Jan Felix

The interleukin-36 receptor (IL-36R) drives proinflammatory signaling and is therapeutically targeted by spesolimab in acute skin inflammation. Beyond its three canonical agonists (IL-36α, IL-36β, and IL-36γ), IL-36R was recently proposed to be activated by IL-37. Together with antagonism by IL-36R antagonist (IL-36RA) and IL-38, this creates a complex structure-function landscape that remains poorly understood. Here, we report the cryo–electron microscopy (cryo-EM) structure of human IL-36γ bound to IL-36R and the shared co-receptor IL-1R accessory protein (IL-1RAcP). IL-36γ engages IL-36R with low affinity, enabling high-affinity IL-1RAcP recruitment. IL-37 exhibits the opposite binding signature, binding IL-36R with high affinity but IL-1RAcP weakly. Functional comparison of IL-36R engagement by IL-36γ and IL-37 confirmed their common proinflammatory signature, with IL-36γ exhibiting markedly higher potency, consistent with its more stable signaling complex. Structural comparison of cytokine-bound versus antibody-antagonized IL-36R revealed that spesolimab acts as an allosteric antagonist. This study therefore provides a structural and mechanistic blueprint for IL-36R activation by distinct cytokines.

Influenza B viruses are antigenically diverse and contribute significantly to the annual influenza burden. Here we report influenza B virus neutralizing single-domain antibodies that target highly conserved regions of the hemagglutinin and neuraminidase. Structural studies by single particle electron cryo-microscopy (cryo-EM) revealed that one of these single-domain antibodies prevents the conformational transition of the viral hemagglutinin to the post-fusion state by targeting a quaternary epitope spanning two protomers in the hemagglutinin-stem region. A second single-domain antibody broadly inhibits influenza B neuraminidase activity, including an oseltamivir-resistant neuraminidase, and its complex with neuraminidase elucidated by single particle cryo-EM established that it binds to residues in the neuraminidase catalytic site. Head-to-tail fusions of these single-domain antibodies led to bispecific binders that further improved the neutralization breadth and potency against influenza B viruses. These single-domain antibodies, fused to a human IgG1-Fc domain, fully protected female mice against an otherwise lethal influenza B virus challenge. Our findings underscore the potential of engineered single-domain antibodies to help control influenza B virus infections.

Activation of Anaplastic lymphoma kinase (ALK) and leukocyte tyrosine kinase (LTK) by their cognate cytokines ALKAL2 and ALKAL1 plays important roles in development, metabolism, and cancer. Recent structural studies revealed ALK/LTK-cytokine assemblies with distinct stoichiometries. Structures of ALK-ALKAL2 and LTK-ALKAL1 complexes with 2:1 stoichiometry determined by X-ray crystallography contrasted the 2:2 ALK-ALKAL2 complexes determined by cryo-EM and X-ray crystallography. Here, we show based on reanalysis of the cryo-EM data deposited in EMPIAR-10930 that over half of the ALK-ALKAL2 particles in the dataset are classified into 2D and 3D classes obeying a 2:1 stoichiometry besides the originally reported structure displaying 2:2 stoichiometry. Unlike particles representing the 2:2 ALK-ALKAL2 complex, particles for the 2:1 ALK-ALKAL2 complex suffer severely from preferred orientations that resulted in cryo-EM maps displaying strong anisotropy. Here, we show that extensive particle orientation rebalancing in cryoSPARC followed by 3D refinement with Blush regularization in RELION constitutes an effective strategy for avoiding map artifacts relating to preferred particle orientations and report a 3D reconstruction of the 2:1 ALK-ALKAL2 complex to 3.2 Å resolution from EMPIAR-10930. This new cryo-EM structure together with the crystal structures of ALK-ALKAL2 and LTK-ALKAL1 complexes with 2:1 stoichiometry reconciles a common receptor dimerization mode for ALK and LTK and provides direct evidence for the presence of an ALK-ALKAL2 complex with 2:1 stoichiometry next to the reported 2:2 stoichiometric assembly in the EMPIAR-10930 dataset. Finally, our analysis emphasizes the importance of public deposition of raw cryo-EM data to allow reanalysis and interpretation.

