Targeting innate immunity holds promise in cancer immunotherapy, particularly in improving checkpoint inhibitors. However, the use of agonists of the promising innate receptors TLRs and STING1-4 is facing challenges. Here we examined the antitumour function of the α-kinase 1 (ALPK1) receptor for bacterial ADP-heptose (ADP-Hep)5-7. Treatment of mice with ADP-Hep induced multiple proinflammatory factors including CXCL10 and CCL2, and stimulated Alpk1-dependent antitumour immunity. Mice bearing a gain-of-function ALPK1(T237M) disease variant8 also rejected grafted tumours. Using medicinal chemistry, we identified a more potent analogue, UDSP-Hep. In contrast to ADP-Hep, UDSP-Hep distinguished Alpk1 polymorphism, which correlates with mouse susceptibility to bacteria-associated colitis9-12. UDSP-Hep exhibited a stronger Alpk1-mediated antitumour effect and synergized with checkpoint inhibitors. The effect required CD8+ T cells, dendritic cells (DCs) and macrophages, and was sensitive to antibodies that block CXCL10 or CCL2 function. ALPK1 agonists activated DCs for cross-presentation, promoting tumour-specific T cell expansion in the tumour-draining lymph nodes. ALPK1 has wider expression than STING in non-immune cells with a distinct inflammatory signature. UDSP-Hep is differentiated from STING agonists in stimulating tumour-cell antigen presentation, macrophage-DC cross-priming and protective memory T cell differentiation, and it does not induce T cell apoptosis. Our study elucidates the antitumour effect of ALPK1 agonism and suggests the potential of ALPK1 agonists in cancer immunotherapy.

Bacterial small molecule metabolites such as adenosine-diphosphate- d - glycero -β- d - manno -heptose (ADP-heptose) and their derivatives act as effective innate immune agonists in mammals. We show that functional nucleotide-diphosphate-heptose biosynthetic enzymes (HBEs) are distributed widely in bacteria, archaea, eukaryotes, and viruses. We identified a conserved STT R5 motif as a hallmark of heptose nucleotidyltransferases that can synthesize not only ADP-heptose but also cytidine-diphosphate (CDP)– and uridine-diphosphate (UDP)–heptose. Both CDP- and UDP-heptoses are agonists that trigger stronger alpha-protein kinase 1 (ALPK1)–dependent immune responses than ADP-heptose in human and mouse cells and mice. We also produced ADP-heptose in archaea and verified its innate immune agonist functions. Hence, the β- d - manno -heptoses are cross-kingdom, small-molecule, pathogen-associated molecular patterns that activate the ALPK1-dependent innate immune signaling cascade.

Cytotoxic lymphocyte-derived granzyme A (GZMA) cleaves GSDMB, a gasdermin-family pore-forming protein1,2, to trigger target cell pyroptosis3. GSDMB and the charter gasdermin family member GSDMD4,5 have been inconsistently reported to be degraded by the Shigella flexneri ubiquitin-ligase virulence factor IpaH7.8 (refs. 6,7). Whether and how IpaH7.8 targets both gasdermins is undefined, and the pyroptosis function of GSDMB has even been questioned recently6,8. Here we report the crystal structure of the IpaH7.8-GSDMB complex, which shows how IpaH7.8 recognizes the GSDMB pore-forming domain. We clarify that IpaH7.8 targets human (but not mouse) GSDMD through a similar mechanism. The structure of full-length GSDMB suggests stronger autoinhibition than in other gasdermins9,10. GSDMB has multiple splicing isoforms that are equally targeted by IpaH7.8 but exhibit contrasting pyroptotic activities. Presence of exon 6 in the isoforms dictates the pore-forming, pyroptotic activity in GSDMB. We determine the cryo-electron microscopy structure of the 27-fold-symmetric GSDMB pore and depict conformational changes that drive pore formation. The structure uncovers an essential role for exon-6-derived elements in pore assembly, explaining pyroptosis deficiency in the non-canonical splicing isoform used in recent studies6,8. Different cancer cell lines have markedly different isoform compositions, correlating with the onset and extent of pyroptosis following GZMA stimulation. Our study illustrates fine regulation of GSDMB pore-forming activity by pathogenic bacteria and mRNA splicing and defines the underlying structural mechanisms.

Chemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) is widely used to study protein-protein interactions (PPI), protein structures, and even protein dynamics. However, structural information provided by CXMS is still limited, partly because most CXMS experiments use lysine-lysine (K-K) cross-linkers. Although superb in selectivity and reactivity, they are ineffective for lysine deficient regions. Herein, we develop aromatic glyoxal cross-linkers (ArGOs) for arginine-arginine (R-R) cross-linking and the lysine-arginine (K-R) cross-linker KArGO. The R-R or K-R cross-links generated by ArGO or KArGO fit well with protein crystal structures and provide information not attainable by K-K cross-links. KArGO, in particular, is highly valuable for CXMS, with robust performance on a variety of samples including a kinase and two multi-protein complexes. In the case of the CNGP complex, KArGO cross-links covered as much of the PPI interface as R-R and K-K cross-links combined and improved the accuracy of Rosetta docking substantially.

Immune recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors often activates proinflammatory NF-κB signalling 1 . Recent studies indicate that the bacterial metabolite d -glycero-β- d -manno-heptose 1,7-bisphosphate (HBP) can activate NF-κB signalling in host cytosol 2 – 4 , but it is unclear whether HBP is a genuine PAMP and the cognate pattern recognition receptor has not been identified. Here we combined a transposon screen in Yersinia pseudotuberculosis with biochemical analyses and identified ADP-β- d -manno-heptose (ADP-Hep), which mediates type III secretion system-dependent NF-κB activation and cytokine expression. ADP-Hep, but not other heptose metabolites, could enter host cytosol to activate NF-κB. A CRISPR–Cas9 screen showed that activation of NF-κB by ADP-Hep involves an ALPK1 (alpha-kinase 1)–TIFA (TRAF-interacting protein with forkhead-associated domain) axis. ADP-Hep directly binds the N-terminal domain of ALPK1, stimulating its kinase domain to phosphorylate and activate TIFA. The crystal structure of the N-terminal domain of ALPK1 and ADP-Hep in complex revealed the atomic mechanism of this ligand–receptor recognition process. HBP was transformed by host adenylyltransferases into ADP-heptose 7-P, which could activate ALPK1 to a lesser extent than ADP-Hep. ADP-Hep (but not HBP) alone or during bacterial infection induced Alpk1 -dependent inflammation in mice. Our findings identify ALPK1 and ADP-Hep as a pattern recognition receptor and an effective immunomodulator, respectively.

Nature 535, 111–116 (2016); doi:10.1038/nature18590 In this Article, owing to a typesetter error the ‘received date’ was incorrectly shown as ‘6 March 2015’ instead of ‘6 March 2016’; this has been corrected in the online versions of the paper.