New Structural and Functional Aspects of the Type I Interferon-Receptor Interaction Revealed by Comprehensive Mutational Analysis of the Binding Interface

Abstract

Type I interferons bind to two cell surface receptors, ifnar1 and ifnar2, as the first step in the activation of several signal transduction pathways that elicit an antiviral state and an anti-proliferative response. Here, we quantitatively mapped the complete binding region of ifnar2 on interferon (IFN)␣2 by 35 individual mutations to alanine and isosteric residues. Of the six "hot-spot" residues identified (Leu-30, Arg-33, Arg-144, Ala-145, Met-148, and Arg-149), four are located on the E-helix, which is located at the center of the binding site flanked by residues on the A-helix and the AB-loop. The contribution of residues of the D-helix, which have been previously implicated in binding, proved to be marginal for the interaction with the extracellular domain of ifnar2. Interestingly, the ifnar2 binding site overlaps the largest continuous hydrophobic patch on IFN␣2. Thus, hydrophobic interactions seem to play a significant role stabilizing this interaction, with the charged residues contributing toward the rapid association of the complex. Relating the anti-viral and anti-proliferative activity of the various interferon mutants with their affinity toward ifnar2 results in linear function over the whole range of affinities investigated, suggesting that ifnar2 binding is the rate-determining step in cellular activation. Dose-time analysis of the anti-viral response revealed that shortening the incubation time of low-level activation cannot be compensated by higher IFN doses. Considering the strict dependence of the cellular response on affinity, these results suggest that for maintaining transcription of IFN-responsive genes over a longer time period, low but continuous signaling through the IFN receptor is essential. Type I interferons (IFN)1 are a family of homologous cytokines, which potently elicit an anti-viral and anti-proliferative state in cells. All human type I IFNs (IFN␣, ␤, , and ) bind to a cell surface receptor consisting of two transmembrane proteins, ifnar1 (1) and ifnar2 (2), which associate upon binding. Binding of IFN to its receptor mediates activation of numerous genes (3) through different signal transduction pathways (reviewed in Ref. 4). Intriguingly, different expression profiles are induced by different IFNs, apparently through the same receptor, with particular differences between IFN␣ and IFN␤ (3, 5). However, the molecular mechanisms underlying the functional differences between IFNs are still unknown. Type I IFNs belong to the class of helical cytokines (6) and are built by five helices. The structure of several type I IFNs have been resolved, such as murine IFN␤ (7), human IFN␣2 (8, 9), human IFN␤ (10), and ovine IFN (11). Mutational studies have revealed functionally important residues on IFNs (reviewed in Ref. 12), which can be ascribed to distinct functional epitopes that interact with ifnar1 and ifnar2 (13, 14). The binding site for ifnar2 was mainly mapped on the AB-loop and D-helix and the binding site for ifnar1 on the C-helix. However, comparison with homologous cytokines...

Structure of the interferon-receptor complex determined by distance constraints from double-mutant cycles and flexible docking

“…The respective binding sites of IFN-␣2 and ifnar2 were previously mapped by means of site-directed mutagenesis (7,10). Here, we extended these studies by carrying out a systematic DMC analysis of the interface.…”

“…IFN-␣2 and ifnar2-extracellular domain (EC) were expressed in Escherichia coli and purified, and their concentrations were determined as described (7,24).…”

“…All measurements were carried out by using 20 mM Hepes (pH 7.5), 150 mM NaCl, and 0.01% Triton X-100 as a running buffer. Ifnar2-EC was immobilized to the surface by using the non-neutralizing antiifnar2-EC mAb 46.10 followed by cross-linking with a second mAb (117.7) (gift from Daniela Novick, Weizmann Institute) (7,25). The binding curves were evaluated with BIAEVALUATION software (Biacore, Uppsala) by using a simple one-to-one kinetic model.…”

Two Interferons Alpha Influence Each Other during Their Interaction with the Extracellular Domain of Human Type Interferon Receptor Subunit 2

“…Differences in K D values between IFN-α2c and CM3 were observed for IFNAR2-EC mutants I105A and M48V (data not shown). The importance of those amino acids for interaction with helices A (45-50) and E (102-106), as well as loop AB(78-82) of IFN-α2 has also previously been shown by Piehler et al ( , 2000. We used native electrophoresis to study differences in binding between CM3 with and without 6-histidine-tag and IFN-α2c with or without 6-histidine-tag and the IFNAR2-EC mutants I47A, I105A and M48V.…”

“…Interestingly enough, these differences had not been observed in the same experiments with IFN-α21b (data not show). Since it was confirmed that helix C is not involved in interaction with IFNAR2-EC (19,23), these results suggested that mutation in helix C (CM3) could affect IFN-IFNAR2-EC indirectly. The presence or absence of Nterminal 6-histidine tag had no effect on behavior of CM3.…”