. 2017 Aug 4;429(16):2571-2589.
Epub 2017 Jun 23.
Dynamic Modulation of Binding Affinity as a Mechanism for Regulating Interferon Signaling
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Dynamic Modulation of Binding Affinity as a Mechanism for Regulating Interferon Signaling
J Mol Biol
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How structural dynamics affects cytokine signaling is under debate. Here, we investigated the dynamics of the type I interferon (IFN) receptor, IFNAR1, and its effect on signaling upon binding IFN and IFNAR2 using a combination of structure-based mechanistic studies, in situ binding, and gene induction assays. Our study reveals that IFNAR1 flexibility modulates ligand-binding affinity, which, in turn, regulates biological signaling. We identified the hinge sites and key interactions implicated in IFNAR1 inter-subdomain (SD1-SD4) movements. We showed that the predicted cooperative movements are essential to accommodate intermolecular interactions. Engineered disulfide bridges, computationally predicted to interfere with IFNAR1 dynamics, were experimentally confirmed. Notably, introducing disulfide bonds between subdomains SD2 and SD3 modulated IFN binding and activity in accordance with the relative attenuation of cooperative movements with varying distance from the hinge center, whereas locking the SD3-SD4 interface flexibility in favor of an extended conformer increased activity.
conformational flexibility; elastic network models; interferon binding affinity; regulation of cytokine signaling; structural dynamics.
Copyright © 2017 Elsevier Ltd. All rights reserved.
Conflict of interest statement
Conflict of interest
The authors do not have a conflict of interest.
Fig. 1. Heterotrimeric complex of IFNa2, IFNAR1 and IFNAR2 in the presence of lipid bilayer
The structure is composed of the experimentally resolved complex (PDB id:
) comprised of IFNAR1 EC subdomains SD1–3, IFNα2 and IFNAR2, the IFNAR1-SD4 domain, and two transmembrane (TM) helices at the C-termini of the receptors, embedded into a POPC lipid bilayer constructed 3SE3 in silico.
Fig. 2. Conformational flexibility of IFNAR1 EC domain
Each panel shows two conformations, compact/bent (
orange) and extended ( gray) sampled upon global fluctuations along the modes 1 ( a) and 2 ( b). The left and right panels display the front and side views. Mode 1 involves a bending around a central hinge at the interface between SD2 and SD3; Mode 2 induces an orthogonal bending by flexing at SD3-SD4 interfacial hinge center. The decrease in the distance between N23 on SD1 ( red sphere) and T407 on SD4 ( magenta) in the bent forms is consistent with that observed in FRET experiments 21. Residue pairs making SD2-SD3 inter-subdomain contacts, G133-F238, L134-R241, Y163-E293 and E111-F290, are shown by cyan, green, magenta and blue/gray spheres, respectively in ( a). The SD3-SD4 interfacial pair, Q268-Q328, is colored green in ( b); see also the respective Animation 1 and 2.
Fig. 3. Intrinsic ability of IFNAR1 structure to adapt to functional interactions
The change observed experimentally between the unbound (
green) and bound ( cyan) structures of IFNAR1 agrees with the changes intrinsically favored by ANM global modes. The conformer colored orange is obtained upon moving the unbound form ( green) along a global ANM mode (ANM mode 4). See also Animation 3. This conformer resembles that of the bound IFNAR1 ( cyan), indicating that the receptor (unbound) structure favors this functional change in conformation (i.e., unbound IFNAR1 is predisposed to take on the bound conformation). The RMSD between the experimentally resolved unbound and bound forms is 4.1 Å; that between the ANM-driven conformer and experimentally resolved bound form is 2.3 Å, i.e. the movement along mode 4 helps the unbound IFNAR1 move toward the conformation that is stabilized upon binding the substrate (IFNα2).
