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Abstract Bacteroidales (syn. Bacteroidetes) are prominent members of the human gastrointestinal ecosystem mainly due to their efficient glycan-degrading machinery, organized into gene clusters known as polysaccharide utilization loci (PULs). A single PUL was reported for catabolism of high-mannose (HM) N-glycan glyco-polypeptides in the gut symbiont Bacteroides thetaiotaomicron, encoding a surface endo-β-N-acetylglucosaminidase (ENGase), BT3987. Here, we discover an ENGase from the GH18 family in B. thetaiotaomicron, BT1285, encoded in a distinct PUL with its own repertoire of proteins for catabolism of the same HM N-glycan substrate as that of BT3987. We employ X-ray crystallography, electron microscopy, mass spectrometry-based activity measurements, alanine scanning mutagenesis and a broad range of biophysical methods to comprehensively define the molecular mechanism by which BT1285 recognizes and hydrolyzes HM N-glycans, revealing that the stabilities and activities of BT1285 and BT3987 were optimal in markedly different conditions. BT1285 exhibits significantly higher affinity and faster hydrolysis of poorly accessible HM N-glycans than does BT3987. We also find that two HM-processing endoglycosidases from the human gut-resident Alistipes finegoldii display condition-specific functional properties. Altogether, our data suggest that human gut microbes employ evolutionary strategies to express distinct ENGases in order to optimally metabolize the same N-glycan substrate in the gastroinstestinal tract.

Abstract Immunoglobulin G (IgG) antibodies contain a complex N-glycan embedded in the hydrophobic pocket between its heavy chain protomers. This glycan contributes to the structural organization of the Fc domain and determines its specificity for Fcγ receptors, thereby dictating distinct cellular responses. The variable construction of this glycan structure leads to highly-related, but non-equivalent glycoproteins known as glycoforms. We previously reported synthetic nanobodies that distinguish IgG glycoforms. Here, we present the structure of one such nanobody, X0, in complex with the Fc fragment of afucosylated IgG1. Upon binding, the elongated CDR3 loop of X0 undergoes a conformational shift to access the buried N-glycan and acts as a ‘glycan sensor’, forming hydrogen bonds with the afucosylated IgG N-glycan that would otherwise be sterically hindered by the presence of a core fucose residue. Based on this structure, we designed X0 fusion constructs that disrupt pathogenic afucosylated IgG1-FcγRIIIa interactions and rescue mice in a model of dengue virus infection.

Bacterial pathogens have evolved intricate mechanisms to evade the human immune system, including the production of immunomodulatory enzymes. Here, the authors establish the mechanisms of recognition and specific deglycosylation of IgG antibodies by the multi-modular enzymes EndoS and EndoS2

EndoE is a multi-domain glycoside hydrolase of the human pathogen Enterococcus faecalis. Here, the authors present crystal structures of EndoE and provide biochemical insights into the molecular basis of EndoE’s substrate specificity and catalytic mechanism.

To understand how hCEACAM1 transitions between monomeric and dimeric states that are required for its activity, Gandhi et al. investigate hCEACAM1 homodimeric, monomeric, and transition states and their behavior in solution. This study suggests the flexibility of the GFCC’ face and its role in governing the formation of hCEACAM1 dimers and selective heterodimers.

AlfC transfucosidase is used to modulate fucosylation of glycans decorating monoclonal antibodies. Herein, structural and biophysical characterization reveals the enzymatic mechanism of AlfC and a blueprint for the design of AlfC mutants with novel specificities and functions.

Human gut bacteria depolymerize glycans into their sugar components, which otherwise cannot be processed by their host. Here, the authors characterise the endo-β-N-acetylglucosaminidase EndoBT-3987 from the Gram-negative symbiont Bacteroides thetaiotaomicron and present the crystal structures of ligand-free EndoBT-3987, a substrate bound complex and product complexes.

