C&SP28: Dynamic modulation of interferon binding affinity as a mechanism to regulate interferon receptor signaling

A. Collaborating Investigators: Gideon Schreiber,1 Ivet Bahar,2 James R Faeder2

B. Institutions: 1Weizmann Institute and 2University of Pittsburgh

C. Funding Status of Project: ISF-I-Core- Center for Integrated Structural Cell Biology 2013-2018; ISF grant: Protein binding and catalysis in crowded environments: from in vitro to in vivo (2014-2018)


Fig VIII.8. Intrinsic flexibility of interferon receptor IFNAR1, predicted by ProDy ANM, is (a) essential to facilitating the formation of ternary complex with IFN and IFNAR2, (b) explains the distance changes observed in FRET. Pairs of residues making interdomain contacts, identified by GNM to control IFNAR1 global motions.

D. Biomedical Research Problem:

Type I interferons (IFNs) are multifunctional cytokines that mediate/induce diverse cellular responses, including both innate and adaptive immune responses, stimulation of antiviral responses, and cancer surveillance, upon forming a ternary complex with two surface receptors, IFNAR1 and IFNAR2 (Fig VIII.8a).95-97 The activities of IFN-a subtypes correlate with their affinities to bind to IFNAR1 and IFNAR2.98 While the Schreiber lab made seminal contributions to understanding the molecular basis of IFNARs,95,96,98-105 the mechanism of regulation of differential IFN activities through interactions with IFNAR1 and 2,102 remains unclear. Our integrated computational (TR&D1) and experimental preliminary studies described below point to the significance of the intrinsic dynamics in modulating binding affinity.

E. Methods and Procedures: We adopted a closely integrated computational/experimental strategy that yielded promising results, which we are currently further pursuing: (i) we retrieved and analyzed existing structures, including a model constructed for human IFNAR1 ectodomain (EC) in the unbound state using data from two PDB structures,105,106 (ii) we determined, using the GNM core function, the hinge sites that control the relative movements of IFNAR1 EC four subdomains SD1-SD4, (iii) we identified 5 residue pairs near those regions, which exhibit large fluctuations in their inter-residue distances, <(DRij)2>, during global motions; we hypothesized that those at the interface between subdomains SD3 and SD4 would be critical to enabling subdomain rearrangements that modulate, if not optimize, IFN binding (Fig VIII.8b), and obstructing their adaptability by locking the distance between those pairs, and in particular the pair with the largest moment arm (e.g. G162-F267) with respective to the hinge site, could impair the function, (iv) in vitro binding experiments and gene induction activity assays by the Schreiber lab confirmed that hindering the conformational flexibility of IFNAR1 at those regions (by cysteine-trapping) decreased binding affinity, and suppressed activity, and the relative strengths of these effects matched our predicted rank-ordered list, (v) ANM-predicted global (energetically softest) mode of motion also showed a highly cooperative flexing movement of the entire IFNAR1 EC (Fig VIII.8c), with end-to-end distance changes in accord with FRET experiments107, (vi) ANM examination of the murine IFNAR1 structure resolved106 in IFNb-bound state, demonstrated that this structure has the intrinsic ability to readily reconfigure toward the human counterpart. This result is important: it shows that the seemingly different human and murine structures are simply alternative easily exchangeable conformers, stabilized by the different sequences and/or bound substrates, and that IFNAR1 EC favors such structural shifts as required for function, consistent with ProDy predictions. This is another illustration of the adaptability of proteins to different bound states or to sequence variations/mutations via their softest modes of motion.108 Further cross-linking, fluorescence quenching and gene induction experiments will be conducted in the Schreiber lab, in close coordination with TR&D1 computational studies at the Bahar lab.


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