Fig. 4. IFNAR1 ternary complex attached to the membrane retains its adaptability to functional interactions
Global fluctuations of ternary complex formed by IFNAR1, IFNa2 and IFNAR2 (colored
cyan, light violet and pink, respectively) modeled in the presence of lipid bilayer. The left and right diagrams display alternative conformations. The lower two diagrams display the same conformations, from a different perspective (90° rotation around the vertical axis). The distance between N23 ( red sphere) and T407 ( magenta sphere) decreases by ~10 Å in bent conformation with respect to the starting conformer; see also Animation 4.
Fig. 5. Formation of a disulfide bridge in the mutant Q268C-Q328C, and its effect on IFNAR1 structure and dynamics
(a) Overlay of human ( green) and mouse ( tan) IFNAR1. Residues 268 and 328, shown in CPK space filling, are within disulfide bridge-forming distance in the human, and the homologous positions are ~30 Å apart in the mouse. (b) Disulfide bridge 268C-328C detected by mass spectrometry. The purified receptor was digested with chymotrypsin as described in Materials and Methods, and subjected to MS analysis. The resulting mass data searched with MassMatrix indicates that 193–172 and 268–328 form disulfide bonds (color code from blue (no disulfide) to red (high confidence disulfide)). The data are a representative of one of the two independent experiments done. (c) Monitoring SD4 movement by electron transfer from a fluorescence dye (AT655) covalently bonded to residue N349C. Fluorescence quenching by neighboring W347 is abrogated upon IFN binding. No abrogation of quenching is observed in Q268C-Q328C double mutant, suggesting that the latter does not undergo (upon IFN binding) a conformational change that affects the relative positions of N349 and W347. The data shown are a representative of one of the three independent experiments done. (d) and (e) MD trajectories generated for IFNAR1-IFNa2-IFNAR2 using wild-type and double mutant Q268C-Q328C IFNAR1, shortly designated as WT and SD34. SD4 movement is severely restricted in SD34 (e) compared to the WT (d). The diagrams show the initial IFNAR1 conformation ( white) and its 40 th ns snapshots ( cyan) in the presence of IFN and IFNAR2 (not shown for clarity). During the course of simulations SD1-SD4 distance is reduced by more than 10 Å in the WT; whereas it remains unchanged in the double mutant. The positions of W347 and N349 are shown in both cases. (f)
(top) Time evolution of the C a distances between N23 (SD1) and T407 (SD4) in the simulations of IFNAR1 ( left) and the ternary complex (IFNAR1+IFNa2+IFNAR2) ( right). The original distance of 107 Å fluctuates in the range 85–115 Å approximately, in the WT; whereas it is confined to a narrower range, 105–115 Å, in SD34. ( bottom) W347-N349 distance increases up to 12 Å in the WT; whereas it remains < 10 Å in SD34.
Fig. 6. Activity of the 268C-328C disulfide mutant of IFNAR1
(a) shows binding of the IFNa2 mutant YNS to purified WT and mutant IFNAR1 as determined by SPR (see also Fig. S2). (b) Antiviral activity after addition of the indicated amounts of IFN for 4 h before ECMV was added for 14 h. Cell survival was determined by crystal violet. (c) Gene expressions of stably transfected Huh7 cells after 16 hours treatment with indicated amounts of IFNα2. qPCR was then performed for IFI6 and MX1 genes. The data presented are the relative expression levels compared to those of untreated cells, normalized against HPRT1. (d) Gene induction, similar to panel C but with single cysteine receptors receptor mutations monitored. (e) Fold change in gene expression using the Fluidigm system (see Methods). Cells were treated as in C and cDNAs (50ng/ml) were preamplified with all the primers polled and analyzed with the BioMark real-time PCR instrument. The presented data are averages from 5 (b), 6 (c), 3 (d), 2 (e) independent experiments.
