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• W2023689744 abstract "Photoaffinity labeling of receptors by bound agonists can provide important spatial constraints for molecular modeling of activated receptor complexes. Secretin is a 27-residue peptide hormone with a diffuse pharmacophoric domain that binds to the secretin receptor, a prototypic member of the Class B family of G protein-coupled receptors. In this work, we have developed, characterized, and applied two new photolabile probes for this receptor, with sites for covalent attachment in peptide positions 12 and 14, surrounding the previously most informative site of affinity labeling of this receptor. The [Tyr10,(BzBz)Lys12]rat secretin-27 probe covalently labeled receptor residue Val6, whereas the [Tyr10,(BzBz)Lys14]rat secretin-27 probe labeled receptor residue Pro38. When combined with previous photoaffinity labeling data, there are now seven independent sets of constraints distributed throughout the peptide and receptor amino-terminal domain that can be used together to generate a new molecular model of the ligand-occupied secretin receptor. The aminoterminal domain of this receptor presented a stable platform for peptide ligand interaction, with the amino terminus of the peptide hormone extended toward the transmembrane helix domain of the receptor. This provides clear insights into the molecular basis of natural ligand binding and supplies testable hypotheses regarding the molecular basis of activation of this receptor. Photoaffinity labeling of receptors by bound agonists can provide important spatial constraints for molecular modeling of activated receptor complexes. Secretin is a 27-residue peptide hormone with a diffuse pharmacophoric domain that binds to the secretin receptor, a prototypic member of the Class B family of G protein-coupled receptors. In this work, we have developed, characterized, and applied two new photolabile probes for this receptor, with sites for covalent attachment in peptide positions 12 and 14, surrounding the previously most informative site of affinity labeling of this receptor. The [Tyr10,(BzBz)Lys12]rat secretin-27 probe covalently labeled receptor residue Val6, whereas the [Tyr10,(BzBz)Lys14]rat secretin-27 probe labeled receptor residue Pro38. When combined with previous photoaffinity labeling data, there are now seven independent sets of constraints distributed throughout the peptide and receptor amino-terminal domain that can be used together to generate a new molecular model of the ligand-occupied secretin receptor. The aminoterminal domain of this receptor presented a stable platform for peptide ligand interaction, with the amino terminus of the peptide hormone extended toward the transmembrane helix domain of the receptor. This provides clear insights into the molecular basis of natural ligand binding and supplies testable hypotheses regarding the molecular basis of activation of this receptor. A detailed understanding of the molecular basis for agonist binding to receptors and their activation can provide key insights for the rational design and refinement of receptor-active drugs. The Class B family of guanine nucleotide-binding protein (G protein) 1The abbreviations used are: G proteinguanine nucleotide-binding proteinLys-Cendoproteinase Lys-CHAhemagglutinin(BzBz)Lysp-benzoylbenzoyl-l-lysineCHOChinese hamster ovarySecRsecretin receptorMES4-morpholineethanesulfonic acidr.m.s.d.root mean square deviation.-coupled receptors includes several very important potential drug targets, including receptors for parathyroid hormone, calcitonin, vasoactive intestinal polypeptide, and glucagon (1Ulrich C.D. Holtmann M.H. Miller L.J. Gastroenterology. 1998; 114: 382-397Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). The secretin receptor was the first member of this family to be cloned and is prototypic of this group of receptors (2Ishihara T. Nakamura S. Kaziro Y. Takahashi T. Takahashi K. Nagata S. EMBO J. 1991; 10: 1635-1641Crossref PubMed Scopus (369) Google Scholar). All of the natural ligands for the Class B G protein-coupled receptors, like secretin, represent moderately long linear peptide hormones that, by first principles, are quite flexible and provide substantial challenges for determination of the appropriate conformation for receptor docking. Although several of these ligands have had solution structures established by NMR (3Clore G.M. Nilges M. Brunger A. Gronenborn A.M. Eur. J. Biochem. 