The inflammasome signaling pathways are activated by infections and sterile stimulation, which lead to the maturation of inflammatory caspases that promote the secretion of inflammatory cytokines such as IL-1b and IL-18. The recognition and cleavage of the gasdermin family members by caspases trigger the activation of their pore-forming activities that lead to pyroptotic cell death. A prominent example is the targeting of gasdermin D (GSDMD) by inflammatory caspases-1/4/5/11 as an essential step in initiating pyroptosis following inflammasome activation. Previous work has identified cleavage site signatures in caspase substrates such as GSDMD and inflammatory cytokines, but it is unclear if these are the sole determinants for caspase engagement. Here we describe structural studies of a complex between caspase-1 (CASP1) and the full-length GSDMD, which reveals that the cleavage site-containing linker in GSDMD adopts a long loop structure that engages the CASP1 active site. In addition, an exosite is observed between the caspase-1 L2 and L2’ loops and a hydrophobic pocket within the GSDMD C-terminal domain distal to its N-terminal domain. The exosites endows a novel function for the GSDMD C-terminal domain as a caspase-recruitment module, in addition to its role in autoinhibition. The dual site recognition may allow stringent substrate selectivity while facilitating efficient cleavage and pyroptosis upon inflammasome activation. Such mode of tertiary structure recognition may be applicable to other physiological substrates of caspases.

Plants and animals detect pathogen infection via intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) that directly or indirectly recognize pathogen effectors and activate an immune response. How effector sensing triggers NLR activation remains poorly understood. Structure-function studies of these complexes are hampered by low levels in native tissue, our inability to express them recombinantly, and their instability in solution. We overcame sample limitation problems and solved a 3.8 Å resolution cryo-EM structure of the activated ROQ1, an NLR native to N. benthamiana with a Toll-like interleukin-1 receptor (TIR) domain, bound to the Xanthomonas effector XopQ. ROQ1 directly binds to both the predicted active site and surface residues of XopQ while forming a tetrameric resistosome that brings together the TIR domains for downstream immune signaling. Our results suggest a mechanism for the direct recognition of effectors by NLRs leading to the oligomerization-dependent activation of a plant resistosome and signaling by the TIR domain.

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors recognize pathogen effectors to trigger cell death and confer disease resistance. The Toll/interleukin-1 receptor (TIR) domains of plant NLRs can hydrolyze nicotinamide adenine dinucleotide in its oxidized form (NAD+), which is required for NLR-mediated immune signaling. The Cryo-EM structures of the RPP1 and Roq1 resistosomes show that formation of two asymmetric dimers of TIR domains is critical for the NADase activity. However, the structural mechanism underlying TIR-catalyzed NAD+ cleavage remains unknown. Here, we report a crystal structure of RPP1-TIR in complex with NAD+. The TIR domain forms a tetramer in an asymmetric unit, which is nearly identical with that seen in the RPP1 resistosome. The NAD+ is bound to the catalytic center between the asymmetric head-to-tail TIR homodimers, with the adenosine group contacting one TIR monomer (TIRb) and the phosphate groups and the nicotinamide ribose contacting the other TIR (TIRa). The nicotinamide-ribose bond of NAD+ has been cleaved, and the free nicotinamide stacks against the adenosine group. The carboxylate oxygen of the catalytic Glu158 interacts with the C-2 and C-3 hydroxyl groups of the nicotinamide ribose, and the interactions are highly conserved in the cADPR-bound ADP-ribosyl cyclase CD38. Our study reveals NAD+ recognition mechanism of a plant TIR domain and provides insight into NAD+ hydrolysis catalyzed by the TIR protein.

Most T cells found in jawed vertebrates express functional heterodimeric receptors (TCRs) on their surface formed by either α and β or γ and δ chains. Each chain possesses two domains, an amino-terminal variable domain (V) and a constant domain (C) on the carboxy-terminus (V-C pattern). In most cases, the ability of T cells to recognize diverse antigens relies on the surface (or paratope) located within Vα – Vβ or Vγ – Vδ segments. Recent genomic studies of non-eutherian mammals identified clusters of genes that resemble the classical TCR loci but surprisingly contain an additional variable segment. The functional product common for marsupials and monotremes called ‘μ chain’ was predicted to contain two variable (Vμ and Vμj) and one constant (Cμ) domains. Single cells analysis of blood and spleen from Monodelphis domestica showed that some of the splenic T cells co-express the μ and γ chains suggesting that both polypeptides could form a novel type of T cell receptor, the γμTCR. Using obtained sequences, we generated and structurally characterized two different γμTCRs. Here, we present the novel and unusual architecture of a third lineage of T cell receptor found in marsupials and monotremes [1].

[1] Morrissey K.A., Wegrecki M., Praveena T., Hansen V.L., Bu L., Sivaraman K.K., Darko S., Douek D.C., Rossjohn J., Miller R.D., Le Nours J. The molecular assembly of the marsupial T cell receptor defines a third T cell lineage. SCIENCE, 371, 1383-1388, 2021.

