Binding of interferon reduces the force of unfolding for interferon receptor 1

Abstract

Differential signaling of the type I interferon receptor (IFNAR) has been correlated with the ability of its subunit, IFNAR1, to differentially recognize a large spectrum of different ligands, which involves intricate conformational re-arrangements of multiple interacting domains. To shed light onto the structural determinants governing ligand recognition, we compared the force-induced unfolding of the IFNAR1 ectodomain when bound to interferon and when free, using the atomic force microscope and steered molecular dynamics simulations. Unexpectedly, we find that IFNAR1 is easier to mechanically unfold when bound to interferon than when free. Analysis of the structures indicated that the origin of the reduction in unfolding forces is a conformational change in IFNAR1 induced by ligand binding.

Fig 1. Forced unfolding of IFNAR1-EC by AFM.

(A) The C-terminal of IFNAR1-EC immobilized onto a mica surface through a flexible linker and an AFM tip interacting with the protein (B) Traces representative of different families of unfolding curves. (C) I. A scatter plot representing the phase space of the system in the ΔL_C vs. F plane. II. Corresponding contour plot (D) I. Histogram of contour length changes fitted by two Gaussians centred at 16 nm and 36 nm with a p-value < 10⁻⁴. II. The force histogram corresponding to the events in I fitted with Gaussians centred at 40 pN and 90 pN.

Fig 2. Influence of IFNα2 binding on IFNAR1 unfolding.

Unfolding of IFNAR1-EC on its own (A and B,) or in the presence of 4 μM WT- IFNα2 (K_D = 1.5 μM, C and D), the high affinity mutant YNS (K_D = 0.03 μM, E and F) and the low affinity mutant NLYY (no measureable binding affinity [46], G and H). The influence of the ligands is described either by contour plots (C,E,G) or force histograms (D,F,H) each representing 3–4 experiments.

Fig 3. Coarse-grained MD simulations of IFNAR1-EC and contact changes affected by YNS binding.

(A) Histograms of unfolding forces by domains (SD1 –blue, SD2 –green, SD3 –red), with the top graph representing unfolding of the domain on its own and the bottom graph showing the unfolding of the domain in the presence of YNS. (B). Traces of steered MD of IFNAR1-EC domains on its own (I) or with its ligand (II). I and II each consist of 60 traces with colours as in A. C. Residues that have lost contacts with other residues upon interaction with YNS are coloured in cyan whereas those that have gained contacts are coloured in magenta.