Interleukin-36 receptor (IL-36R) is activated by IL-36α, IL-36β, and IL-36γ to elicit pro-inflammatory signaling and is targeted in acute skin inflammation by the approved antibody spesolimab. IL-37 was recently proposed as a new IL-36R agonist. Such diverse agonist repertoire together with the antagonistic IL-36Ra and IL-38 create a fascinating structure-function landscape for IL-36R, albeit one that is poorly understood. Here, we elucidate how IL-36R grapples IL-36γ with low affinity to enable facile recruitment of the shared receptor IL-1RAcP with high-affinity. In contrast, IL-36R interacts with IL-37 via the exact opposite binding signature. Comparative interrogation of IL-36R activation by IL-36γ and IL-37 confirmed their common pro-inflammatory signature and distinguished IL-36γ as markedly more pro-inflammatory. Structural comparisons of cytokine-activated versus spesolimab-antagonized IL-36R revealed spesolimab’s mode of action as an allosteric antagonist. Collectively, our study provides the structural and mechanistic blueprint of IL-36R activation by distinct cytokines and will facilitate its therapeutic targeting. One Sentence Summary: Structural blueprint for IL-36R activation by cognate cytokines and its antagonism by spesolimab in generalized pustular psoriasis.

Anaplastic lymphoma kinase (ALK) and leukocyte tyrosine kinase (LTK) are receptor tyrosine kinases activated by their cognate ligands ALKAL2 and ALKAL1 resulting in pleiotropic signalling outputs in development, metabolism and cancer. Structural studies by X-ray crystallography and electron cryo-microscopy (cryo-EM) recently revealed distinct assemblies corresponding to ALK-ALKAL2 and LTK-ALKAL1 assemblies with either 2:1 stoichiometry versus ALK-ALKAL2 complexes with 2:2 stoichiometry. Here, we report the reanalysis of cryo-EM data deposited by Reshetnyak et al. (EMPIAR-10930) and show that over half of the particles in the dataset correspond to ALK-ALKAL2 complexes obeying a 2:1 stoichiometry besides the originally reported structure displaying 2:2 stoichiometry. Our workflow critically relied on extensive particle orientation rebalancing in cryoSPARC followed by 3D refinement with Blush regularization in RELION leading to a 3D reconstruction of the 2:1 ALK-ALKAL2 complex to 3.2 Å resolution. This cryo-EM structure together with the reported crystal structures of ALK-ALKAL2 and LTK-ALKAL1 complexes with 2:1 stoichiometry, reconcile a common receptor dimerization mode for ALK and LTK poised for the facile juxtapositioning of their intracellular kinase domains for signalling.

Activation of Anaplastic lymphoma kinase (ALK) and leukocyte tyrosine kinase (LTK) by their cognate cytokines ALKAL2 and ALKAL1 play important roles in development, metabolism, and cancer. Recent structural studies revealed ALK/LTK-cytokine assemblies with distinct stoichiometries. Structures of ALK-ALKAL2 and LTK-ALKAL1 complexes with 2:1 stoichiometry determined by X-ray crystallography (De Munck et al. Nature, 2021) contrasted the 2:2 ALK-ALKAL2 complexes determined by cryo-EM (Reshetnyak et al., Nature, 2021) and X-ray crystallography (Li et al ., Nature, 2021). Here, we show based on reanalysis of the cryo-EM data deposited in EMPIAR- 10930 that over half of the ALK-ALKAL2 particles in the dataset are classified into 2D classes obeying a 2:1 stoichiometry besides the originally reported structure displaying 2:2 stoichiometry. Unlike particles representing the 2:2 ALK-ALKAL2 complex, particles for the 2:1 ALK-ALKAL2 complex suffer severely from preferred orientations that resulted in cryo-EM maps displaying strong anisotropy. Here, we show that extensive particle orientation rebalancing in cryoSPARC followed by 3D refinement with Blush regularization in RELION constitutes an effective strategy for avoiding map artefacts relating to preferred particle orientations and report a 3D reconstruction of the 2:1 ALK-ALKAL2 complex to 3.2 Å resolution from EMPIAR-10930. This new cryo-EM structure together with the crystal structures of ALK-ALKAL2 and LTK-ALKAL1 complexes with 2:1 stoichiometry reconciles a common receptor dimerization mode for ALK and LTK and provides direct evidence for the presence of an ALK-ALKAL2 complex with 2:1 stoichiometry next to the reported 2:2 stoichiometric assembly in the EMPIAR-10930 dataset. Finally, our analysis emphasizes the importance of public deposition of raw cryo-EM data to allow reanalysis and interpretation.

