FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2085228715> ?p ?o ?g. }

• W2085228715 endingPage "7775" @default.
• W2085228715 startingPage "7763" @default.
• W2085228715 abstract "Complement factor 5a (C5a) is an anaphylatoxin that acts by binding to a G protein-coupled receptor, the C5aR. The relative orientation of this ligand-receptor pair is investigated here using the novel technique of disulfide trapping by random mutagenesis (DTRM) and molecular modeling. In the DTRM technique, an unpaired cysteine is introduced in the ligand, and a library of randomly mutagenized receptors is screened to identify mutants that introduce a cysteine at a position in the receptor that allows functional interactions with the ligand. By repeating this analysis at six positions of C5a, we identify six unique sets of intermolecular interactions for the C5a-C5aR complex, which are then compared with an independently developed computational three-dimensional model of the complex. This analysis reveals that the interface of the receptor N terminus with the cysteine-containing ligand molecules is selected from a variety of possible receptor conformations that exist in dynamic equilibrium. In contrast, DTRM identifies a single position in the second extracellular loop of the receptor that interacts specifically with a cysteine probe placed in the C-terminal tail of the C5a ligand. Complement factor 5a (C5a) is an anaphylatoxin that acts by binding to a G protein-coupled receptor, the C5aR. The relative orientation of this ligand-receptor pair is investigated here using the novel technique of disulfide trapping by random mutagenesis (DTRM) and molecular modeling. In the DTRM technique, an unpaired cysteine is introduced in the ligand, and a library of randomly mutagenized receptors is screened to identify mutants that introduce a cysteine at a position in the receptor that allows functional interactions with the ligand. By repeating this analysis at six positions of C5a, we identify six unique sets of intermolecular interactions for the C5a-C5aR complex, which are then compared with an independently developed computational three-dimensional model of the complex. This analysis reveals that the interface of the receptor N terminus with the cysteine-containing ligand molecules is selected from a variety of possible receptor conformations that exist in dynamic equilibrium. In contrast, DTRM identifies a single position in the second extracellular loop of the receptor that interacts specifically with a cysteine probe placed in the C-terminal tail of the C5a ligand. One of the most biologically important families of receptors is the G protein-coupled receptors (GPCRs), 2The abbreviations used are: GPCRG protein-coupled receptorC5acomplement factor 5aC5aRcomplement factor 5a receptorTMtransmembrane helixECextracellular loopNTN terminusDTRMdisulfide trapping by random mutagenesisPIPES1,4-piperazinediethanesulfonic acid. which eukaryotic cells use to sense signals as diverse as light, odorants, small molecules, and polypeptide hormones (1Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1236) Google Scholar). The GPCRs, which number ∼1000 in the human genome (2Fredriksson R. Schioth H.B. Mol. Pharmacol. 2005; 67: 1414-1425Crossref PubMed Scopus (468) Google Scholar), have a shared topology made up of seven transmembrane domains (TMs) linked by intracellular and extracellular (EC) loops, along with an extracellular N terminus and intracellular C terminus (Fig. 1). These receptors also have a shared mechanism of action, whereby the activated receptor serves as a guanosine exchange factor on the α subunit of a heterotrimeric G protein, thus transmitting the signal to the interior of the cell. Many drugs are directed against GPCRs, including adrenergic, muscarinic, dopaminergic, serotonergic, GABAergic, and histaminergic receptors. Taken together, drugs acting on GPCRs make up an estimated 50% of the current pharmacopoeia (3Drews J. Science. 