protein domains were used for crystal form I as follows: one for the MBP·maltose complex, and the other for the CRFR1 ECD. Six TLS groups were used for crystal form II as follows: one for each of the two MBP·maltose complexes, one for each of the two CRFR1 ECDs, and one for each of the two CRF peptides. Because of the low resolution of crystal form III, a single TLS group comprising the contents of the asymmetric unit was used. Water molecules were added to the form II structure using the ARP feature of CCP4 (37Collaborative Computational Project, Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19879) Google Scholar) in combination with Refmac5. Structure validation was performed with Procheck (42Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). The refinement statistics are summarized in Table 1. Amino Acid Sequence Alignments, Structure Analysis, and Figure Preparation—Amino acid sequence alignments were performed with ClustalW (43Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56679) Google Scholar) and the results displayed with ESpript (44Gouet P. Courcelle E. Stuart D.I. Metoz F. Bioinformatics (Oxf.). 1999; 15: 305-308Crossref PubMed Scopus (2560) Google Scholar). Structural alignments were performed using the align command in PyMol (45DeLano W. Py MOL Molecular Graphics System. Version 0.94, DeLano Scientific, Palo Alto, CA2002Google Scholar), with the alignments based on the core SCR fold of the ECD excluding the N-terminal α-helix, loop 1, and loop 2. Accessible surface area calculations were performed with the program Areaimol in the CCP4 suite (37Collaborative Computational Project, Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19879) Google Scholar). Structure figures were prepared wit

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Molecular Recognition of Corticotropin-releasing Factor by Its G-protein-coupled Receptor CRFR1