Interleukin-1 (IL-1) family cytokines are potent mediators of inflammation, acting to coordinate local and systemic immune responses to a wide range of stimuli. Aberrant signaling by IL-1 family cytokine members, however, is linked to myriad inflammatory syndromes, autoimmune conditions and cancers. As such, blocking the inflammatory signals inherent to IL-1 family signaling is an established and expanding therapeutic strategy. While several FDA-approved IL-1 inhibitors exist, including an Fc fusion protein, a neutralizing antibody, and an antagonist cytokine, none specifically targets the co-receptor IL-1 receptor accessory protein (IL-1RAcP). Most IL-1 family cytokines form productive signaling complexes by binding first to their cognate receptors – IL-1RI for IL-1α and IL-1β; ST2 for IL-33; and IL-36R for IL-36α, IL-36β and IL-36γ – after which they recruit the shared secondary receptor IL-1RAcP to form a ternary cytokine/receptor/co-receptor complex. Recently, IL-1RAcP was identified as a biomarker for both AML and CML. IL-1RAcP has also been implicated in tumor progression in solid tumors and an anti-IL1RAP antibody (nadunolimab, CAN04) is in phase II clinical studies in pancreatic cancer and non-small cell lung cancer (NCT03267316). As IL-1RAcP is common to all of the abovementioned IL-1 family cytokines, targeting this co-receptor raises the possibility of selective signaling inhibition for different IL-1 family cytokines. Indeed, previous studies of IL-1β and IL-33 signaling complexes have revealed that these cytokines employ distinct mechanisms of IL-1RAcP recruitment even though their overall cytokine/receptor/co-receptor complexes are structurally similar. Here, using functional, biophysical, and structural analyses, we show that antibodies specific for IL-1RAcP can differentially block signaling by IL-1 family cytokines depending on the distinct IL-1RAcP epitopes that they engage. Our results indicate that targeting a shared cytokine receptor is a viable therapeutic strategy for selective cytokine signaling inhibition.

Endoglycosidase S only recognizes one particular type of glycan within IgG antibodies but the molecular basis for this high specificity is not fully understood. Here, the authors present the crystal structure of product-bound Endoglycosidase S, revealing the determinants for its glycan specificity.

Bacterial flagella are cell surface protein appendages that are critical for motility and pathogenesis. Flagellar filaments are tubular structures constructed from thousands of copies of the protein flagellin, or FliC, arranged in helical fashion. Individual unfolded FliC subunits traverse the filament pore and are folded and sorted into place with the assistance of the flagellar capping protein complex, an oligomer of the FliD protein. The FliD filament cap is a stool-like structure, with its D2 and D3 domains forming a flat head region, and its D1 domain leg-like structures extending perpendicularly from the head towards the inner core of the filament. Here, using an approach combining bacterial genetics, motility assays, electron microscopy and molecular modeling, we define, in numerous Gram-negative bacteria, which regions of FliD are critical for interaction with FliC subunits and result in the formation of functional flagella. Our data indicate that the D1 domain of FliD is its sole functionally important domain, and that its flexible coiled coil region comprised of helices at its extreme N- and C-termini controls compatibility with the FliC filament. FliD sequences from different bacterial species in the head region are well tolerated. Additionally, head domains can be replaced by small peptides and larger head domains from different species and still produce functional flagella.

Bacterial flagellar filaments are composed almost entirely of a single protein—flagellin—which can switch between different supercoiled states in a highly cooperative manner. Here the authors present near-atomic resolution cryo-EM structures of nine flagellar filaments, and begin to shed light on the molecular basis of filament switching.

The insect IMD signalling pathway detects invading pathogens. Here the authors show that ticks have an alternative IMD system that lacks peptidoglycan receptors, IMD and FADD, and is instead reliant on interaction of the E3 ligase XIAP with the E2 conjugating enzyme Bendless.

Interleukin-1 (IL-1) family cytokines are key signaling molecules in both the innate and adaptive immune systems, mediating inflammation in response to a wide range of stimuli. The basic mechanism of signal initiation is a stepwise process in which an agonist cytokine binds its cognate receptor. Together, this cytokine-receptor complex recruits an often-common secondary receptor. Intracellularly, the Toll/IL-1 Receptor (TIR) domains of the two receptors are brought into close proximity, initiating an NF-κB signal transduction cascade. Due to the potent inflammatory response invoked by IL-1 family cytokines, several physiological mechanisms exist to inhibit IL-1 family signaling, including antagonist cytokines and decoy receptors. The numerous cytokines and receptors in the IL-1 superfamily are further classified into four subfamilies, dependent on their distinct cognate receptors—the IL-1, IL-33, and IL-36 subfamilies share IL-1RAcP as their secondary receptor, while IL-18 subfamily utilizes a distinct secondary receptor. Here, we describe how structural biology has informed our understanding of IL-1 family cytokine signaling, with a particular focus on molecular mechanisms of signaling complex formation and antagonism at the atomic level, as well as how these findings have advanced therapeutics to treat some chronic inflammatory diseases that are the result of dysregulated IL-1 signaling.