2000; 287: 1960-1964Crossref PubMed Scopus (2285) Google Scholar). G protein-coupled receptor complement factor 5a complement factor 5a receptor transmembrane helix extracellular loop N terminus disulfide trapping by random mutagenesis 1,4-piperazinediethanesulfonic acid. The structural basis of receptor-ligand interaction is of great interest in both basic and applied pharmacology. In the case of GPCRs, a central question is how receptors with a single common topology can be activated by such a wide variety of ligands. A related question is how specificity is built into these receptors, so that each is activated only by the appropriate ligands. Many GPCRs are activated by polypeptide ligands, including chemokines such as SDF-1 (CXCL12), physiologic modulators such as angiotensin II, glycoprotein hormones such as luteinizing hormone, and chemotactic factors such as complement factor 5a (C5a). These ligands are too large to fit entirely in an interhelical cleft of a GPCR, so their bulk must serve some adaptive function other than receptor activation per se. In some cases, the ligand is recruited by its affinity for the receptor extracellular domains, and a second discrete domain of the ligand then activates the switch mechanism. Alternatively, some large ligands activate their receptors by interacting with large extracellular domains (as in the case of glycoprotein hormone receptors) (4Fan Q.R. Hendrickson W.A. Nature. 2005; 433: 269-277Crossref PubMed Scopus (481) Google Scholar), or by serving as a protease to cleave the N terminus of the receptor (as in the case of the thrombin receptor) (5Ji T.H. Grossmann M. Ji I. J. Biol. Chem. 1998; 273: 17299-17302Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar). In this study we examine the nature of the interaction between C5a and the N-terminal segment of its receptor, C5aR. C5a is a 74-amino acid polypeptide that is proteolytically elaborated by the serum complement cascade at sites of inflammation; it serves as a chemotactic factor for neutrophils, which express the C5aR (6Gerard N.P. Gerard C. Nature. 1991; 349: 614-617Crossref PubMed Scopus (569) Google Scholar). As a potent anaphylatoxin, C5a has been examined as a possible therapeutic target in sepsis, arthritis, and other inflammatory states (7Allegretti M. Moriconi A. Beccari A.R. Di Bitondo R. Bizzarri C. Bertini R. Colotta F. Curr. Med. Chem. 2005; 12: 217-236Crossref PubMed Scopus (76) Google Scholar). The C5a/C5aR interaction has previously been described by a two-site model (8Siciliano S.J. Rollins T.E. DeMartino J. Konteatis Z. Malkowitz L. Van Riper G. Bondy S. Rosen H. Springer M.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1214-1218Crossref PubMed Scopus (221) Google Scholar, 9Mollison K.W. Mandecki W. Zuiderweg E.R. Fayer L. Fey T.A. Krause R.A. Conway R.G. Miller L. Edalji R.P. Shallcross M.A. Lane B. Fox J.L. Greer J. Carter G.W. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 292-296Crossref PubMed Scopus (119) Google Scholar, 10DeMartino J.A. Van Riper G. Siciliano S.J. Molineaux C.J. Konteatis Z.D. Rosen H. Springer M.S. J. Biol. Chem. 1994; 269: 14446-14450Abstract Full Text PDF PubMed Google Scholar). The first interaction is between the N terminus of the C5aR and undetermined components of the C5a ligand; the C-terminal tail of C5a then enters an interhelical pocket of the C5aR to form the second-site interaction. As evidence for the role of the first-site interaction, truncation of the N terminus reduces the affinity of the receptor for full-length ligand, but preserves the efficacy of hexapeptide analogs of the C5a C-terminal tail (8Siciliano S.J. Rollins T.E. DeMartino J. Konteatis Z. Malkowitz L. Van Riper G. Bondy S. Rosen H. Springer M.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1214-1218Crossref PubMed Scopus (221) Google Scholar). Conversely, the small molecule L-584,020 competes with full-length C5a but not with hexapeptides (8Siciliano S.J. Rollins T.E. DeMartino J. Konteatis Z. Malkowitz L. Van Riper G. Bondy S. Rosen H. Springer M.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1214-1218Crossref PubMed Scopus (221) Google Scholar), suggesting that L-584,020 inhibits binding but not activation. Furthermore, NMR spectroscopy has shown that the structure of the isolated C5aR N terminus is altered by incubation with C5a, suggesting that the N terminus of the receptor interacts with the ligand (11Chen Z. Zhang X. Gonnella N.C. Pellas T.C. Boyar W.C. Ni F. J. Biol. Chem. 1998; 273: 10411-10419Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Because hexapeptide analogs such as W5Cha are full agonists for the C5aR, the first site is apparently not a key component of the switch mechanism (8Siciliano S.J. Rollins T.E. DeMartino J. Konteatis Z. Malkowitz L. Van Riper G. Bondy S. Rosen H. Springer M.