Systematic Mutational Mapping of Sites on Human Interferon-β-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.

In Vivo Pharmacology and Toxicology Evaluation of Polyethylene Glycol-Conjugated Interferon β-1a

“…PEGylated Interferon ␤-1a Pharmacology and Toxicology 993 ing site (Karpusas et al, 1998;Runkel et al, 2000;Baker et al, 2006). Using the same site-specific N-terminal PEGylation procedure, a similar PEG-IFN ␤-1a molecule has been synthesized previously with 20-kDa mPEG-propionaldehyde.…”

“…PEG-conjugated (PEGylated) IFN ␤-1a (PEG-IFN ␤-1a) was developed by attaching 20-kDa methoxy-PEG-O-2-methylpropionaldehyde to the ␣-amino group of the N terminus of IFN ␤-1a (Baker et al, 2006), with the aim of providing a less frequent dosing regimen and improved convenience for patients with MS. The N terminus of IFN ␤-1a is not critical for binding to the type 1 IFN receptor (Runkel et al, 2000). In vitro evaluation demonstrated that PEG-IFN ␤-1a retained significant in vitro activity (approximately 50%) in antiviral and antiproliferative assays compared with the unmodified protein, whereas PEG-IFN ␤-1a in vivo efficacy was enhanced compared with IFN ␤-1a in a mouse tumor angiogenesis model (Baker et al, 2006).…”

Inquiring into the Differential Action of Interferons (IFNs): an IFN-α2 Mutant with Enhanced Affinity to IFNAR1 Is Functionally Similar to IFN-β

“…In light of our findings, it was interesting to reanalyze the work of Runkel et al (44) describing the loss of IFN-␤-specific activities for mutants with mutations at positions 86 and 92. These mutations, which are located at the IFNAR1 binding site on interferon (20,40,43), apparently reduce the binding affinity for IFNAR1, and hence it is not surprising that the mutants now have the characteristics of IFN-␣. Our data also demonstrate that the binding affinity towards IFNAR1 is directly related to the antiproliferative activity of IFN.…”

The HLA-DR2 haplotype is associated with an increased proliferative response to the immunodominant CD4+ T-cell epitope in human interferon-β

“…The Dhelix is not directly involved in receptor binding, and amino-acid substitutions have been made in this region without affecting the activity of the overall molecule. 16 We have identified amino acids within this peptide epitope as critical for the in vitro proliferative response. Multiple changes could be made in this region that could potentially reduce T-cell responses with minimal disruption to bioactivity.…”

“…In addition, multiple alanine-substituted peptides were constructed that were based on published sequences, where the changes coincided with the discovered epitope regions, but did not significantly affect the activity of the mutated IFN-b molecule. 16 A set of 103 community donors was tested with the parent and modified epitope regions (Figure 2). This set of donors is independent of the original set of 87 donors tested with the IFN-b peptide set reported in Figure 1a.…”