Functional insights from the crystal structure of the N-terminal domain of the prototypical toll receptor.

Drosophila melanogaster Toll is the founding member of an important family of pathogen-recognition receptors in humans, the Toll-like receptor (TLR) family. In contrast, the prototypical receptor is a cytokine-like receptor for Spätzle (Spz) protein and plays a dual role in both development and immunity. Here, we present the crystal structure of the N-terminal domain of the receptor that encompasses the first 201 amino acids at 2.4 Å resolution. To our knowledge, the cysteine-rich cap adopts a novel fold unique to Toll-1 orthologs in insects and that is not critical for ligand binding. However, we observed that an antibody directed against the first ten LRRs blocks Spz signaling in a Drosophila cell-based assay. Supplemented by point mutagenesis and deletion analysis, our data suggests that the region up to LRR 14 is involved in Spz binding. Comparison with mammalian TLRs reconciles previous contradictory findings about the mechanism of Toll activation.This work is financed by the Wellcome Trust Award (RG47206). We thank Dr. Martin Moncrieffe for helpful discussions and Ms. Irina Ogay from the Baculovirus Facility, Department of Biochemistry, Cambridge, for protein expression. We are grateful to Dr. Katherine Stott, from the Biophysics Facility, for her aid with analytical ultracentrifugation. Thanks to Dr. Dimitri Chirdgaze, from the Crystallographic X-ray Facility, for his assistance. We are grateful to Prof. Abel Moreno for help with capillary crystallization. We thank the staff at beamlines IO3 at Diamond Light Source, England, and ID23EH1 at the ESRF, Grenoble, France, for help with the data collection.This is the final version of the article. It first appeared from Elsevier (Cell Press) via http://dx.doi.org/10.1016/j.str.2012.11.00

The molecular basis for recognition of bacterial ligands at equine TLR2, TLR1 and TLR6.

TLR2 recognises bacterial lipopeptides and lipoteichoic acid, and forms heterodimers with TLR1 or TLR6. TLR2 is relatively well characterised in mice and humans, with published crystal structures of human TLR2/1/Pam3CSK4 and murine TLR2/6/Pam2CSK4. Equine TLR4 is activated by a different panel of ligands to human and murine TLR4, but less is known about species differences at TLR2. We therefore cloned equine TLR2, TLR1 and TLR6, which showed over 80% sequence identity with these receptors from other mammals, and performed a structure-function analysis. TLR2/1 and TLR2/6 from both horses and humans dose-dependently responded to lipoteichoic acid from Staphylococcus aureus, with no significant species difference in EC50 at either receptor pair. The EC50 of Pam2CSK4 was the same for equine and human TLR2/6, indicating amino acid differences between the two species' TLRs do not significantly affect ligand recognition. Species differences were seen between the responses to Pam2CSK4 and Pam3CSK4 at TLR2/1. Human TLR2/1, as expected, responded to Pam3CSK4 with greater potency and efficacy than Pam2CSK4. At equine TLR2/1, however, Pam3CSK4 was less potent than Pam2CSK4, with both ligands having similar efficacies. Molecular modelling indicates that the majority of non-conserved ligand-interacting residues are at the periphery of the TLR2 binding pocket and in the ligand peptide-interacting regions, which may cause subtle effects on ligand positioning. These results suggest that there are potentially important species differences in recognition of lipopeptides by TLR2/1, which may affect how the horse deals with bacterial infections.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

LPS ligand and culture additives improve production of monomeric MD-1 and 2 in Pichia pastoris by decreasing aggregation and intermolecular disulfide bonding.

Myeloid differentiation proteins MD-1 and MD-2 have both been shown to form a heterogeneous collection of oligomers when expressed in absence of their respective receptor, RP105 and TLR4. The biological relevance of these oligomers is not clear. Only monomeric proteins have been found to be active and able to trigger an immune response to endotoxin by modulating the TLR4 pathway. In this study, we produced variants of MD-1 and MD-2 in Pichia pastoris. To minimize the time and expense of initial expression tests, small-scale cultures have been set up to allow the rapid identification of the highest expressing clone and the optimal expression conditions. The expression vectors used, the site of linearization and the locus of integration affected the yield of transformation. Next we screened culture additives and found that they significantly increased the fraction of monomeric proteins secreted in the culture medium (up to 15% of the total MD protein produced). We confirmed their presence by size-exclusion chromatography. Optimal anti-aggregation agents were protein-dependent except for LPS that presented stabilizing effects for all MD proteins. Contrary to previous reports, this study suggests that MD-1 can bind to LPS.WELLCOME TRUST; ward Number RG47206.This is the final version of the article. It first appeared from Elsevier at http://dx.doi.org/10.1016/j.pep.2010.11.018

Identification of key residues that confer Rhodobacter sphaeroides LPS activity at horse TLR4/MD-2.