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1214-1218Crossref PubMed Scopus (221) Google Scholar, 11Chen Z. Zhang X. Gonnella N.C. Pellas T.C. Boyar W.C. Ni F. J. Biol. Chem. 1998; 273: 10411-10419Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 12Hagemann I.S. Narzinski K.D. Floyd D.H. Baranski T.J. J. Biol. Chem. 2006; 281: 36783-36792Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 13Mery L. Boulay F. Eur. J. Haematol. 1993; 51: 282-287Crossref PubMed Scopus (22) Google Scholar, 14Mery L. Boulay F. J. Biol. Chem. 1994; 269: 3457-3463Abstract Full Text PDF PubMed Google Scholar). Instead, the first site confers “address”-like specificity on the interaction. The second site involves the C terminus of C5a and an interhelical pocket of the C5aR (15Gerber B.O. Meng E.C. Dotsch V. Baranski T.J. Bourne H.R. J. Biol. Chem. 2001; 276: 3394-3400Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 16Higginbottom A. Cain S.A. Woodruff T.M. Proctor L.M. Madala P.K. Tyndall J.D. Taylor S.M. Fairlie D.P. Monk P.N. J. Biol. Chem. 2005; 280: 17831-17840Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Hexapeptide analogs of the C5a C terminus can serve as full agonists or as antagonists for the receptor, albeit with decreased potency (17Kawai M. Quincy D.A. Lane B. Mollison K.W. Or Y.S. Luly J.R. Carter G.W. J. Med. Chem. 1992; 35: 220-223Crossref PubMed Scopus (39) Google Scholar), and this activity can be modulated by making mutations in the interhelical pocket (15Gerber B.O. Meng E.C. Dotsch V. Baranski T.J. Bourne H.R. J. Biol. Chem. 2001; 276: 3394-3400Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 18DeMartino J.A. Konteatis Z.D. Siciliano S.J. Van Riper G. Underwood D.J. Fischer P.A. Springer M.S. J. Biol. Chem. 1995; 270: 15966-15969Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), suggesting that the receptor switch mechanism is actuated by the “message” delivered as a consequence of the first-site interaction providing the correct address (19Kolakowski Jr., L.F. Lu B. Gerard C. Gerard N.P. J. Biol. Chem. 1995; 270: 18077-18082Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In a recent study, we used random saturation mutagenesis of the C5aR to demonstrate that no single residue of the N terminus is responsible for ligand affinity. Rather, the affinity is built from multiple individually weak interactions (12Hagemann I.S. Narzinski K.D. Floyd D.H. Baranski T.J. J. Biol. Chem. 2006; 281: 36783-36792Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). In our random mutagenesis experiments, mutations were introduced into multiple positions within the receptor region of interest; receptors that retained signaling ability despite their multiple mutations were selected by a functional screen in Saccharomyces cerevisiae. Surprisingly, we found that positions 24–30 of the C5aR showed a marked propensity to change to cysteine in functional mutants. Because human C5a contains an unpaired cysteine that is solvent-accessible, we reasoned that the cysteine in the ligand could serve as a disulfide-trapping “bait” to identify interacting regions of the receptor, using a library of mutant receptors containing cysteines as “prey.” We refer to this technique as “disulfide trapping by random mutagenesis,” or DTRM. In principle, it should be generally applicable to a wide range of protein-protein interactions. In the work described here, we use DTRM to study the interaction between C5a and the C5aR. Specifically, we introduce an unpaired cysteine at various positions in the C5a ligand, then identify regions of the receptor that are susceptible to forming disulfide bonds with the mutant ligand. The results provide a series of constraints on the receptor-ligand interaction, allowing it to be described at a new level of molecular detail. Yeast Strains—Yeast strain BY1142 (20Baranski T.J. Herzmark P. Lichtarge O. Gerber B.O. Trueheart J. Meng E.C. Iiri T. Sheikh S.P. Bourne H.R. J. Biol. Chem. 1999; 274: 15757-15765Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) has the genotype MATα far1Δ1442 tbt1-1 PFUS1-HIS3 can1 ste14::trp1::LYS2 ste3Δ1156 gpa1(41)-Gαi3 lys2 ura3 leu2 trp1 his3 ade2. This strain expresses a chimeric Gα subunit: the first 41 residues of the endogenous yeast Gα subunit Gpa1 are substituted for the first 33 residues of human Gαi3. The result is a predominantly human Gα subunit that can be activated by mammalian receptors, but can also signal to downstream components of the yeast mitogen-activated protein kinase pathway (21Dohlman H.G. Thorner J.W. Annu. Rev. Biochem. 