Fig. 7. Intrinsic ability of IFNAR1 structure to adapt to functional interactions
(a) IFNAR1 crystal structures resolved for mouse and human are easily exchangeable conformers favored by their common architecture. Superposition of human (unbound, green) and mouse (bound, tan; (PDB id: ) structures yields an RMSD of 10.2 Å 3WCY (b) Movements along the global modes reduce the RMSD to 6.5 Å. The diagrams represent the human ( orange) and mouse ( yellow) structures deformed along their ANM modes 1 and 3, respectively. (c) MD simulations of IFNAR1 confirm the adaptability of the receptor to sample the conformational space between the human and mouse structures. The figure displays a snapshot ( violet) from MD simulations. The simulations were initiated from human IFNAR1 crystal structure in the bound state ( cyan). SD4 undergoes a large displacement to reorient similarly to its conformation in the mouse structure. The distance between N23 ( red sphere) and T407 ( magenta sphere) decreases by approximately 20 Å during this reorientation.
Fig. 8. GNM-based identification of key sites (global hinges) and key interactions in IFNAR1 EC domain
(a) Distribution of residue movements along global modes 1 ( left) and 2 ( right), predicted by the GNM. Hinge residues are located at the crossover ( y = 0) between positive- and negative-direction motions: E199 (mode 1) and Q100 and Q302 (mode 2). Other key residues near the hinge centers (at y ≈ 0) include E111 and K240-L247 (mode 1) and D15, T127, Q215, Q268 and Q328 (mode 2). (b) Location of key residues and interactions on the structure. The effect of locking these inter-subdomain interactions (by engineered disulfide bridges) on IFNAR1 binding and signaling properties is examined experimentally (see Table 1). Fig. S3 and Animations 5 and 6 show the inter-residue distance changes at three pairs of residues with large distances in structure. See also the zoom-in view for double mutants I162-E293 and Y163-E293 in Fig. S4. (c) Inter-residue distance fluctuations, shown for residue pairs separated by <15 Å in the folded structure. Blue circles highlight sequentially distant pairs (G133-F238 (and L134-R241) and I162-E293) distinguished by large distance fluctuations due to the SD2-SD3 global hinge motions.
Fig. 9. Activity of 8 disulfide mutants of IFNAR1 located between domains SD1-SD2, SD2-SD3 and SD3-SD4
(a) Signal emitted from 1.5 nm I 125-labeled WT IFNα2 after competing with cold IFN at different concentrations. Y-axis represents the fraction of gamma signal relative to the signal in the absence of cold competitor. (b) and (c) IFI6 and MX1 gene expressions respectively of transfected Huh7 cells after 16 hours treatment with indicated amounts of IFNα2. The data presented are the relative expression levels compared to those of untreated cells, normalized against HPRT1. (d) Antiviral activity of stably transfected Huh7 cells after addition of the indicated amounts of IFN for 4 h before ECMV was added for 14 h. Cell survival was determined by crystal violet. (e) Binding of 100 nM of the IFNa2 mutant YNS to purified WT and mutant IFNAR1 receptors as monitored by SPR. The presented data are averages from 3 (a), 4 (b – d), 2 (e) independent experiments.
Fig. 10. Activity of the disulfide mutants of IFNAR1
(a) Fold change in gene expression using the fluidigm system (see methods). Transfected Huh7 Cells were treated for 16 h with indicated amounts of IFNα2 and cDNAs (50ng/ml) were preamplified with all the primers polled and analyzed with the BioMark real-time PCR instrument relative to HPRT1. Color code is blue to red, from no induction to maximal induction. (b) Summary of experimentally determined fold-change (relative to WT) of the data shown in Fig. 6 and Fig. 9 for the different examined activities of IFNAR1 disulfide bond mutations. K D values were determined by SPR from 6 different analyte concentrations (e.g. see Fig. S2). The error bars represent 2x standard deviation of the relative mutant to wild-type data. Absolute values are provided in Table S1.
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Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, Non-U.S. Gov't
Gene Expression Regulation*
Interferons / metabolism*
Receptor, Interferon alpha-beta / metabolism*
Receptor, Interferon alpha-beta