Active site of C3a anaphylatoxin: contributions of the lipophilic and orienting residues

Unson, Cecilia G.; Erickson, Bruce W.; Hugli, Tony E.

doi:10.1021/bi00299a001

Cited by 33 publications

(14 citation statements)

References 23 publications

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“…In particular, knowledge of the crystal structure of C3a (34) facilitated accurate modeling of the C5a protein (26), and NMR studies have confirmed that the structures in solution of C3a (35) and C5a (24) are highly similar. Examination of C3a structure-activity relationships via synthesis of peptides mimicking the carboxyl terminus has demonstrated the crucial importance of this region for C3a activity (36)(37)(38). However, C3a distinctly differs from C5a in that a synthetic carboxyl-terminal peptide of 21 residues was found to account for essentially the full biologic potency of the C3a molecule (38).…”

Section: Resultsmentioning

confidence: 99%

Identification of receptor-binding residues in the inflammatory complement protein C5a by site-directed mutagenesis.

Mollison

Mandecki

Zuiderweg

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

114

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C5a is an inflammatory mediator potentially involved in a number of diseases. To help define which of its 74 residues are important for receptor binding and response triggering, changes in the amino acid sequence of C5a were introduced by site-directed mutagenesis. Synthetic C5a-encoding genes incorporating point mutations were expressed in Escherichia coli, and the mutant proteins were purified to homogeneity. Modifications of the C5a molecule causing parallel reductions in binding to polymorphonuclear leukocyte membranes and in stimulation of polymorphonuclear leukocyte locomotion (chemokinesis) suggest that carboxyl-terminal residues Lys-68, Leu-72, and Arg-74 interact with the receptor. Substitutions in the disulfide-linked core of C5a revealed involvement of Arg-40 or nearby residues, because potency losses were associated with only localized conformational changes as detected by NMR. Surprisingly, a substitution at core residue Ala-26, which did not alter C5a core structure, appeared from NMR results to reduce potency by causing a long-distance conformational change centered on residue His-15. Thus, at least three discontinuous regions of the C5a molecule appear to act in concert to achieve full potency.

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Section: Resultsmentioning

confidence: 99%

Identification of receptor-binding residues in the inflammatory complement protein C5a by site-directed mutagenesis.

Mollison

Mandecki

Zuiderweg

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

114

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“…Removal of Arg77 from the C-terminal end of C3a abolishes C3aR affinity and most biologic activities, and substitution with Lys77 gave at least 300 times less activity. Blockage with amide on carboxyl group of Arg77 in C3a was at least 1,000 times less active~Caporale et Erickson et al, 1981;Unson et al, 1984!. Therefore, we selected charged residues in C3aR as candidates for site-directed mutagenesis as a means to define the primary determinants involved in C3a binding.…”

Section: Discussionmentioning

confidence: 99%

Identification of ligand effector binding sites in transmembrane regions of the human G protein‐coupled C3a receptor

et al. 1999

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The human C3a anaphylatoxin receptor~C3aR! is a G protein-coupled receptor~GPCR! composed of seven transmembrane a-helices connected by hydrophilic loops. Previous studies of chimeric C3aR0C5aR and loop deletions in C3aR demonstrated that the large extracellular loop2 plays an important role in noneffector ligand binding; however, the effector binding site for C3a has not been identified. In this study, selected charged residues in the transmembrane regions of C3aR were replaced by Ala using site-directed mutagenesis, and mutant receptors were stably expressed in the RBL-2H3 cell line. Ligand binding studies demonstrated that R161A~helix IV!, R340A~helix V!, and D417Ã helix VII! showed no binding activity, although full expression of these receptors was established by flow cytometric analysis. C3a induced very weak intracellular calcium flux in cells expressing these three mutant receptors. H81Ã helix II! and K96A~helix III! showed decreased ligand binding activity. The calcium flux induced by C3a in H81A and K96A cells was also consistently reduced. These findings suggest that the charged transmembrane residues Arg161, Arg340, and Asp417 in C3aR are essential for ligand effector binding and0or signal coupling, and that residues His81 and Lys96 may contribute less directly to the overall free energy of ligand binding. These transmembrane residues in C3aR identify specific molecular contacts for ligand interactions that account for C3a-induced receptor activation.

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“…in the peptide sequences [32,33]. The con formation of active peptides were altered by residue replacements [34,35] in an attempt to understand structure-function relation ships as they pertain to the C3a molecule.…”

Section: Fragment C-n-y-i-t-e-l-r-r-q-h-a-r-a-s-h-l-g-l-a-r (C3a 57-7mentioning

confidence: 99%

Biochemistry and Biology of Anaphylatoxins

Hugli¹

1986

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156

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The molecular architecture of anaphylatoxins has been explored on several levels. Primary, secondary and tertiary structural parameters that dictate function of the C3a, C4a and C5a molecules are being elucidated with the aid of comparative sequence analyses, physical measurements and organic syntheses. Although C3a, C4a and C5a are biologically distinct mediators, as defined by their unique receptor systems, a common genetic origin is apparent from conserved features in their primary structures. Evidence is now available which suggests that similarities in the folding pattern of the anaphylatoxins may dictate a concensus conformation for each factor. We have learned from synthetic peptide studies that the binding (e.g. effector) site in anaphylatoxin molecules exists as a linear sequence contained in the C-terminal portion of the polypeptide. What is also evident is that a preferred conformation is defined for the binding site requiring a proper side chain orientation for optimal bioactivity. It is proposed that folding of the native structure stabilizes this conformation at the binding site. The binding site in C3a contains the essential residues LGLAR folded in an irregular or pseudo-β-turn and stabilized by an adjacent α-helical segment. It is proposed that the a-helical segment influences orientation of the side chain residues in the ‘binding site’. A similar model is evolving for C5a based on synthetic C5a peptides that express both spasmogenic and chemotactic activities. This helix turn model promises to be representative of an essential structural feature that determines anaphylatoxin activity. We believe that these models contribute significantly to our understanding of the molecular relationships between structure and function for these humoral mediators of inflammation.

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Active site of C3a anaphylatoxin: contributions of the lipophilic and orienting residues

Cited by 33 publications

References 23 publications

Identification of receptor-binding residues in the inflammatory complement protein C5a by site-directed mutagenesis.

Identification of receptor-binding residues in the inflammatory complement protein C5a by site-directed mutagenesis.

Identification of ligand effector binding sites in transmembrane regions of the human G protein‐coupled C3a receptor

Biochemistry and Biology of Anaphylatoxins

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