The molecular determinants underpinning how hexaacylated lipid A and tetraacylated precursor lipid IVa activate Toll-like receptor 4 (TLR4) are well understood, but how activation is induced by other lipid A species is less clear. Species specificity studies have clarified how TLR4/MD-2 recognises different lipid A structures, for example tetraacylated lipid IVa requires direct electrostatic interactions for agonism. In this study, we examine how pentaacylated lipopolysaccharide from Rhodobacter sphaeroides (RSLPS) antagonises human TLR4/MD-2 and activates the horse receptor complex using a computational approach and cross-species mutagenesis. At a functional level, we show that RSLPS is a partial agonist at horse TLR4/MD-2 with greater efficacy than lipid IVa. These data suggest the importance of the additional acyl chain in RSLPS signalling. Based on docking analysis, we propose a model for positioning of the RSLPS lipid A moiety (RSLA) within the MD-2 cavity at the TLR4 dimer interface, which allows activity at the horse receptor complex. As for lipid IVa, RSLPS agonism requires species-specific contacts with MD-2 and TLR4, but the R2 chain of RSLA protrudes from the MD-2 pocket to contact the TLR4 dimer in the vicinity of proline 442. Our model explains why RSLPS is only partially dependent on horse TLR4 residue R385, unlike lipid IVa. Mutagenesis of proline 442 into a serine residue, as found in human TLR4, uncovers the importance of this site in RSLPS signalling; horse TLR4 R385G/P442S double mutation completely abolishes RSLPS activity without its counterpart, human TLR4 G384R/S441P, being able to restore it. Our data highlight the importance of subtle changes in ligand positioning, and suggest that TLR4 and MD-2 residues that may not participate directly in ligand binding can determine the signalling outcome of a given ligand. This indicates a cooperative binding mechanism within the receptor complex, which is becoming increasingly important in TLR signalling.This work was supported by a project grant from the Horserace Betting Levy Board to CEB and a Horserace Betting Levy Board Veterinary Research Training Scholarship to KLI. This work was also supported by a Wellcome Trust program grant to NJG and CEB. CEB is a BBSRC Research Development Fellow.This is the final version of the article. It first appeared from PLOS at http://dx.doi.org/10.1371/journal.pone.0098776

IIV-6 Inhibits NF-kappaB Responses in Drosophila

The host immune response and virus-encoded immune evasion proteins pose constant, mutual selective pressure on each other. Virally encoded immune evasion proteins also indicate which host pathways must be inhibited to allow for viral replication. Here, we show that IIV-6 is capable of inhibiting the two Drosophila NF-kappaB signaling pathways, Imd and Toll. Antimicrobial peptide (AMP) gene induction downstream of either pathway is suppressed when cells infected with IIV-6 are also stimulated with Toll or Imd ligands. We find that cleavage of both Imd and Relish, as well as Relish nuclear translocation, three key points in Imd signal transduction, occur in IIV-6 infected cells, indicating that the mechanism of viral inhibition is farther downstream, at the level of Relish promoter binding or transcriptional activation. Additionally, flies co-infected with both IIV-6 and the Gram-negative bacterium, Erwinia carotovora carotovora, succumb to infection more rapidly than flies singly infected with either the virus or the bacterium. These findings demonstrate how pre-existing infections can have a dramatic and negative effect on secondary infections, and establish a Drosophila model to study confection susceptibility

Three-tier regulation of cell number plasticity by neurotrophins and Tolls in Drosophila

Cell number plasticity is coupled to circuitry in the nervous system, adjusting cell mass to functional requirements. In mammals, this is achieved by neurotrophin (NT) ligands, which promote cell survival via their Trk and p75NTR receptors and cell death via p75NTR and Sortilin. Drosophila NTs (DNTs) bind Toll receptors instead to promote neuronal survival, but whether they can also regulate cell death is unknown. In this study, we show that DNTs and Tolls can switch from promoting cell survival to death in the central nervous system (CNS) via a three-tier mechanism. First, DNT cleavage patterns result in alternative signaling outcomes. Second, different Tolls can preferentially promote cell survival or death. Third, distinct adaptors downstream of Tolls can drive either apoptosis or cell survival. Toll-6 promotes cell survival via MyD88-NF-kappa B and cell death via Wek-Sarm-JNK. The distribution of adaptors changes in space and time and may segregate to distinct neural circuits. This novel mechanism for CNS cell plasticity may operate in wider contexts

Critical residues involved in Toll-like receptor 4 activation by cationic lipid nanocarriers are not located at the lipopolysaccharide-binding interface.