2001; 70: 703-754Crossref PubMed Scopus (356) Google Scholar). Furthermore, the mitogen-activated protein kinase mating cascade is engineered so that G protein activation causes transcription of the PFUS1-HIS3 reporter. Thus, G protein or receptor activity allows the yeast to grow on histidine-deficient medium. Strain BY1173 has the genotype MATa ura3 leu2 trp1 his3 can1 gpa1Δ:ade2Δ::3xHA far1Δ::ura3Δ fus1::PFUS1-HIS3 LEU2::PFUS1-LacZ sst2Δ::ura3Δ ste2Δ::G418R trp1::GPA1/Gαi1 (22Brown A.J. Dyos S.L. Whiteway M.S. White J.H. Watson M.A. Marzioch M. Clare J.J. Cousens D.J. Paddon C. Plumpton C. Romanos M.A. Dowell S.J. Yeast. 2000; 16: 11-22Crossref PubMed Scopus (152) Google Scholar); in this strain, the final 5 residues of Gpa1 are replaced by the corresponding residues of human Gαi1, again to provide a signaling assay for mammalian receptors. The integrated PFUS1-LacZ reporter causes G protein activation to elicit β-galactosidase expression. Yeast were transformed by the standard lithium acetate procedure and grown at 30 °C. Construction of Mutant Ligands—Point mutations were introduced into the wild-type C5a ligand by site-directed mutagenesis using Pfu Turbo polymerase (Stratagene). Plasmid construction was verified by sequencing (Protein and Nucleic Acid Chemistry Laboratory, Washington University School of Medicine, St. Louis, MO). β-Galactosidase Assay—Yeast BY1173 transformed with wild-type C5aR and ligand plasmids or empty vector was grown in suspension at 30 °C under selective conditions. To assess yeast density after overnight growth, the A600 of each culture was measured using a Spectronic-20 spectrophotometer (Bausch & Lomb). All cultures were assayed at A600 of ∼0.15. For each culture, a standard quantity of yeast (15 μl divided by the A600) was seeded into a 96-well microtiter plate containing yeast growth medium (to 100 μl) and lysis/substrate buffer containing the final concentrations of 0.5% Triton X-100, 1 mg/ml chlorophenol red β-d-galactopyranoside, and 25 mm PIPES, pH 6.8. The quantity of yeast was sufficiently small that there was no turbidity visible in the wells. The assay plate was sealed and incubated at 37 °C. After 1 h, color development was halted by addition of Na2CO3 to 0.2 m, and A570 was measured on a Bio-Rad Model 680 microplate reader. Library Construction and Screening—Libraries of mutant C5aRs were generated as described previously (12Hagemann I.S. Narzinski K.D. Floyd D.H. Baranski T.J. J. Biol. Chem. 2006; 281: 36783-36792Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 23Klco J.M. Wiegand C.B. Narzinski K. Baranski T.J. Nat. Struct. Mol. Biol. 2005; 12: 320-326Crossref PubMed Scopus (138) Google Scholar). Briefly, the nucleotide sequence of the region of interest (NT or EC2) was flanked by silent restriction sites, then replaced by a noncoding “stuffer” sequence to prevent wild-type contamination of the library. Double-stranded oligonucleotides doped with mutations at a frequency of 20% (Integrated DNA Technologies, Coralville, IA) were subcloned into the appropriate sites of C5aR to create libraries of >105 mutant receptors. For the NT library, residues 2–32 of the C5aR were mutagenized; for the EC2 library, the mutations spanned residues 175–199. We screened the libraries for receptors whose behavior suggested that they made disulfide interactions with cysteine-containing ligands. To accomplish this, yeast BY1142 was cotransformed with a receptor library (ADE2 vector) and with a C5a mutant ligand plasmid (URA3 vector), then grown under Ade-Ura- selection. After 2 days of growth, colonies were replica-plated onto His-Ura- plates with 1 mm Ade to assay them for receptor signaling. Functional receptors conferred histidine prototrophy and allowed growth on this medium; furthermore, the strength of signaling was assessed by replica plating on media containing increasing concentrations of 3-amino-1,2,4-triazole, a competitive inhibitor of the HIS3 gene product. Growth on 3-amino-1,2,4-triazole was considered to be receptor-dependent if colonies maintained their white color, indicating continued adenine prototrophy. Colonies that lost their receptor plasmid became reliant on scarce environmental