1988; 171: 479-484Crossref PubMed Scopus (39) Google Scholar), it is unclear that these are relevant to the conformations of these peptides when bound to the receptor. guanine nucleotide-binding protein endoproteinase Lys-C hemagglutinin p-benzoylbenzoyl-l-lysine Chinese hamster ovary secretin receptor 4-morpholineethanesulfonic acid root mean square deviation. Additionally, the amino-terminal domain of receptors in this family are large (greater than 120 residues), with complex topology, including three conserved disulfide bonds that are critical for ligand binding (1Ulrich C.D. Holtmann M.H. Miller L.J. Gastroenterology. 1998; 114: 382-397Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 4Asmann Y.W. Dong M.Q. Ganguli S. Hadac E.M. Miller L.J. Mol. Pharmacol. 2000; 58: 911-919Crossref PubMed Scopus (39) Google Scholar). Attempts to gain insight into these structures have come from studies in which the non-glycosylated amino-terminal regions of the parathyroid hormone receptor and the corticotropin-releasing factor receptor have been produced in Escherichia coli, denatured, refolded, and had their disulfide bonds determined directly (5Grauschopf U. Lilie H. Honold K. Wozny M. Reusch D. Esswein A. Schafer W. Rucknagel K.P. Rudolph R. Biochemistry. 2000; 39: 8878-8887Crossref PubMed Scopus (106) Google Scholar, 6Perrin 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 N.S. Vale W.W. J. Biol. Chem. 2001; 276: 31528-31534Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Of interest, the pattern of disulfide bonding for the amino terminus of the corticotropin-releasing factor receptor determined under these conditions was different from that evaluated previously by mutagenesis of the entire intact receptor (7Qi L.J. Leung A.T. Xiong Y. Marx K.A. Abou-Samra A.B. Biochemistry. 1997; 36: 12442-12448Crossref PubMed Scopus (69) Google Scholar). The recent analysis by Taylor et al. (8Taylor W.R. Munro R.E.J. Petersen K. Bywater R.P. Comput. Biol. Chem. 2003; 27: 103-114Crossref PubMed Scopus (15) Google Scholar) using ab initio modeling and careful evaluation of all permutations of disulfide bond connectivity identified a number of plausible patterns, with no unique solution. Thus, even the constraints for molecular modeling of disulfide bonding are not as certain as would be optimal. Using the direct approach of intrinsic photoaffinity labeling, we have previously established five distinct spatial approximations between residues in various positions throughout the pharmacophoric domain of secretin and its receptor (9Dong M. Wang Y. Pinon D.I. Hadac E.M. Miller L.J. J. Biol. Chem. 1999; 274: 903-909Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 10Dong M. Wang Y. Hadac E.M. Pinon D.I. Holicky E. Miller L.J. J. Biol. Chem. 1999; 274: 19161-19167Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 11Dong M. Asmann Y.W. Zang M. Pinon D.I. Miller L.J. J. Biol. Chem. 2000; 275: 26032-26039Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 12Dong M. Zang M.W. Pinon D.I. Li Z. Lybrand T.P. Miller L.J. Mol. Endocrinol. 2002; 16: 2490-2501Crossref PubMed Scopus (35) Google Scholar, 13Zang M.W. Dong M.Q. Pinon D.I. Ding X.Q. Hadac E.M. Li Z.J. Lybrand T.P. Miller L.J. Mol. Pharmacol. 2003; 63: 993-1001Crossref PubMed Scopus (36) Google Scholar). Of note, four of these five constraints, experimentally established for secretin peptide residues 6, 18, 22, and 26 (of 27 residues), covalently labeled residues within the first 36 residues at the distal end of the amino terminus of the receptor (9Dong M. Wang Y. Pinon D.I. Hadac E.M. Miller L.J. J. Biol. Chem. 1999; 274: 903-909Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 10Dong M. Wang Y. Hadac E.M. Pinon D.I. Holicky E. Miller L.J. J. Biol. Chem. 1999; 274: 19161-19167Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 11Dong M. Asmann Y.W. Zang M. Pinon D.I. Miller L.J. J. Biol. Chem. 2000; 275: 26032-26039Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 12Dong M. Zang M.W. Pinon D.I. Li Z. Lybrand T.P. Miller L.J. Mol. Endocrinol. 