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  1. Evaluation Summary:

    This manuscript is of interest to colleagues in immunity and signal transduction. Cytokines are small protein signaling molecules with a diverse range of activities in inflammation and immune system function. This manuscript reports the cryo-EM structure of the cytokine interleukin-27 (IL-27) bound to soluble domains of two receptor subunits, IL-27Rα and gp130. IL-27 is a composite cytokine consisting of the protein p28 bound to EBI3, which resembles soluble cytokine receptors such as the receptors for IL-6, IL-11 or CNTF. IL-27 signals predominantly via STAT1 and plays an important role in immune homeostasis. The data provide a detailed molecular view of how IL-27 binds to its receptor.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  2. Reviewer #1 (Public Review):

    Caveney et al. set out to use the technique of cryo-electron microscopy to deduce how the cytokine interleukine-27 (IL-27) interacts with its heterodimeric receptor composed of gp130 and IL-27Rα. Here they find that the quaternary structure of the human IL-27/gp130/IL27Rα complex contains three interface sites that are characteristic of other gp130 family cytokines. This work indicates that the interface between EBI3 and IL-27p28, the two subunits that make up the heterodimeric cytokine IL-27, is highly hydrophobic in nature, providing an indication of how these subunits pair for efficient secretion. A conserved tryptophan residue was identified in the IL-27p28 subunit that is also found in IL-6 and viral IL-6 that facilitates its interaction with domain 1 of gp130. Importantly, the results indicate that the interaction of IL-27 with its receptor closely resembles the interaction of IL-6 with its receptor rather than the other related heterodimeric cytokines IL-12 and IL-23. Whereas IL-27p28 lies centrally allowing it to interact with all receptor components, like IL-6, each subunit of IL-12 and IL-23 engages a separate subunit of the receptor. Moreover, the overall architecture of the IL-27-IL-27R complex also helps to explain previous findings that indicated IL-27p28 can block IL-6 and why soluble IL-27p28 cannot induce signaling through the receptor in the absence of the EBI3 subunit.

    Limitations associated with the study include an inability to purify a soluble receptor complex composed of human IL-27, IL-27Rα domains 1-2, and gp130 domains 1-3 due to dissociation during size-exclusion chromatography that the authors based on the low affinity of gp130 for binding IL-27. To deal with this purification issue, the addition of a flexible linker of 20 amino acids was required to connect IL-27p28 to gp130 to facilitate purification of the cytokine-receptor complex. While this linker creates an artificial connection between these proteins, the authors conclude that it does not restrict the binding of IL-27p28 to gp130. Furthermore, while the authors highlight the amino acids important for the interaction between the IL-27-IL-27R subunits and suggest that they are plausible sites to target to antagonize this interaction the study does not set forth to test this idea here. Moving forward, it will be of interest to see if monoclonal antibodies can be identified or designed to block these key interactions between the cytokine and receptor subunits.

    Overall, the conclusions of this paper are supported by the data, and the data provide valuable information for the design and testing of therapeutics to block or agonize the interaction of this important anti-inflammatory cytokine with its cognate receptor.

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  3. Reviewer #2 (Public Review):

    The cytokine IL-27 is structurally interesting because its receptor-like subunit EBI3 can also form other heterodimeric cytokines, namely IL-35, in which EBI3 binds to p35 (the cytokine-like subunit of IL-12). In the cytokine IL-39, EBI3 binds to p19 (the cytokine-like subunit of IL-23). Moreover, the four-helical protein p28 has been described to interact with the IL-6R. In this case, p28 assembles a receptor complex consisting of a homodimer of gp130, which predominantly signals via STAT3 and therefore elicits intracellular responses different from IL-27. It would be highly desirable that the structure of the complex of cytokine IL-27 in complex with the two receptor subunits gp130 and WSX-1 would explain the structural flexibility of p28 and EBI3. The resolution of the presented structure, which has been obtained by cryogenic-electron microscopy, is 3.47 Å, which might not be high enough to yield such a detailed molecular explanation.

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  4. Reviewer #3 (Public Review):

    Cytokines are small proteins that play key roles in inflammation and immune homeostasis. In order to elicit proper signaling, cytokines must interact with a broad repertoire of receptors in complex ways, often requiring several stabilizing binding partners. This work reveals a structure of the four-part cytokine/receptor complex, composed of the heterodimeric cytokine, interleukin 27, bound to soluble fragments of two different receptors, IL-27Ra and gp130 thus providing insight into signaling mechanisms and a blueprint for targeted therapeutic intervention

    Strengths:

    The expression and purification of active membrane protein receptors can be an extremely difficult process. Furthermore, the reconstitution of stable multi-component complexes can be especially difficult. The authors employ a clever strategy to increase the stability of IL-27/IL-27Ra and gp130 by tethering gp130 and p28 via a flexible linker.

    A thorough comparative analysis of existing cytokine/receptor structures is done, which clearly lays out the key differences and similarities between IL-27/IL-27Ra/gp130 and other complexes categorizing it with IL-6 instead of IL-12 which helps reconcile existing signaling data.

    The map and model were provided, which is appreciated. The map supports the reported resolution of 3.5Å and modeled density is consistent.

    Figures are clear, consistent, and well-thought-out.

    Weaknesses:

    Other than the structure, no new comparison structures or additional biochemistry or cell biology experiments were done to support the more speculative/future directions discussion, however, a comparative analysis was done with existing structures (see above).

    The aim was to determine the molecular details of IL-27 engaged with its receptors and the conclusions are a straightforward comparative analysis of this structure with other cytokine/receptor structures.

    This is a clever strategy for obtaining a multi-part soluble monodisperse cytokine/receptor complex suitable for structural analysis which can be applied to other similar complexes, or this complex engaged with potential therapeutics. The molecular details of the structure will aid the design of function-altering mutations which can be used to reveal more specifics about the signaling mechanisms, as well as guide the rational design of potential therapeutics.

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