adenine, therefore either failing to grow or producing a red pigment that was cause for exclusion from our screen. Plasmids conferring ligand-dependent growth on 3-amino-1,2,4-triazole were selected for further analysis. The mutant receptor plasmids were isolated from these colonies by plasmid rescue, sequenced, and resubjected to the growth assay to confirm their phenotypes. Receptors were also assayed for their ability to be activated by C5a C27R. Those receptors that were activated by the cysteine-containing ligand but not by the “Cys-less” ligand C5a C27R were considered to be hits in our screen. Statistical Methods—The number of cysteines expected at each position under the null hypothesis (absence of any selective pressure favoring cysteine) was calculated as described in the supplementary materials. Expected and actual values were compared by the two-tailed Fisher's exact test (GraphPad Software) with expectation values rounded to the nearest integer. Molecular Modeling—The building of the three-dimensional model of the complex between C5aR and C5a, which followed general procedures developed earlier (24Nikiforovich G.V. Marshall G.R. Biochemistry. 2003; 42: 9110-9120Crossref PubMed Scopus (44) Google Scholar, 25Nikiforovich G.V. Marshall G.R. Biophys. J. 2005; 89: 3780-3789Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 26Matsumoto M.L. Narzinski K. Kiser P.D. Nikiforovich G.V. Baranski T.J. J. Biol. Chem. 2007; 282: 3105-3121Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar), is described in detail elsewhere. 3Nikiforovich, G. V., Marshall, G. R., and Baranski, T. J. (2008) Biochemistry, in press. Briefly, energy calculations were performed by minimizing the energy of atom-atom interactions between the molecular fragments, such as fragments of C5a or C5aR, involved in each calculation. During minimization, the fragments were allowed to move as rigid bodies in six dimensions each (three rotations and three translations). The dihedral angles of the side chains (but not of the backbone) were also involved in minimization. Also, at each step of minimization (several times during the process), the side chains were repacked to yield optimal positions before starting minimization itself. C5aR—A three-dimensional model of the TM region of human C5aR was developed earlier (26Matsumoto M.L. Narzinski K. Kiser P.D. Nikiforovich G.V. Baranski T.J. J. Biol. Chem. 2007; 282: 3105-3121Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar) using the x-ray structure of rhodopsin as a template with the transmembrane helices defined as Ile38–Ala63 (TM1), Asn71–Glu98 (TM2), Ala107–Val138 (TM3), Ala150–Phe172 (TM4), Glu199–Phe224 (TM5), Arg236–Phe267 (TM6), and Leu281–Tyr300 (TM7). All realistic low-energy conformations of the EC loops were restored independently according to a modeling procedure developed earlier (25Nikiforovich G.V. Marshall G.R. Biophys. J. 2005; 89: 3780-3789Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The EC loops were defined as EC1 (His99–Gly106), EC2 (Leu173–Arg198), and EC3 (Leu268–Lys280); a total of 29 low-energy options for the conformers of the “package” EC1 + EC2 + EC3 were found. The package was mounted onto the TM region of C5aR and the “most open” conformation of the loops (that with the largest distance between the “tips” of EC2 (Cα177) and EC3 (Cα276)) was selected for the three-dimensional model of the TM region + EC loops of C5aR used in further docking of C5a and its fragments. C5a-(59–74) and Docking—First, fragment C5a-(65–69) was docked within an opening in the three-dimensional model that allowed access to Glu199, Arg206, and Asp282, which are the residues suggested as possible contacts to C5a by site-directed mutagenesis (see e.g. Ref. 7Allegretti M. Moriconi A. Beccari A.R. Di Bitondo R. Bizzarri C. Bertini R. Colotta F. Curr. Med. Chem. 2005; 12: 217-236Crossref PubMed Scopus (76) Google Scholar). The backbone conformation of C5a-(65–69) was extracted from the NMR/calculation data on YSFKPMPLaR, a known agonist of C5aR (27Finch A.M. Vogen S.M. Sherman S.A. Kirnarsky L. Taylor S.M. Sanderson S.D. J. Med. Chem. 1997; 40: 877-884Crossref PubMed Scopus (50) Google Scholar, 28Vogen S.M. Finch A.M. Wadi S.K. Thatcher J. Monk P.N. Taylor S.M. Sanderson S.D. J. Pept. Res. 1999; 53: 8-17Crossref PubMed Scopus (20) Google Scholar). Docking was performed by a systematic search on the six-dimensional grid (three translations and three rotations) that covered a total of 2,268 starting orientations of C5a-(65–69) within the TM region of C5aR. Twenty low-energy orientations of C5a-(65–69) without steric clashes with the EC loops (Cα-Cα distances ≥ 4 Å) were selected for further consideration. Then, all low-energy conformations of fragment C5a-(59–74) were obtained assuming that (i) backbone conformations of C5a-(65–69) remained the same; (ii) the backbone conformation of C5a-(59–62) was the same as revealed by NMR data for C5a (the PDB entry 1KJS (29Zuiderweg E.R. Nettesheim D.G. Mollison K.W. Carter G.W. Biochemistry. 