Structure-function studies in a series of carboxyl-terminal octapeptide analogs of anaphylatoxin C5a

Kawai, Megumi; Quincy, David A.; Lane, Benjamin C.; Mollison, Karl W.; Or, Yat Sun; Luly, Jay R.; Carter, George W.

doi:10.1021/jm00080a004

Cited by 40 publications

(35 citation statements)

References 2 publications

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“…It is therefore counted among the anaphylatoxins, as well. This implicates the C-terminus as a receptor-interacting structure as confirmed by deletion proteins (Gerard et al, 1979;Wexler et al, 1985;Bohnsack et al, 1991), synthetic C-terminal analogue peptides Kawai et al, 1992;Or et al, 1992;Ember et al, 1992;) and rhC5a mutants (Mollison et al, 1989). In additition to the C-terminal residue Arg74, Lys68 and Leu72 (and apparently aromatic residues in position 67) are also involved in receptor interaction.…”

mentioning

confidence: 86%

Site‐specific mutagenesis of residues in the human C5a anaphylatoxin which are involved in possible interaction with the C5a receptor

Bubeck

Grötzinger

Winkler

et al. 1994

European Journal of Biochemistry

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To check and clarify existing data on receptor-interacting residues in the human C5a anaphylatoxin, we tested mutant C5 a proteins obtained by site-directed mutagenesis of a recombinant human C5a (rhC5a) cDNA clone for structural and functional integrity. Amino acid positions in three different regions of the molecule were investigated: Arg74 at the C-terminus, Arg40 and Pro45 located in the core region, and Lysl4 and Lysl9, Lys20 in the N-terminus. Des-Arg74-rhC5a displayed only a residual 3 -4% functional activity in the myeloperoxidase-release assay from human granulocytes while retaining the three-dimensional solution structure of wild-type (wt) -rhC5 a as shown by circular dichroism (CD) spectroscopy. Des-Arg74-rhC5a was able to activate the human C5 a receptor transiently expressed in Xenopus oocytes, but was inactive in the heterologous guinea pig (gp) ileum-contraction assay. These results reveal profound differences between the guinea pig and human C5a-receptor ligand-binding characteristics. Exchange of the core residue Arg40 by a glycine did not significantly affect functional C5a activity, in contrast to a previous observation [Mollison, K. W., Mandecki, W., Zuiderweg, E. P., 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. (1989) Identification of receptor-interacting residues in the inflammatory complement protein C5 a by sitedirected mutagenesis, Proc. Nutl Acad. Sci. USA 86, 292-2961, nor did exchange of the conserved Pro45 residue by the C3a analogue glutamic acid, a mutation expected to alter the whole geometry of the loop connecting helix I11 -helix IV (including Arg40) of the C5a molecule. Thus, participation of this loop in receptor interaction appears unlikely. While exchange of the N-terminal Lysl4 residue by alanine did not significantly affect functional activity, a double replacement of Lysl9 and Lys20 by alanine residues reduced activity more than 30-fold. These results c o n f m Lysl9 a n d or Lys20 as a putative receptor-interacting site, although we could not obtain a CD spectrum of this important mutant due to poor expression.The human C5a anaphylatoxin is a small protein of 74 amino acids which mediates a variety of cellular activation and release processes. It is generated at sites of inflammation with concomitant complement-system activation by proteolytic cleavage from the a chain of the complement factor C5. The major target cells are the granulocyte and macrophage, leading to chemotactic attraction of neutrophils, induction of the oxidative burst and release of lysosomal enzymes, histamine and interleukins. All responses are mediated by interaction with the membrane-bound C5a receptor, which has recently been cloned (for review, see Kohl and Bitter-Suermann, 1993).Identification of receptor-interacting residues in the human C5 a molecule are essential for the rational development of receptor antagonists which are of considerable therapeutic

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confidence: 86%

Site‐specific mutagenesis of residues in the human C5a anaphylatoxin which are involved in possible interaction with the C5a receptor

Bubeck

Grötzinger

Winkler

et al. 1994

European Journal of Biochemistry

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“…In support of this claim, mutations in the ligand C-terminal tail inhibit ligand binding and receptor activation (19). Synthetic hexapeptides derived from the tail of C5a can compete with the full-length ligand and serve as full agonists (18,20), indicating that the tail interacts with the receptor. Receptor residues Ile 116 , Arg 206 , and Val 286 have been identified as points of interaction with the C5a C-terminal tail (21,22).…”

Section: G Protein-coupled Receptors (Gpcrs)mentioning

confidence: 99%

“…In support of this claim, mutations in the ligand C-terminal tail inhibit ligand binding and receptor activation (19). Synthetic hexapeptides derived from the tail of C5a can compete with the full-length ligand and serve as full agonists (18,20) * This work was supported by National Institutes of Health Grant R01 GM63720 (to T. J. B.).…”

