The caspase-4/11-GSDMD pyroptosis axis recognizes cytosolic lipopolysaccharide for antibacterial defenses. Shigella flexneri employs an OspC3 effector to block pyroptosis by catalyzing NAD+-dependent arginine ADP-riboxanation of caspase-4/11. Here, we identify Ca2+-free calmodulin (CaM) that binds and stimulates OspC3 ADP-riboxanase activity. Crystal structures of OspC3–CaM and OspC3–caspase-4 binary complexes reveal unique CaM binding to an OspC3 N-terminal domain featuring an ADP-ribosyltransferase-like fold and specific recognition of caspase-4 by an OspC3 ankryin repeat domain, respectively. CaM–OspC3–caspase-4 ternary complex structures show that NAD+ binding reorganizes the catalytic pocket, in which D231 and D177 activate the substrate arginine for initial ADP-ribosylation and ribosyl 2′-OH in the ADP-ribosylated arginine, respectively, for subsequent deamination. We also determine structures of unmodified and OspC3-ADP-riboxanated caspase-4. Mechanisms derived from this series of structures covering the entire process of OspC3 action are supported by biochemical analyses in vitro and functional validation in S. flexneri-infected mice.
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The atomic coordinates and structure factors generated in this study have been deposited in the Protein Data Bank (PDB) under the accession codes 7WR0, 7WR1, 7WR2, 7WR3, 7WR4, 7WR5 and 7WR6. Structural coordinates for the previously determined p30 form of caspase-11, the p20/p10 forms of caspase-4 and caspase-11, ADP-ribose-bound Af1521, myosin V IQ motif-bound CaM, diphtheria toxin and cholera toxin, under the PDB accession codes 6KMT, 6KMZ, 6KMU, 2BFQ, 2IX7, 1TOX and 2A5F, respectively, are also used in this study. Source data are provided with this paper.
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We thank Y. Chen (Institute of Biophysics, CAS) for assistance with ITC experiments, S. Gao (Institute of Tibetan Plateau Research, CAS) for assistance with ICP-MS experiments and the staff of BL19U1 beamlines at National Center for Protein Science Shanghai and Shanghai Synchrotron Radiation Facility for assistance during data collection. This work was supported by CAS Strategic Priority Research Program of the Chinese Academy of Sciences XDB29020202 (J.D.) and XDB37030202 (F.S.), National Key Research and Development Programs of China 2017YFA0504000 (J.D.) and 2017YFA0505900 (F.S.), the NSFC Basic Science Center Project No. 81788104 (F.S.) and Excellent Young Scholar Program No. 81922043 (J.D.), the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences 2019-I2M-5-084 (F.S.) and a grant from the Youth Innovation Promotion Association CAS to J.D.
The authors declare no competing interests.
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Extended Data Fig. 1 Biochemical and structural characterization of unmodified and OspC3-modified caspase-4.
a, b, Mapping the region in OspC3 responsible for modification and inactivation of caspase-4/11. a, CASP4-/- HeLa cells stably expressing Flag-caspase-4 were transfected with 3xHA-OspC3 (full-length (FL) or an indicated truncation mutant), and then electroporated with LPS. ATP-based cell viability was measured 2 h post-electroporation. Data are means (bars) of three individual replicates (circles). Anti-Flag immunoprecipitates were subjected to anti-ADPR and anti-Flag immunoblotting. Expression of OspC3 was analyzed by anti-HA immunoblotting with tubulin as the loading control. b, Caspase-4/11-p30-C/A was reacted with NAD+ and OspC3 (FL or an indicated truncation mutant). The reactions were subjected to native/SDS-PAGE analyses. Control, fully ADP-riboxanated caspase-4/11 obtained by co-expression with OspC3 in E. coli. c, Gel-filtration chromatography and native/SDS-PAGE analyses of unmodified and OspC3-modified caspase-4/11-p30-C/A. d, Crystal packing of unmodified caspase-4-p30-C/A and superimposition with structure of CASP4-p20/p10 dimer. CASP4-p20/p10 dimer and neighboring symmetric caspase-4-p30 molecules around an asymmetric unit are colored as indicated. The superimposition indicates that CASP-p30 did not form the antiparallel dimer as CASP4-p20/p10 in the crystal. e, Gel-filtration chromatography combined with SDS-PAGE analyses of the complex between OspC3-modified caspase-4/11-p30-C/A and Af1521. f, Structural comparison of Af1521 bound with ADP-riboxanated arginine (this study) with Af1521 bound with ADP-ribose (PDB code: 2BFQ). g, Structural comparison of unmodified and OspC3-modified caspase-4-p30-C/A. Data in a–c, e are representative of three independent experiments.