1989; 28: 172-185Crossref PubMed Scopus (97) Google Scholar, 30Zhang X. Boyar W. Galakatos N. Gonnella N.C. Protein Sci. 1997; 6: 65-72Crossref PubMed Scopus (21) Google Scholar)); (iii) the backbone conformations of C5a-(63–65) and -(70–74) might vary, but the distance between Cα59 and Cα74 was kept greater than about 22 Å (otherwise it would be impossible to extend the structure to the entire C5a). The obtained conformations of C5a-(59–74) were checked as to possible steric clashes with C5aR for all selected low-energy orientations of C5a-(65–69) and, in turn, subjected to energy minimization. Four types of low-energy orientations of C5a-(59–74) possessed no steric clashes with C5aR and were used for placing the entire C5a within C5aR. Entire Complex of C5a and C5aR—Independently, energy calculations were performed for the N-terminal segment 8–41 of C5aR (NT 8–41) resulting in 185 low-energy conformations of the peptide backbone. (The N-terminal segment 1–7 was not included due to difficulties in modeling the carbohydrate moiety at the N-glycosylated site.) When mounted onto the three-dimensional model of C5aR (onto the N-terminal residues 38–41 of TM1), only 44 conformations of NT 8–41 possessed no steric clashes with C5aR. Then, possible orientations of the entire structure of C5a were restored by overlapping the rigid core C5a-(1–62) from PDB entry 1KJS with each of the four selected orientations of C5a-(59–74) within C5aR. It was found that only one specific orientation of C5a satisfied the requirements of spatial proximity of positions 15, 18, 19, 20, 27, and 46 in C5a to specific residues in the N-terminal segment 8–41 of C5aR, as was suggested by available site-directed mutagenesis data (31Bubeck P. Grotzinger J. Winkler M. Kohl J. Wollmer A. Klos A. Bautsch W. Eur. J. Biochem. 1994; 219: 897-904Crossref PubMed Scopus (39) Google Scholar, 32Toth M.J. Huwyler L. Boyar W.C. Braunwalder A.F. Yarwood D. Hadala J. Haston W.O. Sills M.A. Seligmann B. Galakatos N. Protein Sci. 1994; 3: 1159-1168Crossref PubMed Scopus (42) Google Scholar). Seventeen conformations of NT 8–41 (out of 185) and 11 conformations of the EC1 + EC2 + EC3 loop package (out of 29) did not show steric clashes with this particular orientation of C5a; all these conformations together with the selected orientation of C5a and the TM region of C5aR make up the final model of the C5a-C5aR complex. Generation of Mutant C5a Ligands with Unpaired Sulfhydryls—The wild-type C5a ligand contains seven cysteine residues, of which six (Cys-21, -22, -34, -47, -54, and -55) are involved in intramolecular disulfide bonds that stabilize an α-helical bundle (Fig. 2A). The seventh cysteine (Cys27) has an outwardly directed side chain (29Zuiderweg E.R. Nettesheim D.G. Mollison K.W. Carter G.W. Biochemistry. 1989; 28: 172-185Crossref PubMed Scopus (97) Google Scholar, 30Zhang X. Boyar W. Galakatos N. Gonnella N.C. Protein Sci. 1997; 6: 65-72Crossref PubMed Scopus (21) Google Scholar) that was previously used for disulfide trapping analysis (12Hagemann I.S. Narzinski K.D. Floyd D.H. Baranski T.J. J. Biol. Chem. 2006; 281: 36783-36792Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). The data in our earlier study showed that in the receptor-ligand complex, Cys27 of C5a is positioned close to residues 24–30 of the C5aR. To apply DTRM to this system, we moved the unpaired cysteine to eight positions in C5a (Fig. 2, B and C). In each of these bait mutants, Cys27 was replaced with Arg; this residue was chosen because it replaces Cys27 in rodent C5a sequences, which are otherwise highly conserved relative to human. The sites in the C5a ligand were chosen based on their surface accessibility, previous data demonstrating potential roles in receptor