2002; 16: 2490-2501Crossref PubMed Scopus (35) Google Scholar). Therefore, this extensive set of data was only able to support a very limited and focused molecular model of this most distal region of the secretin receptor, and even that conformation was not firmly established (12Dong M. Zang M.W. Pinon D.I. Li Z. Lybrand T.P. Miller L.J. Mol. Endocrinol. 2002; 16: 2490-2501Crossref PubMed Scopus (35) Google Scholar). Only the most recent study, labeling through peptide residue 13, established a covalent bond with a distinct region of the amino terminus of the receptor, at residue 103 (13Zang M.W. Dong M.Q. Pinon D.I. Ding X.Q. Hadac E.M. Li Z.J. Lybrand T.P. Miller L.J. Mol. Pharmacol. 2003; 63: 993-1001Crossref PubMed Scopus (36) Google Scholar). This provided adequate information to propose a preliminary model of the entire amino-terminal domain of this receptor (13Zang M.W. Dong M.Q. Pinon D.I. Ding X.Q. Hadac E.M. Li Z.J. Lybrand T.P. Miller L.J. Mol. Pharmacol. 2003; 63: 993-1001Crossref PubMed Scopus (36) Google Scholar). As part of the current project, we have extensively refined and expanded that model, and have experimentally tested it by performing two additional series of photoaffinity labeling experiments with new probes on either side of this most informative site. These probes incorporated photolabile residues into positions 12 or 14. Both bound specifically and saturably to the secretin receptor where they stimulated full biological responses. Each of these probes also covalently labeled this receptor in single, unique, and distinct residues. These residues were quite close to the sites predicted by the working model, and were subsequently incorporated into this model to further refine it. The seven photoaffinity labeling cross-links, together with the three disulfide bonds in the receptor amino-terminal domain, constitute a significant set of topological constraints for the ligand-receptor complex. Although there is still considerable conformational flexibility in both peptide and receptor, this set of ten constraints reduces dramatically the number of viable three-dimensional models for the peptide-receptor complex that must be considered. The resulting models provide additional insight into the nature of the ligand-receptor interaction for Class B G protein-coupled receptors and suggest new experiments to further test the conformation and the molecular basis of agonist-induced activation of this receptor. Materials—The solid-phase oxidant, N-chlorobenzenesulfonamide (IODO-BEADs), cyanogen bromide, and m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester were purchased from Pierce Chemical Co. Phenylmethylsulfonyl fluoride, 3-isobutyl-1-methylxanthine, and N-(2-aminoethyl-1)-3-aminopropyl glass beads were from Sigma. Endoproteinase Lys-C (Lys-C) and the 12CA5 monoclonal antibody against the hemagglutinin (HA) epitope were from Roche Applied Science. Soybean trypsin inhibitor was from Worthington. Endoglycosidase F was prepared in our laboratory, as we described previously (14Pearson R.K. Miller L.J. Hadac E.M. Powers S.P. J. Biol. Chem. 1987; 262: 13850-13856Abstract Full Text PDF PubMed Google Scholar). All other reagents were of analytical grade. Peptides—The photolabile secretin probes used in this study were [Tyr10,(BzBz)Lys12]rat secretin-27 ((BzBz)Lys12 probe) and [Tyr10, (BzBz)Lys14]rat secretin-27 ((BzBz)Lys14 probe). They were designed to incorporate a photolabile residue p-benzoylbenzoyl-l-lysine ((BzBz)Lys) to replace Arg12 and Gln14 of secretin, respectively. Like photolabile secretin probes used previously (9Dong M. Wang Y. Pinon D.I. Hadac E.M. Miller L.J. J. Biol. Chem. 1999; 274: 903-909Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 10Dong M. Wang Y. Hadac E.M. Pinon D.I. Holicky E. Miller L.J. J. Biol. Chem. 1999; 274: 19161-19167Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 11Dong M. Asmann Y.W. Zang M. Pinon D.I. Miller L.J. J. Biol. Chem. 2000; 275: 26032-26039Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 12Dong M. Zang M.W. Pinon D.I. Li Z. Lybrand T.P. Miller L.J. Mol. Endocrinol. 