Identification of receptor-binding residues in the inflammatory complement protein C5a by site-directed mutagenesis
| Autor: | W. Mandecki, M A Shallcross, R G Conway, Erik R. P. Zuiderweg, Loan N. Miller, R P Edalji, Karl W. Mollison, Fey Ta, L Fayer, R. A. Krause |
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| Rok vydání: | 1989 |
| Předmět: |
Conformational change
Neutrophils Protein Conformation Swine Molecular Sequence Data Chemokinesis In Vitro Techniques Biology Sensitivity and Specificity Mice Protein structure Sequence Homology Nucleic Acid Animals Humans Amino Acid Sequence Binding site Site-directed mutagenesis Receptor Receptor Anaphylatoxin C5a Peptide sequence Binding Sites Multidisciplinary Mutagenesis Complement C5 Recombinant Proteins Receptors Complement Genes Biochemistry Mutation Cattle Research Article |
| Zdroj: | Proceedings of the National Academy of Sciences. 86:292-296 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.86.1.292 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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