binding, and inspection of our initial models of the docked C5a ligand (12Hagemann I.S. Narzinski K.D. Floyd D.H. Baranski T.J. J. Biol. Chem. 2006; 281: 36783-36792Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). To verify that the ligands were functional, we coexpressed them with the C5aR in a S. cerevisiae expression system with a LacZ (β-galactosidase) reporter. The mutants were generally able to activate the C5aR with efficacy similar to the wild-type ligand (Fig. 2B), suggesting that they were properly folded and directed through the secretory pathway in yeast. In particular, the C27R/N64C ligand showed full functionality, indicating that N-linked glycosylation is not essential for C5a function. The C27R/D24C and C27R/S66C ligands showed some decrease in efficacy relative to wild-type. Immunoblotting showed the ligands to be expressed at levels roughly similar to wild-type, although differences in the level of expression could account for some of the variation in function (data not shown). Individual mutant ligands were used to screen libraries of C5aR mutants generated by random saturation mutagenesis (denoted by letters from “A”to“E”), where changes had been randomly introduced into receptor regions of interest. We hypothesized that some randomly mutated receptors would contain cysteine side chains at positions suitable for forming a disulfide bond with the mutant ligand, thereby complementing the affinity lost via receptor mutagenesis. Use of C5a C27R/D24C to Trap Interactions with the C5aR NT—As an initial test of the DTRM technique, we screened a library of N-terminal mutant C5aRs with the ligand C5a C27R/D24C (Fig. 3). Relative to our earlier work with the wild-type ligand (12Hagemann I.S. Narzinski K.D. Floyd D.H. Baranski T.J. J. Biol. Chem. 2006; 281: 36783-36792Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar), we predicted that moving the unpaired cysteine by a small number of amino acid positions would cause a correspondingly small displacement in the receptor positions where a disulfide cross-link could form. When the N-terminal library was screened against ligand C27R/D24C in yeast BY1142 (an engineered yeast strain in which C5a receptor signaling allows growth on selective media; see “Experimental Procedures”), we identified 11 receptors meeting our screening criteria, in that they were activated by C5a C27R/D24C but were much less strongly activated by C5a C27R (Fig. 3, the A series of receptors). We interpret these receptors as being those whose ligand affinity depends upon the presence of a cysteine in the ligand. We noted that all of the A receptors, with the exception of A20, contained at least one and usually two cysteine residues. These cysteines were concentrated in two bands within the N terminus: positions 10–12 and 23–27 (Fig. 3). We wished to determine whether these cysteines exceeded the number that would have arisen by chance in the absence of selective pressure favoring cysteine insertion. To this end, we calculated the probability of incorporating a cysteine at each position, and multiplied this by the number of selected receptors to estimate the expected number of cysteines in the set as a whole. At receptor positions 10–12, most of the cysteines were able to arise via a single nucleotide mutation, making them somewhat likely to occur even by chance. In the absence of selective pressure favoring cysteines, a set of 11 randomly selected receptors would have been expected" @default.
• W2085228715 created "2016-06-24" @default.
• W2085228715 creator A5004313839 @default.
• W2085228715 creator A5031896350 @default.
• W2085228715 creator A5061557305 @default.
• W2085228715 creator A5081155797 @default.
• W2085228715 creator A5088837180 @default.
• W2085228715 date "2008-03-01" @default.
• W2085228715 modified "2023-10-05" @default.
• W2085228715 title "Structure of the Complement Factor 5a Receptor-Ligand Complex Studied by Disulfide Trapping and Molecular Modeling" @default.
• W2085228715 cites W1506918918 @default.
• W2085228715 cites W1533894940 @default.
• W2085228715 cites W1546290898 @default.
• W2085228715 cites W1904288160 @default.
• W2085228715 cites W1963523454 @default.
• W2085228715 cites W1968001320 @default.
• W2085228715 cites W1972084384 @default.
• W2085228715 cites W1973220865 @default.
• W2085228715 cites W1978958492 @default.
• W2085228715 cites W1995089537 @default.
