title: Systematic mutational mapping of sites on human interferon-beta-1a that are important for receptor binding and functional activity.
abstract: A systematic mutational analysis of human interferon-beta-1a (IFN-beta) was performed to identify regions on the surface of the molecule that are important for receptor binding and for functional activity. The crystal structure of IFN-beta-1a was used to design a panel of 15 mutant proteins, in each of which a contiguous group of 2-8 surface residues was mutated, in most instances to alanine. The mutants were analyzed for activity in vitro in antiviral and in antiproliferation assays, and for their ability to bind to the type I IFN (ifnar1/ifnar2) receptor on Daudi cells and to a soluble ifnar2 fusion protein (ifnar2-Fc). Abolition of binding to ifnar2-Fc for mutants A2, AB1, AB2, and E established that the ifnar2 binding site on IFN-beta comprises parts of the A helix, the AB loop, and the E helix. Mutations in these areas, which together define a contiguous patch of the IFN-beta surface, also resulted in reduced affinity for binding to the receptor on cells and in reductions in activity of 5-50-fold in functional assays. A second receptor interaction site, concluded to be the ifnar1 binding site, was identified on the opposite face of the molecule. Mutations in this region, which encompasses parts of the B, C, and D helices and the DE loop, resulted in disparate effects on receptor binding and on functional activity. Analysis of antiproliferation activity as a function of the level of receptor occupancy allowed mutational effects on receptor activation to be distinguished from effects on receptor binding. The results suggest that the binding energy from interaction of IFN-beta with ifnar2 serves mainly to stabilize the bound IFN/receptor complex, whereas the binding energy generated by interaction of certain regions of IFN-beta with ifnar1 is not fully expressed in the observed affinity of binding but instead serves to selectively stabilize activated states of the receptor.
authors.affiliation: Biogen, Inc., 14 Cambridge Center, Cambridge, Massachusetts 02142, USA.
authors.forename: L
authors.initials: L
authors.lastname: Runkel
authors.forename: C
authors.initials: C
authors.lastname: deDios
authors.forename: M
authors.initials: M
authors.lastname: Karpusas
authors.forename: M
authors.initials: M
authors.lastname: Betzenhauser
authors.forename: C
authors.initials: C
authors.lastname: Muldowney
authors.forename: M
authors.initials: M
authors.lastname: Zafari
authors.forename: C D
authors.initials: CD
authors.lastname: Benjamin
authors.forename: S
authors.initials: S
authors.lastname: Miller
authors.forename: P S
authors.initials: PS
authors.lastname: Hochman
authors.forename: A
authors.initials: A
authors.lastname: Whitty
mesh_terms: D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism; D019556:COS Cells; D004252:DNA Mutational Analysis; D005822:Genetic Vectors / Q000378:metabolism; D006131:Growth Inhibitors / Q000502:physiology; D006801:Humans; D000068556:Interferon beta-1a; D016899:Interferon-beta / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D053633:Receptor, Interferon alpha-beta; D017471:Receptors, Interferon / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
chemical_list: D000998:Antiviral Agents; D006131:Growth Inhibitors; C507964:IFNAR1 protein, human; C508033:IFNAR2 protein, human; D008565:Membrane Proteins; D017471:Receptors, Interferon; D053633:Receptor, Interferon alpha-beta; D016899:Interferon-beta; D000409:Alanine; D000068556:Interferon beta-1a