2002; 16: 2490-2501Crossref PubMed Scopus (35) Google Scholar, 13Zang M.W. Dong M.Q. Pinon D.I. Ding X.Q. Hadac E.M. Li Z.J. Lybrand T.P. Miller L.J. Mol. Pharmacol. 2003; 63: 993-1001Crossref PubMed Scopus (36) Google Scholar), they both incorporated a Tyr residue at position 10 as a site for radioiodination. Together with other peptides used in this study, i.e. rat secretin-27, [Tyr10]rat secretin-27, and the HA peptide, they were synthesized by solid-phase techniques and purified to homogeneity by reversed-phase high-performance liquid chromatography (15Powers S.P. Pinon D.I. Miller L.J. Int. J. Pept. Protein. Res. 1988; 31: 429-434Crossref PubMed Scopus (99) Google Scholar). The expected molecular masses of the probes were verified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The radioligand used for binding (i.e. [Tyr10]rat secretin-27) and the (BzBz)Lys12 and the (BzBz)Lys14 probes were radioiodinated oxidatively with Na125I (PerkinElmer Life Sciences, Boston, MA). Upon exposure to the solid-phase oxidant, IODO-BEADs, for 15 s, the resulting product was purified by reversed-phase high-performance liquid chromatography to yield specific radioactivities of 2000 Ci/mmol (15Powers S.P. Pinon D.I. Miller L.J. Int. J. Pept. Protein. Res. 1988; 31: 429-434Crossref PubMed Scopus (99) Google Scholar). Receptor-bearing Cell Lines—Chinese hamster ovary (CHO) cell lines expressing the wild type secretin receptor (SecR) (16Ulrich C.D. Pinon D.I. Hadac E.M. Holicky E.L. Chang-Miller A. Gates L.K. Miller L.J. Gastroenterology. 1993; 105: 1534-1543Abstract Full Text PDF PubMed Scopus (68) Google Scholar), the HA-tagged secretin receptor (SecR-HA37 and SecR-HA79) (9Dong M. Wang Y. Pinon D.I. Hadac E.M. Miller L.J. J. Biol. Chem. 1999; 274: 903-909Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and various methionine mutants of the secretin receptor (SecR-A41M, SecR-V16M-HA37, and SecR-V13M-HA37) (10Dong M. Wang Y. Hadac E.M. Pinon D.I. Holicky E. Miller L.J. J. Biol. Chem. 1999; 274: 19161-19167Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 11Dong M. Asmann Y.W. Zang M. Pinon D.I. Miller L.J. J. Biol. Chem. 2000; 275: 26032-26039Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) were utilized as sources of receptor for this study, each having been well characterized previously. These stable CHO cell lines were cultured at 37 °C in a 5% CO2 environment on Falcon tissue culture plasticware in Ham's F-12 medium supplemented with 5% Fetal Clone-2 (HyClone Laboratories, Logan, UT). Cells were passaged twice a week and lifted mechanically before use. Development of a new secretin receptor mutant that incorporated an additional site for CNBr cleavage in a key position was necessary for the current work. This represented mutation of Pro8 to Met (P8M), prepared using an oligonucleotide-directed approach with the Quik-Change™ site-directed mutagenesis kit from Stratagene. The P8M secretin receptor construct was subcloned into the eukaryotic expression vector, pcDNA3 (Invitrogen, Carlsbad, CA), and its sequence was confirmed by direct DNA sequencing (17Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52741) Google Scholar). The P8M secretin receptor construct was expressed transiently in COS cells (American Type Cell Collection, Manassas, VA) after transfection using a modification of the DEAE-dextran method (18Holtmann M.H. Ganguli S. Hadac E.M. Dolu V. Miller L.J. J. Biol. Chem. 1996; 271: 14944-14949Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). These cells were harvested mechanically 72 h after transfection. Plasma membranes from receptor-bearing cells were prepared using a procedure that has been well described, which included cell disruption by Dounce homogenization and sucrose gradient ultracentrifugation (19Hadac E.M. Ghanekar D.V. Holicky E.L. Pinon D.I. Dougherty R.W. Miller L.J. Pancreas. 