• W2085228715 cites W2000023245 @default.
• W2085228715 cites W2002354698 @default.
• W2085228715 cites W2005918263 @default.
• W2085228715 cites W2006993859 @default.
• W2085228715 cites W2013230660 @default.
• W2085228715 cites W2021073687 @default.
• W2085228715 cites W2022659527 @default.
• W2085228715 cites W2023082430 @default.
• W2085228715 cites W2024875771 @default.
• W2085228715 cites W2033388874 @default.
• W2085228715 cites W2043890981 @default.
• W2085228715 cites W2045109604 @default.
• W2085228715 cites W2046271055 @default.
• W2085228715 cites W2058573636 @default.
• W2085228715 cites W2063588043 @default.
• W2085228715 cites W2073034308 @default.
• W2085228715 cites W2076324521 @default.
• W2085228715 cites W2076412935 @default.
• W2085228715 cites W2080210935 @default.
• W2085228715 cites W2091886761 @default.
• W2085228715 cites W2094951122 @default.
• W2085228715 cites W2098437920 @default.
• W2085228715 cites W2102763490 @default.
• W2085228715 cites W2104477830 @default.
• W2085228715 cites W2122967721 @default.
• W2085228715 cites W2135689740 @default.
• W2085228715 cites W2157959080 @default.
• W2085228715 cites W2159001526 @default.
• W2085228715 doi "https://doi.org/10.1074/jbc.m709467200" @default.
• W2085228715 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18195008" @default.
• W2085228715 hasPublicationYear "2008" @default.
• W2085228715 type Work @default.
• W2085228715 sameAs 2085228715 @default.
• W2085228715 citedByCount "24" @default.
• W2085228715 countsByYear W20852287152012 @default.
• W2085228715 countsByYear W20852287152013 @default.
• W2085228715 countsByYear W20852287152014 @default.
• W2085228715 countsByYear W20852287152015 @default.
• W2085228715 countsByYear W20852287152016 @default.
• W2085228715 countsByYear W20852287152018 @default.
• W2085228715 countsByYear W20852287152019 @default.
• W2085228715 countsByYear W20852287152020 @default.
• W2085228715 countsByYear W20852287152021 @default.
• W2085228715 countsByYear W20852287152023 @default.
• W2085228715 crossrefType "journal-article" @default.
• W2085228715 hasAuthorship W2085228715A5004313839 @default.
• W2085228715 hasAuthorship W2085228715A5031896350 @default.
• W2085228715 hasAuthorship W2085228715A5061557305 @default.
• W2085228715 hasAuthorship W2085228715A5081155797 @default.
• W2085228715 hasAuthorship W2085228715A5088837180 @default.
• W2085228715 hasBestOaLocation W20852287151 @default.
• W2085228715 hasConcept C104317684 @default.
• W2085228715 hasConcept C111684460 @default.
• W2085228715 hasConcept C112313634 @default.
• W2085228715 hasConcept C116569031 @default.
• W2085228715 hasConcept C12554922 @default.
• W2085228715 hasConcept C127716648 @default.
• W2085228715 hasConcept C159654299 @default.
• W2085228715 hasConcept C170493617 @default.
• W2085228715 hasConcept C185592680 @default.
• W2085228715 hasConcept C188082640 @default.
• W2085228715 hasConcept C18903297 @default.
• W2085228715 hasConcept C203014093 @default.
• W2085228715 hasConcept C27256138 @default.
• W2085228715 hasConcept C2777924906 @default.
• W2085228715 hasConcept C55493867 @default.
• W2085228715 hasConcept C86803240 @default.
• W2085228715 hasConcept C95444343 @default.
• W2085228715 hasConceptScore W2085228715C104317684 @default.
• W2085228715 hasConceptScore W2085228715C111684460 @default.
• W2085228715 hasConceptScore W2085228715C112313634 @default.
• W2085228715 hasConceptScore W2085228715C116569031 @default.
• W2085228715 hasConceptScore W2085228715C12554922 @default.
• W2085228715 hasConceptScore W2085228715C127716648 @default.
• W2085228715 hasConceptScore W2085228715C159654299 @default.
• W2085228715 hasConceptScore W2085228715C170493617 @default.
• W2085228715 hasConceptScore W2085228715C185592680 @default.
• W2085228715 hasConceptScore W2085228715C188082640 @default.

• next