A mutational analysis of receptor binding sites of interleukin-1β:differences in binding of human interleukin-1β muteins to human and mouse receptors

• Published in: Protein engineering 1994-05, Vol.7 (5), p.663-671
• Main Authors: Grütter, M.G., Oostrum, J.van, Priestle, J.P., Edelmann, E., Joss, U., Feige, U., Vosbeck, K., Schmitz, A.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Biological and medical sciences; Biotechnology; Circular Dichroism; Crystallography, X-Ray; DNA Mutational Analysis; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Humans; Interleukin-1 - chemistry; Interleukin-1 - genetics; Interleukin-1 - metabolism; interleukin-1β; Methods. Procedures. Technologies; Mice; Models, Molecular; Molecular Sequence Data; Molecular Structure; Mutagenesis, Site-Directed; mutational mapping; Protein engineering; receptor binding; Receptors, Interleukin-1 - chemistry; Receptors, Interleukin-1 - metabolism; species difference; Structure-Activity Relationship
• ISSN: 1741-0126; 0269-2139; 1741-0134; 1460-213X
• DOI: 10.1093/protein/7.5.663

The 3-D crystal structure of interleukin-1β(IL-1β) has been used to define its receptor binding surface by mutational analysis. The surface of IL-1β was probed by site-directed mutagenesis. A total of 27 different IL-1β muteins were constructed, purified and analyzed. Receptor binding measurements on mouse and human cell lines were performed to identify receptor affinities. IL-1β muteins with modified receptor affinity were evaluated for structural integrity by CD spectroscopy or X-ray crystallography. Changes in six surface loops, as well as in the C- and N-termini, yielded muteins with lower binding affinities. Two muteins with intact binding affinities showed 10- to 100-fold reduced biological activity. The surface region involved in receptor binding constitutes a discontinuous area of ∽1000 Å2 formed by discontinuous polypeptide chain stretches. Based on these results, a subdivision into two distinct local areas is proposed. Differences in receptor binding affinities for human and mouse receptors have been observed for some muteins, but not for wild-type IL-1β. This is the first time a difference in binding affinity of IL-1β muteins to human and mouse receptors has been demonstrated