Cell-surface receptor complexes mediated by pro-inflammatory interleukin (IL)-12 and IL-23, both validated therapeutic targets, are incompletely understood due to the lack of structural insights into their complete extracellular assemblies. Furthermore, there is a paucity of structural details describing the IL-12–receptor interaction interfaces, in contrast to IL-23–receptor complexes. Here we report structures of fully assembled mouse IL-12/human IL-23–receptor complexes comprising the complete extracellular segments of the cognate receptors determined by electron cryo-microscopy. The structures reveal key commonalities but also surprisingly diverse features. Most notably, whereas IL-12 and IL-23 both utilize a conspicuously presented aromatic residue on their α-subunit as a hotspot to interact with the N-terminal Ig domain of their high-affinity receptors, only IL-12 juxtaposes receptor domains proximal to the cell membrane. Collectively, our findings will help to complete our understanding of cytokine-mediated assemblies of tall cytokine receptors and will enable a cytokine-specific interrogation of IL-12/IL-23 signaling in physiology and disease.

The adipokine Leptin activates its receptor LEP-R in the hypothalamus to regulate body weight and exerts additional pleiotropic functions in immunity, fertility and cancer. However, the structure and mechanism of Leptin-mediated LEP-R assemblies has remained unclear. Intriguingly, the signaling-competent isoform of LEP-R is only lowly abundant amid several inactive short LEP-R isoforms contributing to a mechanistic conundrum. Here we show by X-ray crystallography and cryo-EM that, in contrast to long-standing paradigms, Leptin induces type I cytokine receptor assemblies featuring 3:3 stoichiometry and demonstrate such Leptin-induced trimerization of LEP-R on living cells via single-molecule microscopy. In mediating these assemblies, Leptin undergoes drastic restructuring that activates its site III for binding to the Ig domain of an adjacent LEP-R. These interactions are abolished by mutations linked to obesity. Collectively, our study provides the structural and mechanistic framework for how evolutionarily conserved Leptin:LEP-R assemblies with 3:3 stoichiometry can engage distinct LEP-R isoforms to achieve signaling.

Cell-surface receptor complexes mediated by pro-inflammatory Interleukin-12 and -23, both validated therapeutic targets, are incompletely understood due to the lack of structural insights into their complete extracellular assemblies. Furthermore, there is a paucity of structural details describing the IL-12:receptor interaction interfaces, in contrast to IL23:receptor complexes. Here we report cryo-EM structures of fully assembled IL-12/IL-23:receptor complexes comprising the complete extracellular segments of the cognate receptors. The structures reveal important commonalities but also surprisingly diverse features. Whereas IL-12 and IL-23 both utilize a conspicuously presented aromatic residue on their α-subunit as a hotspot to interact with the N-terminal Ig-domain of their high affinity receptors, only IL-12 juxtaposes receptor domains proximal to the cell-membrane. Collectively, our findings will enable a cytokine-specific interrogation of IL-12 and IL-23 signaling in physiology and disease.