1996; 13: 130-139Crossref PubMed Scopus (101) Google Scholar). Ligand Binding—This assay was used for characterization of the newly synthesized photolabile probes that were used in the current study. Briefly, increasing concentrations of the (BzBz)Lys12 or (BzBz)Lys14 probe (from 0 to 1 μm) were incubated with a constant amount of radioligand 125I-[Tyr10]rat secretin-27, and 5 μg enriched CHO-SecR plasma membranes for 1 h at room temperature in Krebs-Ringer-HEPES (KRH) medium (25 mm HEPES, pH 7.4, 104 mm NaCl, 5 mm KCl, 1 mm KH2PO4, 1.2 mm MgSO4, 2 mm CaCl2) containing 0.01% soybean trypsin inhibitor, and 0.2% bovine serum albumin. After incubation, bound and free radioligand were separated using a Skatron cell harvester (Molecular Devices, Sunnyvale, CA) with receptor-binding glass-fiber filter mats that had been soaked in 0.3% Polybrene for 1 h, with the bound radioactivity being quantified in a γ-spectrometer. Non-specific binding was determined in the presence of 1 μm secretin and represented less than 20% of total radioligand in the incubation. The same assay was also utilized to characterize the binding activity of the COS cells transiently expressing the P8M secretin receptor construct. Binding curves were analyzed and plotted using the non-linear regression analysis program in the Prism software package (GraphPad Software, San Diego, CA). Binding kinetics were determined by analysis with the LIGAND program of Munson and Rodbard (20Munson P.J. Rodbard D. Anal. Biochem. 1980; 107: 220-239Crossref PubMed Scopus (7772) Google Scholar). Data are reported as the means ± S.E. of duplicate determinations from a minimum of three independent experiments. Biological Activity Assay—This was done by measuring the intracellular cAMP accumulation in CHO-SecR cells in response to stimulation by secretin or the photolabile analogues, representing either the (BzBz)Lys12 or the (BzBz)Lys14 probes, using reagents provided by Diagnostic Products Corp. (Los Angeles, CA). Briefly, Cells grown in 24-well plates were washed in ice-cold phosphate-buffered saline and stimulated by increasing concentrations (0–1 μm) of peptide for 30 min at 37 °C in KRH medium containing 1 mm 3-isobutyl-1-methylxanthine, 0.01% soybean trypsin inhibitor, 0.1% bacitracin, and 0.2% bovine serum albumin. After incubation, cells were lysed by 6% perchloric acid, and cell lysates were then adjusted to pH 6.0 by adding 30% KHCO3 and cleared by centrifugation before being introduced into the assay tubes, as described previously (21Ganguli S.C. Park C.G. Holtmann M.H. Hadac E.M. Kenakin T.P. Miller L.J. J. Pharm. Exp. Ther. 1998; 286: 593-598PubMed Google Scholar). Radioactivity was quantified by scintillation counting in a liquid scintillation Beckman LS6000 counter. Assays were performed in duplicate and repeated in at least three independent experiments. Receptor Photoaffinity Labeling—Covalent labeling of the secretin receptor was achieved as described previously (9Dong M. Wang Y. Pinon D.I. Hadac E.M. Miller L.J. J. Biol. Chem. 1999; 274: 903-909Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In brief, 50 μg of enriched receptor-bearing plasma membranes were incubated with 0.1 nm125I-[Tyr10,(BzBz)Lys12]rat secretin-27 or 125I-[Tyr10,(BzBz)Lys14]rat secretin-27 in the presence of increasing concentrations of secretin (from 0 to 1 μm) in KRH buffer in the dark for 1 h at room temperature. This was then photolyzed for 30 min at 4 °C in a Rayonet photochemical reactor (Southern New England Ultraviolet Company, Hamden, CT) equipped with 3500-Å lamps. Membranes were then washed, solubilized in Laemmli SDS sample buffer, and resolved by 10% SDS-polyacrylamide gels (22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207445) Google Scholar). Labeled proteins were visualized by autoradiography. To scale up labeled receptor for further purification and peptide mapping, larger amounts of receptor-bearing membranes (200 μg) and 125I-[Tyr10,(BzBz)Lys12]rat secretin-27 or 125I-[Tyr10,(BzBz)Lys14]rat secretin-27 (0.5 nm) were incubated in the absence of competing secretin prior to photolysis. Peptide Mapping of the Site of Covalent Labeling—After gel electrophoresis, labeled bands prepared in large scale were cut out, eluted, lyophilized, and ethanol-precipitated, before being used for deglycosylation and peptide mapping by chemical and enzymatic cleavage. Deglycosylation of labeled secretin receptor was performed with endoglycosidase F under the conditions we previously reported (19Hadac E.M. Ghanekar D.V. Holicky E.L. Pinon D.I. Dougherty R.W. Miller L.J. Pancreas. 