Identification of receptor-binding residues in the inflammatory complement protein C5a by site-directed mutagenesis.
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.
Mollison
K W
KW
Immunoscience Research Area, Abbott Laboratories, Abbott Park, IL 60064.
Mandecki
W
W
Zuiderweg
E R
ER
Fayer
L
L
Fey
T A
TA
Krause
R A
RA
Conway
R G
RG
Miller
L
L
Edalji
R P
RP
Shallcross
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003182:Complement C5 / Q000378:metabolism*; D005796:Genes; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009504:Neutrophils / Q000276:immunology; D011487:Protein Conformation; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid; D013552:Swine
D003182:Complement C5; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D011994:Recombinant Proteins