Extended Data Fig. 2 Characterization of the binding between OspC3ARD and caspase-4.
a, ITC profiles of caspase-4-p30-C/A binding to OspC3ARD or OspC3 (WT or an indicated mutant). Data are representative of three independent experiments. b, Structural comparisons of OspC3ARD and caspase-4-p30-C/A in the OspC3ARD–caspase-4 complex with their cognate apo-states. c, d, Alignments of caspase sequences around the OspC3ARD-binding region (c) and ARD sequences in different OspC3 homologues (d). Residues involved in OspC3ARD–caspase-4 binding are highlighted in cyan. Identical residues are in red background and conserved residues are in red. Numbers of the starting residues are indicated on the left. Accession numbers of OspC3 homologues are the same as previously reported19.
Extended Data Fig. 3 Biochemical property and structural mechanism of CaM interaction with OspC3.
a, ITC profiles of CaM binding to OspC3 or OspC3ARD. Calculated dissociation constant (KD) and binding stoichiometry (N) are expressed as means ± SD from three determinations. ND, not detectable. b, c, Activation of OspC3 by Ca2+-loaded CaM. b, Gel-filtration chromatography combined with SDS-PAGE analyses of the complex between OspC3 and Ca2+-loaded CaM. c, Caspase-4-p30-C/A was reacted with NAD+ and OspC3 that had been complexed with Ca2+-free or Ca2+-loaded CaM. The reactions were subjected to native/SDS-PAGE analyses. Control, fully ADP-riboxanated caspase-4 obtained by co-expression with OspC3 in E. coli. d, Measurements of Ca2+ contents in OspC3-bound CaM by ICP-MS. Purified Ca2+-free and Ca2+-loaded CaM were included as controls. Data are means ± SD from three determinations. e, f, Complex formation and activation of MBP-OspC3 by Ca2+-free CaM. e, Gel-filtration chromatography combined with SDS-PAGE analyses of the complex between MBP-OspC3 and Ca2+-free CaM. f, Caspase-4/11-p30-C/A was reacted with NAD+ and the OspC3–CaM or MBP-OspC3–CaM complex. The reactions were subjected to native/SDS-PAGE analyses. Control, fully ADP-riboxanated caspase-4/11 obtained by co-expression with OspC3 in E. coli. g, Structural comparison of OspC3ARD in the CaM complex with its apo-state. h, Cartoon model of Ca2+-free CaM binding to the IQ motif of myosin V (PDB code: 2IX7). On the right are close-up views of structural comparisons of the two lobes in OspC3-bound CaM and those in myosin V-bound CaM. i, Gel-filtration chromatography analyses of complex formation between CaM and OspC3 (WT or an indicated mutant). Shown are elution profiles along with SDS-PAGE analyses of the elution fractions. Data in representative of three (a–c, e, f, i) or two (d) independent experiments.