1996; 13: 130-139Crossref PubMed Scopus (101) Google Scholar). CNBr and endoproteinase Lys-C were used separately and in sequence to cleave the labeled secretin receptor and its fragments, following procedures described previously (9Dong M. Wang Y. Pinon D.I. Hadac E.M. Miller L.J. J. Biol. Chem. 1999; 274: 903-909Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Products of cleavage were resolved on 10% NuPAGE gels using MES buffer system (Invitrogen), with labeled products being visualized by autoradiography. The apparent molecular masses of labeled receptor fragments were determined by interpolation on a plot of the mobility of Multimark™ protein standards (Invitrogen) versus the log values of their apparent masses. Immunoprecipitation of labeled intact and digested HA-tagged receptor constructs using anti-HA monoclonal antibody was performed to determine the identities of some of the labeled fragments using procedures that we described previously (13Zang M.W. Dong M.Q. Pinon D.I. Ding X.Q. Hadac E.M. Li Z.J. Lybrand T.P. Miller L.J. Mol. Pharmacol. 2003; 63: 993-1001Crossref PubMed Scopus (36) Google Scholar). Identification of the Covalently Labeled Residues—Purified CNBr and endoproteinase Lys-C fragments from the radiolabeled secretin receptor were utilized for identification of the residues labeled with the (BzBz)Lys12 and the (BzBz)Lys14 probes by radiochemical Edman degradation sequencing. Receptor fragments containing cysteine residues were immobilized through their sulfhydryl groups utilizing the bifunctional cross-linker, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, and N-(2-aminoethyl-1)-3-aminopropyl glass beads. Edman degradation was manually repeated up to eight cycles, in a manner that has been previously reported in detail (23Ji Z. Hadac E.M. Henne R.M. Patel S.A. Lybrand T.P. Miller L.J. J. Biol. Chem. 1997; 272: 24393-24401Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 24Hadac E.M. Pinon D.I. Ji Z. Holicky E.L. Henne R.M. Lybrand T.P. Miller L.J. J. Biol. Chem. 1998; 273: 12988-12993Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), and the radioactivity released in each cycle was quantified in a γ-spectrometer. Molecular Modeling of the Receptor Amino-terminal Region—Multiple sequence alignment was performed for Class B G protein-coupled receptors to ascertain those family members with significant sequence identity (>30%) and sequence similarity (>50%) to the amino-terminal domain of the secretin receptor. The NCBI, SwissProt, and EMBL databases were then searched for homologues of each of the closely related Class B family amino-terminal domains. Homologues with experimentally determined three-dimensional structures were assessed for suitability as templates in a homology modeling exercise using structure-based sequence alignment of the secretin receptor amino terminus. Three-dimensional homology models for the rat secretin receptor amino terminus were then built. Multiple sequence alignments were generated using the AMPS package (25Livingstone C.D. Barton G.J. Comput. Appl. Biosci. 1993; 9: 745-756PubMed Google Scholar), and FASTA3 was used for all sequence data base searches (26Pearson W.R. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2444-2448Crossref PubMed Scopus (9388) Google Scholar). Structure-based sequence alignments were generated using the program MOE with the Blossum62 scoring matrix (27Chemical Computing Group (2001) MOE 2001.01, Chemical Computing Group, Inc., Montreal, CanadaGoogle Scholar). Homology models were created using Modeler (28Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10613) Google Scholar). Final models were assessed for stereochemical quality and side-chain packing profiles using the PROCHECK (29Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) and QPACK programs (30Gregoret L.M. Cohen F.E. J. Mol. Biol. 1990; 211: 959-974Crossref PubMed Scopus (119) Google Scholar). Ligand Docking to the Receptor Amino-terminal Domain—The NMR-derived solution structure of secretin (3Clore G.M. Nilges M. Brunger A. Gronenborn A.M. Eur. J. Biochem. 