Biology subjects

Deredge, D.

Publications and source records attributed to Deredge, D..

3 recordsLinked to original sources

bioRxiv2021

Biochemical and structural characterization of the fused Bacteroides fragilis NFeoAB domain reveals a role for FeoA

Iron is an essential element for nearly all organisms, and under anoxic and/or reducing conditions, Fe2+ is the dominant form of iron available to bacteria. The ferrous iron transport (Feo) system has been identified as the primary prokaryotic Fe2+ import machinery, and two proteins (FeoA and FeoB) are conserved across most bacterial species. However, how FeoA and FeoB function relative to one another remained enigmatic. In this work we explored the distribution of feoAB operons predicted to encode for a fusion of FeoA tethered to the soluble N-terminal, G-protein domain of FeoB via a connecting linker region. We hypothesized that this fusion might poise FeoA to interact with FeoB in order to affect function. To test this hypothesis, we cloned, expressed, purified, and biochemically characterized the soluble NFeoAB fusion protein from Bacteroides fragilis, a commensal organism implicated in drug-resistant peritoneal infections. Using X-ray crystallography, we determined to 1.50 [A] resolution the structure of BfFeoA, which adopts an SH3-like fold implicated in protein-protein interactions. In combination with structural modeling, small-angle X-ray scattering, and hydrogen-deuterium exchange mass spectrometry, we show that FeoA and NFeoB indeed interact in a nucleotide-dependent manner, and we have mapped the protein-protein interaction interface. Finally, using GTP hydrolysis assays, we demonstrate that BfNFeoAB exhibits one of the slowest known rates of Feo-mediated GTP hydrolysis and is not potassium-stimulated, indicating that FeoA-NFeoB interactions may function to stabilize the GTP-bound form of FeoB. Our work thus reveals a role for FeoA function in the fused FeoAB systems and suggests a broader role for FeoA function amongst prokaryotes.

biochemistry↗

bioRxiv2021

Insights into substrate recognition and specificity for IgG by Endoglycosidase S2

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 site 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) simulation and site-identification by ligand competitive saturation (SILCS) docking followed by reconstruction and long-time 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. 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. Author SummaryThe pathogen Streptococcus pyogenes uses the endoglycosidases S and S2 to cleave the glycans on the Fc portion of IgG antibodies, leading to a decreased cytotoxicity of the antibodies, thereby evading the host immune response. To identify potential structures of the complex of EndoS2 with IgG that could lead to the catalytic hydrolysis of the IgG glycan, molecular modeling and molecular dynamics simulations were applied. The resulting structural models predict that EndoS2 initially interacts through its carbohydrate binding module (CBM) with the IgG with subsequent interactions with the catalytic glycoside hydrolase (GH) domain yielding stable complexes. In the modeled complexes the CBM and the GH interact either simultaneously with the same Fc CH2/CH3 domain or with the two individual Fc CH2/CH3 domains separately to yield potentially catalytically competent species. In addition, apo EndoS2 is shown to assume both open and closed conformations allowing it to directly form either type of complex from which deglycosylation of either mono- or diglycosylated IgG species may occur. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/445403v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@15ffa95org.highwire.dtl.DTLVardef@3c8032org.highwire.dtl.DTLVardef@452bdorg.highwire.dtl.DTLVardef@1d3f85a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Molecular basis of selective cytokine signaling inhibition by antibodies targeting a shared receptor

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 exists, 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-1 and IL-1{beta}; ST2 for IL-33; and IL-36R for IL-36, IL-36{beta} and IL-36{gamma} - 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-1{beta} 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.

immunology↗