Immunoglobulin G
(IgG) glycosylation critically modulates antibody
effector functions.
Streptococcus pyogenes
secretes
a unique endo-β-
N
-acetylglucosaminidase, EndoS2,
which deglycosylates the conserved
N
-linked glycan
at Asn297 on IgG Fc to eliminate its effector functions and evade
the immune system. EndoS2 and specific point mutants have been used
to chemoenzymatically synthesize antibodies with customizable glycosylation
for gain of functions. EndoS2 is useful in these schemes because it
accommodates a broad range of
N
-glycans, including
high-mannose, complex, and hybrid types; however, its mechanism of
substrate recognition is poorly understood. We present crystal structures
of EndoS2 alone and bound to complex and high-mannose glycans; the
broad
N
-glycan specificity is governed by critical
loops that shape the binding site of EndoS2. Furthermore, hydrolytic
experiments, domain-swap chimeras, and hydrogen–deuterium exchange
mass spectrometry reveal the importance of the carbohydrate-binding
module in the mechanism of IgG recognition by EndoS2, providing insights
into engineering enzymes to catalyze customizable glycosylation reactions.
infects half of the world's population, and strains that encode the type IV secretion system for injection of the oncoprotein CagA into host gastric epithelial cells are associated with elevated levels of cancer. CagA translocation into host cells is dependent on interactions between the adhesin protein HopQ and human CEACAMs. Here, we present high-resolution structures of several HopQ-CEACAM complexes and CEACAMs in their monomeric and dimeric forms establishing that HopQ uses a coupled folding and binding mechanism to engage the canonical CEACAM dimerization interface for CEACAM recognition. By combining mutagenesis with biophysical and functional analyses, we show that the modes of CEACAM recognition by HopQ and CEACAMs themselves are starkly different. Our data describe precise molecular mechanisms by which microbes exploit host CEACAMs for infection and enable future development of novel oncoprotein translocation inhibitors and -specific antimicrobial agents.
Summary
Within the interleukin 1 (IL-1) cytokine family, IL-1 receptor accessory protein (IL-1RAcP) is the co-receptor for eight receptor:cytokine pairs, including cytokines IL-1β and IL-33. Unlike for IL-1β, no structure of the IL-33 signaling complex exists that includes both its cognate receptor, ST2, and the shared co-receptor IL-1RAcP, which we now present here. Although the IL-1β and IL-33 complexes shared structural features and engaged identical molecular surfaces of IL-1RAcP, these cytokines had starkly different strategies for co-receptor engagement and signal activation. Our data suggested that IL-1β bound to IL-1RI to properly present the cytokine to IL-1RAcP, while IL-33 bound to ST2 in order to conformationally constrain the cognate receptor in an IL-1RAcP-receptive state. These findings indicated that IL-1 family cytokines use distinct molecular mechanisms to signal through their shared co-receptor, and provide the foundation from which to design new therapies to target IL-33 signaling.
Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) comprise a large family of cell surface adhesion molecules that bind to themselves and other family members to carry out numerous cellular functions, including proliferation, signaling, differentiation, tumor suppression, and survival. They also play diverse and significant roles in immunity and infection. The formation of CEACAM oligomers is caused predominantly by interactions between their N-terminal IgV domains. Although X-ray crystal structures of CEACAM IgV domain homodimers have been described, how CEACAMs form heterodimers or remain monomers is poorly understood. To address this key aspect of CEACAM function, we determined the crystal structures of IgV domains that form a homodimeric CEACAM6 complex, monomeric CEACAM8, and a heterodimeric CEACAM6-CEACAM8 complex. To confirm and quantify these interactions in solution, we used analytical ultracentrifugation to measure the dimerization constants of CEACAM homodimers and isothermal titration calorimetry to determine the thermodynamic parameters and binding affinities of CEACAM heterodimers. We found the CEACAM6-CEACAM8 heterodimeric state to be substantially favored energetically relative to the CEACAM6 homodimer. Our data provide a molecular basis for the adoption of the diverse oligomeric states known to exist for CEACAMs and suggest ways in which CEACAM6 and CEACAM8 regulate the biological functions of one another, as well as of additional CEACAMs with which they interact, both in cis and in trans.CEACAM | X-ray crystallography | isothermal titration calorimetry | analytical ultracentrifugation
Background: An Arg-Gly-Asp (RGD) motif in CagL anchors Helicobacter pylori to host cell integrins. Results: Reduced pH causes a conformational change in CagL that buries the RGD motif. Conclusion: A pH-induced conformational change regulates CagL RGD binding to host cells. Significance: Regulation of the RGD-integrin interaction prevents premature host cell attachment.
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