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confidence: 99%

Random Mutagenesis of the Complement Factor 5a (C5a) Receptor N Terminus Provides a Structural Constraint for C5a Docking

Hagemann¹,

Narzinski

Floyd

et al. 2006

Journal of Biological Chemistry

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The N terminus of G protein-coupled receptors has been implicated in binding to peptide hormones. We have used random saturation mutagenesis to identify essential residues in the N terminus of the human complement factor 5a receptor (C5aR). In a library of N-terminal mutant C5aR molecules screened for activation by C5a, residues 24 -30 of the C5aR showed a marked propensity to mutate to cysteine, most likely indicating that sulfhydryl groups at these positions are appropriately situated to form disulfide interactions with the unpaired Cys 27 of human C5a. This presumptive spatial constraint allowed the ligand to be computationally docked to the receptor to form a model of the C5a/C5aR interaction. When the N-terminal mutant C5aR library was rescreened with C5a C27R, a ligand incapable of disulfide interactions, no individual position in the N terminus was essential for receptor signaling. However, the region 19 -29 was relatively highly conserved in the functional mutants, further demonstrating that this region of the C5aR makes a productive physiologic interaction with the C5a ligand.G protein-coupled receptors (GPCRs) 3 transduce signals from a wide variety of biological stimuli, including small molecules, lipids, peptides, proteins, and environmental cues such as odorants and light (1). As the largest family of cell-surface receptors, they have provided fertile ground for pharmacologic modulation. GPCRs share a secondary structure consisting of seven transmembrane helices (TMs 1-7) joined by three intracellular and three extracellular loops, along with N-and C-terminal domains (Fig. 1). The receptors serve as binary switches that, when activated by ligand, exhibit guanine nucleotide exchange factor activity toward heterotrimeric G proteins (2). Because they are large transmembrane proteins, GPCRs are difficult to study by direct structural methods such as x-ray crystallography. A series of crystal structures of bovine rhodopsin has allowed major progress in the understanding of GPCR structure (3-7) but has not made clear the mechanism by which these receptors serve as switches, because rhodopsin has only been crystallized in its resting state. The mechanism of GPCR activation has, however, been productively explored using a variety of biochemical, biophysical, and computational techniques. Overall, ligand binding by GPCRs appears to induce a rigid-body movement of TM6 away from TM3, exposing new receptor surfaces that allow coupling to downstream effectors (8 -11).Our laboratory has studied the human complement factor 5a receptor (C5aR) (12) as a model system because it, like rhodopsin and many other clinically important receptors, belongs to the large family A of GPCRs (13). The C5aR has 19% amino acid identity with rhodopsin and has loop and transmembrane helix lengths similar to those of rhodopsin. Furthermore, the C5aR can be expressed in Saccharomyces cerevisiae, making it amenable to genetic studies.In addition to its role as a model receptor, the C5aR has intrinsic biological interest. The vertebrate...

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“…Initial analysis showed that the C-terminal arginine of C5a was important for binding affinity and functional activity (Chenoweth & Hugli, 1980;Braunwalder et al, 1992). In addition, a synthetic octapeptide corresponding to the C-terminus of C5a was able to mimic some of the C5a-induced functional responses but with a greatly reduced potency (Kawai et al, 1992). Using a mutant analysis of the full-length protein, 2 groups have identified some of the other amino acids of C5a involved with receptor binding (Mollison et al, , 1991Carney & Hugli, 1993).…”

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confidence: 99%

The pharmacophore of the human C5a anaphylatoxin

Toth¹,

Huwyler²,

Boyar³

et al. 1994

Protein Science

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We have determined which amino acids contribute to the pharmacophore of human C5a, a potent inflammatory mediator. A systematic mutational analysis of this 74-amino acid protein was performed and the effects on the potency of receptor binding and of CSa-induced intracellular calcium ion mobilization were measured. This analysis included the construction of hybrids between C5a and the homologous but unreactive C3a protein and sitedirected mutagenesis. Ten noncontiguous amino acids from the structurally well-defined 4-helix core domain (amino acids 1-63) and the C-terminal arginine-containing tripeptide were found to contribute to the pharmacophore of human C5a. The 10 mostly charged amino acids from the core domain generally made small incremental contributions toward binding affinity, some of which were independent. Substitutions of the C-terminal amino acid Arg 74 produced the largest single effect. We also found the connection between these 2 important regions to be unconstrained.

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Structure-function studies in a series of carboxyl-terminal octapeptide analogs of anaphylatoxin C5a

Cited by 40 publications

References 2 publications

Site‐specific mutagenesis of residues in the human C5a anaphylatoxin which are involved in possible interaction with the C5a receptor

Site‐specific mutagenesis of residues in the human C5a anaphylatoxin which are involved in possible interaction with the C5a receptor

Random Mutagenesis of the Complement Factor 5a (C5a) Receptor N Terminus Provides a Structural Constraint for C5a Docking

The pharmacophore of the human C5a anaphylatoxin

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