Human surfactant protein D is a collectin and member of the C-type lectin superfamily of proteins that forms an essential part of the mammalian innate immune system. The collectins have been recognised to not only bind to invading pathogens, allowing for recognition by immune cells, but also play an important role in activating and regulating the response of both the innate and acquired immune systems. Crystal structures of a biologically and therapeutically active recombinant homotrimeric fragment of human SP-D (hSP-D) complexed with the inner core oligosaccharides from gram negative bacterial human pathogens Haemophilus influenzae, Salmonella enterica sv Minnesota rough strains [1-2] and Escherichia coli provide unique multiple insights into the recognition and binding of bacterial lipopolysaccharide (LPS) by hSP-D. LPS binding is achieved through calcium dependent recognition of the proximal inner core heptose dihydroxyethyl side chain coupled with specific interactions with the binding site flanking residues Arg343 and Asp325 and evidence for an extended binding site for LPS inner cores. Where this preferred mode of binding is precluded by the crystal lattice, oligosaccharide is bound through a terminal core glucose.

The structures thus reveal that hSP-D specifically and preferentially targets the LPS inner core via the innermost conserved heptose (Hep) with the flexibility and versatility to adopt alternative strategies for bacterial recognition, utilising alternative LPS epitopes including terminal or non-terminal sugars, when the preferred inner core Hep is not available for binding. Alongside binding studies of both whole bacteria and LPS the structures also demonstrate that carbohydrate extensions to the core LPS oligosaccharide, previously thought to be targets for collectins, are important in shielding the more vulnerable target sites in the LPS core. Recent structures of hSP-D bound with small synthetic LPS core component ligands which include phosphorylation of specific carbohydrate residues further demonstrate not only the ability to adopt alternative modes of recognition but also that LPS phosphorylation can provide an additional mechanism by which pathogens can efficiently evade a first-line mucosal innate immune defence.

[1] Clark, H. W., Mackay, R. M., Deadman, M. E., Hood, D. W., Madsen, J., Moxon, E. R., Townsend, J. P., Reid, K. B. M., Ahmed, A., Shaw, A. J., Greenhough, T. J. & Shrive, A. K. (2016). Infection & Immunity 84, 1585.

[2] Littlejohn, J. R., da Silva, R. F., Neale, W. A., Smallcombe, C. C., Clark, H. W., Mackay, R. M. A., Watson, A. S., Madsen, J., Hood, D. W., Burns, I., Greenhough, T. J. & Shrive, A. K. (2018). PLOS One 13, e0199175.

Metabolite based T cell immunity is emerging as a major player in antimicrobial immunity, autoimmunity and cancer. Here, small molecule metabolites were identified to be captured and presented by the major histocompatibility complex (MHC) class-I related molecule MR1 to T cells, namely Mucosal Associated Invariant T cells (MAIT) and diverse MR1-restricted T cells. Both MR1 and MAIT T cell receptors (TCR) are evolutionarily conserved in many mammals, suggesting important roles in host immunity. Namely, during infection with riboflavin-producing microorganisms, MR1 trapped riboflavin-based metabolites and presented on the surface of the antigen-presenting cells encountering the MAIT TCR leading to the activation of the MAIT cells. How modifications to these small molecule-metabolites affect presentation by MR1 and MAIT cell activation remains unclear.
To dissect the molecular basis underpinning MR1 antigen capture and MAIT recognition, we chemically synthesized and characterized a large panel of these naturally occurring metabolites, termed “altered metabolite ligands” (AMLs), and investigated functionally and structurally their impact on the MR1-MAIT axis. Through the generation and detailed analysis of 13 high-resolution MAIT TCR-MR1-AML crystal structures, along with biochemical and functional assays, we show that the propensity of MR1-upregulation on the cell surface was related to the nature of MR1-AML interactions. MR1-AML adaptability and a dynamic compensatory interplay at the TCR-AML-MR1 interface impacted the affinity of the TCR-MR1-AML interaction, which ultimately underscored the ability of the AMLs to activate MAIT cells. Here, I will provide a generalized framework for metabolite recognition and modulation of MAIT cells. Further, I will discuss the design, use and implications of “AMLs” for selective and specific tailoring of T cell immune responses.

1. Awad, W. ^#, Ler, G.M.^# et al. (2020). The molecular basis underpinning the potency and specificity of MAIT cell antigens. Nature Immunology 21, 400-411.
2. Salio, M.^#, Awad, W.^# et al. (2020). Ligand-dependent downregulation of MR1 cell surface expression. PNAS, 117(19), 10465–10475.
3. Awad W, et al. (2020). Atypical TRAV1-2- T cell receptor recognition of the antigen-presenting molecule MR1. J. Biol. Chem. 295(42), 14445–14457.