Tissue-resident memory T (Trm) cells are enriched at the sites of previous infection and required for enhanced protective immunity. However, the emergence of Trm cells and their roles in providing protection are unclear in the field of Helicobacter pylori (H. pylori) vaccinology. Here, our results suggest that conventional vaccine strategies are unable to establish a measurable antigen (Ag)-specific memory cell pool in stomach; in comparison, gastric subserous injection of mice with micro-dose of Alum-based H. pylori vaccine can induce a pool of local CD4+ Trm cells. Regional recruitment of Ag-specific CD4+ T cells depends on the engagement of Ag and adjuvant-induced inflammation. Prior subcutaneous vaccination enhanced this recruitment. A stable pool of Ag-specific CD4+ T cells can be detected for 240 days. Two weeks of FTY720 administration in immune mice suggests that these cells do not experience the recirculation. Immunohistochemistry results show that close to the vaccination site, abundant CD4+T cells locate on epithelial niches, independent of lymphocyte cluster. Paradigmatically, Ag-specific CD4+ T cells with a phenotype of CD69+CD103- are preferential on lymphocytes isolated from epithelium. Upon Helicobacter infection, CD4+ Trm cells orchestrate a swift recall response with the recruitment of circulating antigen-specific Th1/Th17 cells to trigger a tissue-wide pathogen clearance. This study investigates the vaccine-induced gastric CD4+ Trm cells in a mice model, and highlights the need for designing a vaccine strategy against H. pylori by establishing the protective CD4+ Trm cells.

Toll-like receptors (TLRs) activate distinct, yet overlapping sets of signaling molecules, leading to inflammatory responses to pathogens. Toll/interleukin-1 receptor (TIR) domains, present in all TLRs and TLR adapters, mediate protein interactions downstream of activated TLRs. A peptide library derived from TLR2 TIR was screened for inhibition of TLR2 signaling. Cell-permeable peptides derived from the D helix and the segment immediately N-terminal to the TLR2 TIR domain potently inhibited TLR2-mediated cytokine production. The D-helix peptide, 2R9, also potently inhibited TLR4, TLR7, and TLR9, but not TLR3 or TNF-α signaling. Cell imaging, co-immunoprecipitation, and in vitro studies demonstrated that 2R9 preferentially targets TIRAP. 2R9 diminished systemic cytokine responses elicited in vivo by synthetic TLR2 and TLR7 agonists; it inhibited the activation of macrophages infected with influenza strain A/PR/8/34 (PR8) and significantly improved the survival of PR8-infected mice. Thus, 2R9 represents a TLR-targeting agent that blocks protein interactions downstream of activated TLRs.

We present here the identification and characterization of an SCP3 (small C-terminal domain phosphatase-3) homologue in smooth muscle and show, for the first time, that it dephosphorylates CaMKII [Ca2+/CaM (calmodulin)-dependent protein kinase II]. SCP3 is a PP2C (protein phosphatase 2C)-type phosphatase that is primarily expressed in vascular smooth muscle tissues and specifically binds to the association domain of the CaMKIIγ G-2 variant. The dephosphorylation is site-specific, excluding the Thr287 associated with Ca2+/CaM-independent activation of the kinase. As a result, the autonomous activity of CaMKIIγ G-2 is not affected by the phosphatase activity of SCP3. SCP3 co-localizes with CaMKIIγ G-2 on cytoskeletal filaments, but is excluded from the nucleus in differentiated vascular smooth muscle cells. Upon depolarization-induced Ca2+ influx, CaMKIIγ G-2 is activated and dissociates from SCP3. Subsequently, CaMKIIγ G-2 is targeted to cortical adhesion plaques. We show here that SCP3 regulates phosphorylation sites in the catalytic domain, but not those involved in regulation of kinase activation. This selective dephosphorylation by SCP3 creates a constitutively active kinase that can then be differentially regulated by other phosphorylation-dependent regulatory mechanisms. [ABSTRACT FROM AUTHOR]

