Ding Jingheng's research group and Shao Feng's team at Beisheng Institute revealed that bacterial effector proteins antagonize host cell pyrosis ...

As an important natural immune response of the body, cell pyrosis plays a key role
in antagonizing and eliminating pathogenic infections.
When gram-negative bacteria invade host cells, the important pathogenic molecular pattern LPS (lipopolysaccharide, also known as endotoxin) in its outer membrane is recognized by the natural immune receptor caspase-4/5/11 in the host cell, and LPS-activated caspase-4/5/11 will further cleave the activated pyrozotic protein GSDMD to release its membrane perforation activity, resulting in cell pyrosis and stimulating the host's antibacterial inflammatory response
。 At the same time, bacteria also employ a variety of strategies to evade the host's immune defenses, such as "injecting" specialized effector proteins into host cells through a unique type III secretion system that interferes with the host's immune defense pathways
.
In 2021, Shao Feng's team at the Beijing Institute of Life Sciences found that the effector protein OspC3 secreted by Shigella can specifically recognize the natural immune receptor caspase-4/11 in host cells, and inactivate caspase-4/11 by catalyzing a new ADP-riboxanation post-translational modification of arginine, a key arginine in the caspase activity center.
Block its activation of downstream GSDMD-mediated cellular pyrozoosis immune defenses
.
However, key scientific questions such as how the effector protein OspC3 specifically recognizes the host target caspase-4/11, how it catalyzes the novel ADP-riboxanation, and the precise molecular mechanism of antagonizing cell pyroptosis need to be further answered
.

On January 9, 2023, the research group of Dacheng Wang/Ding Jingjue of the Institute of Biophysics of the Chinese Academy of Sciences and the team of Shao Feng of Beisheng Institute published a report entitled "Structural mechanisms of calmodulin activation ofShigella" in Nature Structural & Molecular Biology effector OspC3 to ADP-riboxanate caspase-4/11 and block pyroptosis
" 。 This study revealed that the effector protein OspC3 uses calmodulin (CaM) in host cells as a cofactor to activate its enzymatic activity, specifically recognizes the host target caspase-4/11 and catalyzes the novel arginine ADP-riboxalation modification, blocking the complete molecular mechanism of the host cell caspase-4/11-GSDMD pyrosis pathway
.

The researchers first found that OspC3 can effectively modify the two forms of caspase-4/11 without self-shearing in the resting state and caspase-4/11 that has self-shearing after bacterial LPS activation, but if the active center of the activated form of caspase-4/11 is irreversibly occupied by zVAD, a covalent inhibitor of the tetrapeptide sequence of the simulated substrate cleavage site, it will greatly weaken the modification of caspase-4/11 by OspC3.
This indicates that caspase-4/11 in the unbound state of the substrate is the substrate of OspC3 regardless of whether it is activated or
not.
The researchers then found that the ADP-ribose-specific binding protein Af1521 can form a stable 1:1 complex with the modified caspase-4/11 product, and by resolving the high-resolution crystal structure of the complex of Af1521 and the modified caspase-4 product, the researchers clearly observed the precise chemical structure of the ADP-riboxanation modification for the first time.
The side chain guanidine group of the caspase-4 modification site R314 is separated from a terminal N^ω atom and linked to the C1 atom and C2 hydroxyl group on the ADP-ribosyl ribose ring from NAD⁺, forming a novel five-membered oxazolidine ring, which provides direct structural proof
for the new post-translational modification of arginine ADP-riboxanation.

OspC3 and the bacterial effector protein family to which it belongs have a typical bidomain characteristic, with its N-terminal domain and any known protein having no sequence homology, while the C-terminal contains a conserved ankyrin-repeat domain (ARD), which normally mediates protein interactions, so it is speculated that this domain is the substrate recognition domain
of OspC3 。 The researchers further determined that the OspC3 ARD domain specifically recruits the host target caspase-4/11
through a series of hydrogen bond networks and hydrophobic interactions by resolving the crystal structure of the OspC3 ARD domain and caspase-4 substrate complex.

