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From agonist to antagonist: structure and dynamics of innate immune glycoprotein MD-2 upon recognition of variably acylated bacterial endotoxins.

ABSTRACT: The human immune response to an infection by Gram-negative bacteria involves detection of lipopolysaccharides (LPS), also known as endotoxins, which comprise the bacterial outer cell wall. Distinct from mammalian glycolipid structures, LPS have a conserved chemical pattern that is recognized by the pattern recognition receptor complex formed by myeloid differentiation protein 2 (MD-2) and toll-like receptor 4 (TLR4). A remarkable immune-mediated structure-toxicity relationship has been defined that relates to the number of acyl chains in the endotoxin. While there is a clear correlation between endotoxin acylation and elicited agonist or antagonist responses, the 3D structural basis of this relationship remains unclear. In order to explore, at atomic-resolution, the effects of a range of chemically distinct endotoxins on the structure and dynamics of their MD-2·endotoxin complexes, we examined a series of variably acylated lipid A molecules from Escherichia coli and Neisseria meningitidis in complex with human MD-2. Through the application of molecular dynamics simulations, in concert with experimental data, we have identified specific structural and dynamic features of the MD-2-endotoxin complexes that may control dimerization of TLR4 molecules. As dimerization is central to the release of downstream chemical mediators, the results provide a structural foundation for the ability of endotoxins to act as either agonists or antagonists of the TLR4 pathway.

SUBMITTER: DeMarco ML

PROVIDER: S-EPMC3252744 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:The circumventricular organs (CVOs) are blood-brain-barrier missing structures whose activation through lipopolysaccharide (LPS) is a starting point for TLR-driven (Toll-like receptors) neuroinflammation. The aim of this study was to evaluate in the CVO area postrema (AP), subfornical organ (SFO), and median eminence (ME), the inflammatory response to two TLR4 agonists: LPS from Escherichia coli (EC-LPS), the strongest endotoxin molecule described, and LPS from Porphyromonas gingivalis (PG-LPS), a pathogenic bacteria present in the periodontium related to neuroinflammation in neurodegenerative/psychiatric diseases. The response to LPS from the cyanobacteria Rhodobacter sphaeroides (RS-LPS), a TLR4 antagonist with an interesting anti-inflammatory potential, was also assessed. METHODS:LPSs were intraperitoneally administered to Wistar rats and, as indicatives of neuroinflammation in CVOs, the cellular localization of the nuclear factor NF-κB was studied by immunofluorescence, and microglia morphology was quantified by fractal and skeleton analysis. RESULTS:Data showed that EC-LPS increased NF-κB nuclear translocation in the three CVOs studied and PG-LPS only induced NF-κB nuclear translocation in the ME. RS-LPS showed no difference in NF-κB nuclear translocation compared to control. Microglia in the three CVOs showed an ameboid-shape after EC-LPS exposure, whereas PG-LPS only elicited a mild tendency to induce an ameboid shape. On the other hand, RS-LPS produced a markedly elongated morphology described as "rod" microglia in the three CVOs. CONCLUSIONS:In conclusion, at the doses tested, EC-LPS induces a stronger neuroinflammatory response than PG-LPS in CVOs, which might be related to their different potency as TLR4 agonists. The non-reduction of basal NF-κB activation and induction of rod microglia by RS-LPS, a cell morphology only present in severe brain injury and infections, suggests that this molecule must be carefully studied before being proposed as an anti-inflammatory treatment for neuroinflammation related to neurodegenerative/psychiatric diseases.

Project description:The molecular basis for recognition by human P2Y1 receptors of the novel, competitive antagonist 2'-deoxy-N6-methyladenosine 3', 5'-bisphosphate (MRS 2179) was probed using site-directed mutagenesis and molecular modeling. The potency of this antagonist was measured in mutant receptors in which key residues in the transmembrane helical domains (TMs) 3, 5, 6, and 7 were replaced by Ala or other amino acids. The capacity of MRS 2179 to block stimulation of phospholipase C promoted by 2-methylthioadenosine 5'-diphosphate (2-MeSADP) was lost in P2Y1 receptors having F226A, K280A, or Q307A mutations, indicating that these residues are critical for the binding of the antagonist molecule. Mutation of the residues His132, Thr222, and Tyr136 had an intermediate effect on the capacity of MRS 2179 to block the P2Y1 receptor. These positions therefore appear to have a modulatory role in recognition of this antagonist. F131A, H277A, T221A, R310K, or S317A mutant receptors exhibited an apparent affinity for MRS 2179 that was similar to that observed with the wild-type receptor. Thus, Phe131, Thr221, His277, and Ser317 are not essential for antagonist recognition. A computer-generated model of the human P2Y1 receptor was built and analyzed to help interpret these results. The model was derived through primary sequence comparison, secondary structure prediction, and three-dimensional homology building, using rhodopsin as a template, and was consistent with data obtained from mutagenesis studies. We have introduced a "cross-docking" procedure to obtain energetically refined 3D structures of the ligand-receptor complexes. Cross-docking simulates the reorganization of the native receptor structure induced by a ligand. A putative nucleotide binding site was localized and used to predict which residues are likely to be in proximity to agonists and antagonists. According to our model TM6 and TM7 are close to the adenine ring, TM3 and TM6 are close to the ribose moiety, and TM3, TM6, and TM7 are near the triphosphate chain.

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From agonist to antagonist: structure and dynamics of innate immune glycoprotein MD-2 upon recognition of variably acylated bacterial endotoxins.

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