DiC14-amidine is a cationic lipid that was originally designed as a lipid nanocarrier for nucleic acid transport, and turned out to be a Toll-like receptor 4 (TLR4) agonist as well. We found that while E. coli lipopolysaccharide (LPS) is a TLR4 agonist in all species, diC14-amidine nanoliposomes are full agonists for human, mouse and cat receptors but weak horse agonists. Taking advantage of this unusual species specificity, we used chimeric constructs based on the human and horse sequences and identified two regions in the human TLR4 that modulate the agonist activity of diC14-amidine. Interestingly, these regions lie outside the known LPS-binding domain. Competition experiments also support our hypothesis that diC14-amidine interacts primarily with TLR4 hydrophobic crevices located at the edges of the TLR4/TLR4* dimerization interface. We have characterized potential binding modes using molecular docking analysis and suggest that diC14-amidine nanoliposomes activate TLR4 by facilitating its dimerization in a process that is myeloid differentiation 2 (MD-2)-dependent and cluster of differentiation 14 (CD14)-independent. Our data suggest that TLR4 may be activated through binding at different anchoring points, expanding the repertoire of TLR4 ligands to non-MD-2-binding lipids.C.L. is a IEF Marie Curie Action Research Fellow (TLR4-CAT PIEF-GA-2012-326481) and would like to thank the Wiener-Anspach Foundation for financial support. K.L.I. was supported by a HBLB Veterinary Research Training scholarship. This work was supported by program grant support from the Wellcome Trust and the MRC to N.J.G and C.E.B.This is the final version of the article. It first appeared from Springer via http://dx.doi.org/10.1007/s00018-015-1915-

IIV-6 Inhibits NF-κB Responses in Drosophila.

The host immune response and virus-encoded immune evasion proteins pose constant, mutual selective pressure on each other. Virally encoded immune evasion proteins also indicate which host pathways must be inhibited to allow for viral replication. Here, we show that IIV-6 is capable of inhibiting the two Drosophila NF-κB signaling pathways, Imd and Toll. Antimicrobial peptide (AMP) gene induction downstream of either pathway is suppressed when cells infected with IIV-6 are also stimulated with Toll or Imd ligands. We find that cleavage of both Imd and Relish, as well as Relish nuclear translocation, three key points in Imd signal transduction, occur in IIV-6 infected cells, indicating that the mechanism of viral inhibition is farther downstream, at the level of Relish promoter binding or transcriptional activation. Additionally, flies co-infected with both IIV-6 and the Gram-negative bacterium, Erwinia carotovora carotovora, succumb to infection more rapidly than flies singly infected with either the virus or the bacterium. These findings demonstrate how pre-existing infections can have a dramatic and negative effect on secondary infections, and establish a Drosophila model to study confection susceptibility

Saturation of acyl chains converts cardiolipin from an antagonist to an activator of Toll-like receptor-4

Abstract: Cardiolipins (CLs) are tetra-acylated diphosphatidylglycerols found in bacteria, yeast, plants, and animals. In healthy mammals, CLs are unsaturated, whereas saturated CLs are found in blood cells from Barth syndrome patients and in some Gram-positive bacteria. Here, we show that unsaturated but not saturated CLs block LPS-induced NF-κB activation, TNF-α and IP-10 secretion in human and murine macrophages, as well as LPS-induced TNF-α and IL-1β release in human blood mononuclear cells. Using HEK293 cells transfected with Toll-like receptor 4 (TLR4) and its co-receptor Myeloid Differentiation 2 (MD2), we demonstrate that unsaturated CLs compete with LPS for binding TLR4/MD2 preventing its activation, whereas saturated CLs are TLR4/MD2 agonists. As a consequence, saturated CLs induce a pro-inflammatory response in macrophages characterized by TNF-α and IP-10 secretion, and activate the alternative NLRP3 inflammasome pathway in human blood-derived monocytes. Thus, we identify that double bonds discriminate between anti- and pro-inflammatory properties of tetra-acylated molecules, providing a rationale for the development of TLR4 activators and inhibitors for use as vaccine adjuvants or in the treatment of TLR4-related diseases. Graphical abstract