In the enzyme activity experiment of recombinant OspC3 modification of caspase-4/11 in vitro, the researchers found that OspC3 requires almost the same amount as the substrate protein caspase-4/11 to achieve complete modification of the substrate, which is contrary to the classical understanding
of the efficiency of enzyme-catalyzed substrate reactions.
Through co-immunoprecipitation combined with mass spectrometry, the researchers identified that the host's calmodulin CaM formed a stable binary complex with OspC3 in the form of^Ca2+-free, which greatly improved the catalytic efficiency
of OspC3.
Subsequently, the researchers successfully resolved the high-resolution crystal structure of OspC3 and CaM binary complexes, and found that CaM in the^Ca2+ unbound state firmly grasped the N-terminal domain of OspC3 with extensive hydrophobic action through the two subdomains, respectively, while the N-terminal domain of OspC3 exhibited a classical Rossmann folded conformation, with similar structural characteristics and conserved NAD+ with the known ADP-ribosyltransferase domain Bind motifs
.
In order to further elucidate the complete enzymatic mechanism of OspC3 using NAD+ as a donor to catalyze the ADP-riboxanation modification of arginine of caspase-4/11, the researchers successfully resolved the OspC3-CaM-caspase-4 ternary complex and its relationship with 2'-F-NAD+ (non-hydrolyzed NAD⁺).
analogue), it was found that the enzymatic active center of the N-terminus of OspC3 immobilizes the terminal N ω atom of the R314 side chain guanidine group of the active center of caspase-4 by acidic amino acid D231, so that the ADP-ribosyl C1 position is close to the N^δ atom of the R314guanidine group, thereby facilitating the departure of the NAD^{+ nicotinamide} group and the modification site R314^N The first classical ADP-ribosyl modification occurs on ^δ atoms; Another acidic amino acid in the enzyme activity center, D177, is responsible for activating the hydroxyl nucleophilic attack R314 side chain guanidine C atom at the ADP-ribosyl C2 position to undergo a deamination reaction, so that the arginine side chain guanidine group and the ADP-ribose group form a oxazolidine ring
.
Subsequently, the researchers verified the discovery of structural studies at the biochemical, cellular and Shigella infected mice by using site-directed mutations, and fully elucidated the molecular mechanism of OspC3's arginine ADP-riboxanation modification of caspase-4/11 using the host cofactor CaM
.

Through a series of three-dimensional structural analysis and functional experiments, this work revealed that the effector protein OspC3 specifically recognizes the host's innate immune receptor caspase-4/11, and uses host calmodulin CaM as a cofactor to catalyze the new arginine ADP-riboxalation modification and block the complete molecular mechanism of the host cell caspase-4/11-GSDMD pyrosis defense pathway.
It also provides a comprehensive and in-depth understanding of the enzymatic reaction mechanism of ADP-riboxanation, a novel post-translational modification, and provides a new strategy
for the further search and development of novel antimicrobial drugs or bacterial attenuated vaccines.

Figure Molecular mechanism of dysentery effector protein OspC3 antagonistic host cell pyrosis pathway

A.
Model diagram of the effect protein OspC3 blocking of the Caspase-4/11 pyrosis pathway antagonizing host innate immunity; B.
OspC3-CaM-caspase-4-2'-F-NAD⁺ quaternary complex structure and catalytic key residue display; C.
Caspase-4 structure modified by arginine ADP-riboxanation; D.
OpsC3 catalytic key site mutants were validated
in experiments with dysentery bacteria.

Professor Ding Jingjue of the Institute of Biophysics, Chinese Academy of Sciences and Professor Shao Feng of the Institute of Beisheng are the co-corresponding authors of this paper, and Dr.
Hou Yanjie of Ding Jingjue's research group and Zeng Huan, a doctoral student from the Institute of Biophysics of Shao Feng's group, are the co-first authors
of this paper.
The research was supported
by the Strategic Leading Science and Technology Project of the Chinese Academy of Sciences, the Key R&D Program of the Ministry of Science and Technology, the Excellent Youth Project of the Foundation Committee and the Program of the Youth Promotion Council of the Chinese Academy of Sciences.

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(Contributed by: Ding Jingjue Research Group)