1988; 171: 479-484Crossref PubMed Scopus (39) Google Scholar) was manually docked close to its putative binding site in the three-dimensional model, guided by our previous photoaffinity labeling data (9Dong M. Wang Y. Pinon D.I. Hadac E.M. Miller L.J. J. Biol. Chem. 1999; 274: 903-909Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 10Dong M. Wang Y. Hadac E.M. Pinon D.I. Holicky E. Miller L.J. J. Biol. Chem. 1999; 274: 19161-19167Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 11Dong M. Asmann Y.W. Zang M. Pinon D.I. Miller L.J. J. Biol. Chem. 2000; 275: 26032-26039Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 12Dong M. Zang M.W. Pinon D.I. Li Z. Lybrand T.P. Miller L.J. Mol. Endocrinol. 2002; 16: 2490-2501Crossref PubMed Scopus (35) Google Scholar, 13Zang M.W. Dong M.Q. Pinon D.I. Ding X.Q. Hadac E.M. Li Z.J. Lybrand T.P. Miller L.J. Mol. Pharmacol. 2003; 63: 993-1001Crossref PubMed Scopus (36) Google Scholar). The docked complex model was then subjected to 100 steps of in vacuo energy minimization with no constraints, followed by brief (5 ps), low temperature (40 K) molecular dynamics simulations using a generalized Born model. Five constraints (Table I) were applied to impose peptide-receptor contacts determined from previous photoaffinity labeling experiments during the MD simulation, using a harmonic restraining potential with a 20 kcal/mol/Å force constant. The last configuration from the simulation was then energy minimized to generate a final structure for the complex. This same protocol was used to refine additional models that included the three putative disulfide bonds as extra topological constraints (5Grauschopf U. Lilie H. Honold K. Wozny M. Reusch D. Esswein A. Schafer W. Rucknagel K.P. Rudolph R. Biochemistry. 2000; 39: 8878-8887Crossref PubMed Scopus (106) Google Scholar, 6Perrin 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 N.S. Vale W.W. J. Biol. Chem. 2001; 276: 31528-31534Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar).Table IDocking constraints derived from secretin receptor photoaffinity labeling studiesPositions of photolabile residues within secretin peptideCovalently labeled residues within the secretin receptorRef.Phe6Val410Dong M. Wang Y. Hadac E.M. Pinon D.I. Holicky E. Miller L.J. J. Biol. Chem. 1999; 274: 19161-19167Abstract Full Text Full Text PDF PubMed Scopus (67) Google ScholarLeu13Val10313Zang M.W. Dong M.Q. Pinon D.I. Ding X.Q. Hadac E.M. Li Z.J. Lybrand T.P. Miller L.J. Mol. Pharmacol. 2003; 63: 993-1001Crossref PubMed Scopus (36) Google ScholarArg18Arg1412Dong M. Zang M.W. Pinon D.I. Li Z. Lybrand T.P. Miller L.J. Mol. Endocrinol. 2002; 16: 2490-2501Crossref PubMed Scopus (35) Google ScholarLeu22Leu179Dong M. Wang Y. Pinon D.I. Hadac E.M. Miller L.J. J. Biol. Chem. 1999; 274: 903-909Abstract Full Text Full Text PDF PubMed Scopus (82) Google ScholarLeu26Leu3611Dong M. Asmann Y.W. Zang M. Pinon D.I. Miller L.J. J. Biol. Chem. 2000; 275: 26032-26039Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar Open table in a new tab Finally, distance geometry calculations were used to search for alternate three-dimensional structures for the ligand-receptor complex that might also satisfy the photoaffinity labeling cross-links and disulfide bond constraints equally well. Both metric matrix and random embedding methods were used to generate three-dimensional structures. For the random embedding procedure, 75,000 iterations were employed for the embedding process, and 500 independent structures were generated by varying the initial seed value. To test the dependence of final structures on starting model conformations, fully extended and various random, compact initial conformations were used for both the receptor amino-terminal and peptide hormone fragments. After embedding, resultant structures were refined with 200 steps of energy minimization. In addition to the ten experimental constraints from cross-linking and disulfide binding patterns, empirical distance constraints were included to impose trans peptide bond conformations for the entire structure." @default.
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