Extended Data Fig. 4 Structural homology search reveals an ADP-ribosyltransferase (ART) domain-like fold in OspC3-N domain.
a, Dali search results of OspC3-N domain structure (from the MBP-OspC3–CaM complex). b, Cartoon schemes of ART prototypes, including OspC3 (this study) and the ART domains of diphtheria toxin (PDB code: 1TOX) and cholera toxin (PDB code: 2A5F). The conserved central β-sheets are colored in orange and the catalytic triads are shown in sticks. c, Sequence alignment of the N-terminal domains from different OspC3 homologues. The catalytic triad residues are highlighted by magenta arrowheads on top of the sequence. Residues involved in binding to CaM are boxed in green. Identical residues are in red background and conserved residues are in red. Numbers of the starting residues are indicated on the left. Accession number of OspC3 homologues are the same as previously reported19.
Extended Data Fig. 5 Characterization of OspC3–CaM complex binding to and modification of caspase-4/11 and analyses of NAD+ loading into OspC3 catalytic pocket.
a, ITC profiles of caspase-4-p30-C/A binding to OspC3 or OspC3–CaM complex. Calculated dissociation constant (KD) and binding stoichiometry (N) are expressed as means ± SD from three determinations. b, c, CaM binding and caspase-4/11-p30-C/A modification by OspC3 devoid of C-terminal 10 residues (OspC3-de10). b, Gel-filtration chromatography combined with SDS-PAGE analyses of the complex between CaM-bound OspC3-de10 and caspase-4-p30-C/A. c, Caspase-4/11-p30-C/A was reacted with NAD+ and the OspC3–CaM or OspC3-de10–CaM complex. The reactions were subjected to native/SDS-PAGE analyses. Control, fully ADP-riboxanated caspase-4/11 obtained by co-expression with OspC3 in E. coli. d, Structural comparisons of the OspC3ARD–caspase-4-p30-C/A part and the CaM–OspC3-N domain part in the ternary complex with their respective binary complexes in isolation. e, Modeling of NAD+ into the ligand-binding pocket of OspC3-N domain. Residues potentially involved in binding NAD+ are labeled and shown in sticks. Dotted lines represent hydrogen bonds. f, g, Mutagenesis analyses of OspC3 catalytic pocket. f, Caspase-11-p30-C/A was reacted with NAD+ and OspC3 (WT or an indicated mutant). The reactions were subjected to native/SDS-PAGE analyses (note: the amounts of the OspC3–CaM complex loaded onto the SDS gel were 20 times of those used in the reaction to indicate protein quality). Control, fully ADP-riboxanated caspase-11 obtained by co-expression with OspC3 in E. coli. g, CASP4-/- HeLa cells stably expressing Flag-caspase-11 were electroporated with LPS together with purified OspC3 (WT or an indicated mutant). ATP-based cell viability was measured 2 h post-electroporation. Data are means (bars) of three individual replicates (circles), two-tailed unpaired Student’s t-test. Anti-Flag immunoprecipitates were subjected to anti-ADPR and anti-Flag immunoblotting. Loading of OspC3 proteins was analyzed by SDS-PAGE. Data in a–c, f, g are representative of three independent experiments.
Extended Data Fig. 6 Schematic illustration of the proposed mechanism for OspC3-catalyzed arginine ADP-riboxanation.
In the four-step reaction, E326 together with D231 in OspC3 first deprotonates N-ribose 2′-OH of NAD+ to promote scission of the glycosidic bond between the Nam and the ADPR. This generates an oxocarbenium cation of the N-ribose. Subsequently, the ADPR rotates its β-phosphate, bringing the oxocarbenium cation close to the D231-deprotonated arginine. D231 occupies the position of arginine Nω, inducing a crooked conformation of the guanidine and exposing its Nδ for attacking C1 of the N-ribose. Following this initial ADP-ribosylation, D177, now becoming close to N-ribose 2′-OH, not only fixes the flipped N-ribose in a proper position but also acts as a base to activate the 2′-OH to attack the guanidine carbon, thereby completing the catalysis of ADP-riboxanation.
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Hou, Y., Zeng, H., Li, Z. et al. Structural mechanisms of calmodulin activation of Shigella effector OspC3 to ADP-riboxanate caspase-4/11 and block pyroptosis. Nat Struct Mol Biol 30, 261–272 (2023). https://doi.org/10.1038/s41594-022-00888-3