Interleukin-2 druggability is modulated by global conformational transitions controlled by a helical capping switch.

Interleukin-2 (IL-2) is a small α-helical cytokine that regulates immune cell homeostasis through its recruitment to a high-affinity heterotrimeric receptor complex (IL-2Rα/IL-2Rβ/γc). IL-2 has been shown to have therapeutic efficacy for immune diseases by preferentially expanding distinct T cell compartments, and several regulatory T cell (Treg)-biasing anti-IL-2 antibodies have been developed for combination therapies. The conformational plasticity of IL-2 plays an important role in its biological actions by modulating the strength of receptor and drug interactions. Through an NMR analysis of milliseconds-timescale dynamics of free mouse IL-2 (mIL-2), we identify a global transition to a sparse conformation which is regulated by an α-helical capping "switch" at the loop between the A and B helices (AB loop). Binding to either an anti-mouse IL-2 monoclonal antibody (mAb) or a small molecule inhibitor near the loop induces a measurable response at the core of the structure, while locking the switch to a single conformation through a designed point mutation leads to a global quenching of core dynamics accompanied by a pronounced effect in mAb binding. By elucidating key details of the long-range allosteric communication between the receptor binding surfaces and the core of the IL-2 structure, our results offer a direct blueprint for designing precision therapeutics targeting a continuum of conformational states

Facile discovery of surrogate cytokine agonists

Cytokines are powerful immune modulators that initiate signaling through receptor dimerization, but natural cytokines have structural limitations as therapeutics. We present a strategy to discover cytokine surrogate agonists by using modular ligands that exploit induced proximity and receptor dimer geometry as pharmacological metrics amenable to high-throughput screening. Using VHH and scFv to human interleukin-2/15, type-I interferon, and interleukin-10 receptors, we generated combinatorial matrices of single-chain bispecific ligands that exhibited diverse spectrums of functional activities, including potent inhibition of SARS-CoV-2 by surrogate interferons. Crystal structures of IL-2R:VHH complexes revealed that variation in receptor dimer geometries resulted in functionally diverse signaling outputs. This modular platform enabled engineering of surrogate ligands that compelled assembly of an IL-2R/IL-10R heterodimer, which does not naturally exist, that signaled through pSTAT5 on T and natural killer (NK) cells. This “cytokine med-chem” approach, rooted in principles of induced proximity, is generalizable for discovery of diversified agonists for many ligand-receptor systems

A Transmembrane Domain GGxxG Motif in CD4 Contributes to Its Lck-Independent Function but Does Not Mediate CD4 Dimerization.

CD4 interactions with class II major histocompatibility complex (MHC) molecules are essential for CD4+ T cell development, activation, and effector functions. While its association with p56lck (Lck), a Src kinase, is important for these functions CD4 also has an Lck-independent role in TCR signaling that is incompletely understood. Here, we identify a conserved GGxxG motif in the CD4 transmembrane domain that is related to the previously described GxxxG motifs of other proteins and predicted to form a flat glycine patch in a transmembrane helix. In other proteins, these patches have been reported to mediate dimerization of transmembrane domains. Here we show that introducing bulky side-chains into this patch (GGxxG to GVxxL) impairs the Lck-independent role of CD4 in T cell activation upon TCR engagement of agonist and weak agonist stimulation. However, using Forster's Resonance Energy Transfer (FRET), we saw no evidence that these mutations decreased CD4 dimerization either in the unliganded state or upon engagement of pMHC concomitantly with the TCR. This suggests that the CD4 transmembrane domain is either mediating interactions with an unidentified partner, or mediating some other function such as membrane domain localization that is important for its role in T cell activation

A Mechanical Switch Couples T Cell Receptor Triggering to the Cytoplasmic Juxtamembrane Regions of CD3ζζ

The eight-subunit T cell receptor (TCR)-CD3 complex is the primary determinant for T cell fate decisions. Yet how it relays ligand-specific information across the cell membrane for conversion to chemical signals remains unresolved. We hypothesized that TCR engagement triggers a change in the spatial relationship between the associated CD3ζζ subunits at the junction where they emerge from the membrane into the cytoplasm. Using three in situ proximity assays based on ID-PRIME, FRET, and EPOR activity, we determined that the cytosolic juxtamembrane regions of the CD3ζζ subunits are spread apart upon assembly into the TCR-CD3 complex. TCR engagement then triggered their apposition. This mechanical switch resides upstream of the CD3ζζ intracellular motifs that initiate chemical signaling, as well as the polybasic stretches that regulate signal potentiation. These findings provide a framework from which to examine triggering events for activating immune receptors and other complex molecular machines.National Institutes of Health (U.S.) (5R01CA186568

Mutating the CD4 TMD GGxxG motif does not impair dimerization at steady-state.

<p>(A) Grey-scale images of mCherry (top) and GFP (bottom) intensities pre and post mCherry ablation for representative cells. (B) Plot of relative mCherry values pre- and post-ablation for all cells analyzed. The average ablation of all populations was below 10% and did not differ significantly from each other. (C) FRET_E values for CD28^GFP/Ch, PD1^GFP/Ch and CD4T^GFP/Ch cells. Representative of two experiments with independently generated cell lines. (D) FRET_E values for CD4WT^GFP/Ch vs. CD4T^GFP/Ch cells. Concatenated data from two independently generated cells lines is shown because the CD4WT^GFP/Ch cells had a broader range of donor and acceptor levels that limited the number of cells available for subset analysis (methods). (E) FRET_E values for CD4T^GFP/Ch vs. CD4T^TMD-GFP/Ch cells. Representative of two experiments with independently generated cell lines. Matched expression for analysis was based on median pre-bleach intensity. Dots represent single cells and green bars represent median values (*p<0.05, **p<0.001; ***p<0.0001; Mann-Whitney).</p