In its simplest form, bacterial flagellar filaments are composed of flagellin proteins with just two helical inner domains, which together comprise the filament core. Although this minimal filament is sufficient to provide motility in many flagellated bacteria, most bacteria produce flagella composed of flagellin proteins with one or more outer domains arranged in a variety of supramolecular architectures radiating from the inner core. Flagellin outer domains are known to be involved in adhesion, proteolysis and immune evasion but have not been thought to be required for motility. Here we show that in the Pseudomonas aeruginosa PAO1 strain, a bacterium that forms a ridged filament with a dimerization of its flagellin outer domains, motility is categorically dependent on these flagellin outer domains. Moreover, a comprehensive network of intermolecular interactions connecting the inner domains to the outer domains, the outer domains to one another, and the outer domains back to the inner domain filament core, is required for motility. This inter-domain connectivity confers PAO1 flagella with increased stability, essential for its motility in viscous environments. Additionally, we find that such ridged flagellar filaments are not unique to Pseudomonas but are, instead, present throughout diverse bacterial phyla.

Antibodies bind foreign antigens with high affinity and specificity leading to their neutralization and/or clearance by the immune system. The conserved N-glycan on IgG has significant impact on antibody effector function, with the endoglycosidases of Streptococcus pyogenes deglycosylating the IgG to evade the immune system, a process catalyzed by the endoglycosidase EndoS2. Studies have shown that two of the four domains of EndoS2, the carbohydrate binding module (CBM) and the glycoside hydrolase (GH) domain are critical for catalytic activity. To yield structural insights into contributions of the CBM and the GH domains as well as the overall flexibility of EndoS2 to the proteins' catalytic activity, models of EndoS2-Fc complexes were generated through enhanced-sampling molecular-dynamics (MD) simulations and site-identification by ligand competitive saturation (SILCS) docking followed by reconstruction and multi-microsecond MD simulations. Modeling results predict that EndoS2 initially interacts with the IgG through its CBM followed by interactions with the GH yielding catalytically competent states. These may involve the CBM and GH of EndoS2 simultaneously interacting with either the same Fc CH2/CH3 domain or individually with the two Fc CH2/CH3 domains, with EndoS2 predicted to assume closed conformations in the former case and open conformations in the latter. Apo EndoS2 is predicted to sample both the open and closed states, suggesting that either complex can directly form following initial IgG-EndoS2 encounter. Interactions of the CBM and GH domains with the IgG are predicted to occur through both its glycan and protein regions. Simulations also predict that the Fc glycan can directly transfer from the CBM to the GH, facilitating formation of catalytically competent complexes and how the 734 to 751 loop on the CBM can facilitate extraction of the glycan away from the Fc CH2/CH3 domain. The predicted models are compared and consistent with Hydrogen/Deuterium Exchange data. In addition, the complex models are consistent with the high specificity of EndoS2 for the glycans on IgG supporting the validity of the predicted models.

Flagella are crucial for bacterial motility and pathogenesis. The flagellar capping protein (FliD) regulates filament assembly by chaperoning and sorting flagellin (FliC) proteins after they traverse the hollow filament and exit the growing flagellum tip. In the absence of FliD, flagella are not formed, resulting in impaired motility and infectivity. Here, we report the 2.2 Å resolution X-ray crystal structure of FliD from Pseudomonas aeruginosa, the first high-resolution structure of any FliD protein from any bacterium. Using this evidence in combination with a multitude of biophysical and functional analyses, we find that Pseudomonas FliD exhibits unexpected structural similarity to other flagellar proteins at the domain level, adopts a unique hexameric oligomeric state, and depends on flexible determinants for oligomerization. Considering that the flagellin filaments on which FliD oligomers are affixed vary in protofilament number between bacteria, our results suggest that FliD oligomer stoichiometries vary across bacteria to complement their filament assemblies.