Dataset Information

Three-dimensional mapping of differential amino acids of human, murine, canine and equine TLR4/MD-2 receptor complexes conferring endotoxic activation by lipid A, antagonism by Eritoran and species-dependent activities of Lipid IVA in the mammalian LPS sensor system.

ABSTRACT: A literature review concerning the unexpected species differences of the vertebrate innate immune response to lipid IVA was published in CSBJ prior to the present computational study to address the unpaired activity-sequence correlation of prototypic E. coli -type lipid A and its precursor lipid IVA regarding human, murine, equine and canine species. To this end, their sequences and structures of hitherto known Toll-like receptor 4 (TLR4) and myeloid differentiation factor 2 (MD-2) complexes were aligned and their differential side chain patterns studied. If required due to the lack of the corresponding X-ray crystallographic data, three-dimensional models of TLR4/MD-2/ligand complexes were generated using mono and dimeric crystal structures as templates and in silico docking of the prototypic ligands lipid A, lipid IVA and Eritoran. All differential amino acids were mapped to pinpoint species dependency on an atomic scale, i.e. the possible concert of mechanistically relevant side chains. In its most abstract and general form the three-dimensional (3D-) models devise a triangular interface or "wedge" where molecular interactions between TLR4, MD-2 and ligand itself take place. This study identifies two areas in the wedge related to either agonism or antagonism reflecting why ligands like lipid IVA can possess a species dependent dual activity. Lipid IVA represents an imperfect (underacylated and backbone-flipped), low affinity ligand of mammalian TLR4/MD-2 complexes. Its specific but weak antagonistic activity in the human system is in particular due to the loss of phosphate attraction in the wedge-shaped region conferred by nonhomologous residue changes when compared to crystal and modeled structures of the corresponding murine and equine TLR4/MD-2 complexes. The counter-TLR4/MD-2 unit was also taken into account since agonist-mediated dimerization in a defined m-shaped complex composed of two TLR4/MD-2/agonist subunits triggers intracellular signaling during the innate immune response to bacterial endotoxin exposure.

SUBMITTER: Scior T

PROVIDER: S-EPMC3962092 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Three-dimensional mapping of differential amino acids of human, murine, canine and equine TLR4/MD-2 receptor complexes conferring endotoxic activation by lipid A, antagonism by Eritoran and species-dependent activities of Lipid IVA in the mammalian LPS sensor system.

Scior Thomas T Lozano-Aponte Jorge J Figueroa-Vazquez Vianihuini V Yunes-Rojas Julian A JA Zähringer Ulrich U Alexander Christian C

Computational and structural biotechnology journal 20130808

A literature review concerning the unexpected species differences of the vertebrate innate immune response to lipid IVA was published in CSBJ prior to the present computational study to address the unpaired activity-sequence correlation of prototypic E. coli -type lipid A and its precursor lipid IVA regarding human, murine, equine and canine species. To this end, their sequences and structures of hitherto known Toll-like receptor 4 (TLR4) and myeloid differentiation factor 2 (MD-2) complexes wer ...[more]

PMID: 24688739

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Project description:There is literature evidence gathered throughout the last two decades reflecting unexpected species differences concerning the immune response to lipid IVa which provides the opportunity to gain more detailed insight by the molecular modeling approach described in this study. Lipid IVa is a tetra-acylated precursor of lipid A in the biosynthesis of lipopolysaccharide (LPS) in Gram-negative bacteria. Lipid A of the prototypic E. coli-type is a hexa-acylated structure that acts as an agonist in all tested mammalian species by innate immunorecognition via the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD-2) receptor complex. In contrast, lipid IVa is proinflammatory in mouse cells (agonism) but it remains inactive to human macrophages and even antagonizes the action of potent agonists like E. coli-type lipid A. This particular ambivalent activity profile of lipid IVa has been confirmed in other mammalian species: in equine cells Lipid IVa also acts in a weak agonistic manner, whereas being inactive and antagonizing the lipid A-induced activation of canine TLR4/MD-2. Intriguingly, the respective TLR4 amino acid sequences of the latter species are more identical to the human (67%, 68%) than to the murine (62%, 58%) ortholog. In order to address the unpaired activity-sequence dualism for human, murine, canine and equine species regarding the activity of lipid IVa as compared to LPS and lipid A and, we review the literature and computationally pinpoint the differential biological effects of lipid IVa versus LPS and lipid A to specific amino acid residues. In contrast to lipid IVa the structurally related synthetic compound Eritoran (E5564) acts consistently in an antagonistic manner in these mammalian species and serves as a reference ligand for molecular modeling in this study. The combined evaluation of data sets provided by prior studies and in silico homology mapping of differential residues of TLR4/MD-2 complexes lends detailed insight into the driving forces of the characteristic binding modes of the lipid A domain in LPS and the precursor structure lipid IVa to the receptor complex in individual mammalian species.

Project description:The crystal structure of the TLR4-MD-2-LPS complex responsible for triggering powerful pro-inflammatory cytokine responses has recently become available. Central to cell surface complex formation is binding of lipopolysaccharide (LPS) to soluble MD-2. We have previously shown, in biologically based experiments, that a generation 3.5 PAMAM dendrimer with 64 peripheral carboxylic acid groups acts as an antagonist of pro-inflammatory cytokine production after surface modification with 8 glucosamine molecules. We have also shown using molecular modelling approaches that this partially glycosylated dendrimer has the flexibility, cluster density, surface electrostatic charge, and hydrophilicity to make it a therapeutically useful antagonist of complex formation. These studies enabled the computational study of the interactions of the unmodified dendrimer, glucosamine, and of the partially glycosylated dendrimer with TLR4 and MD-2 using molecular docking and molecular dynamics techniques. They demonstrate that dendrimer glucosamine forms co-operative electrostatic interactions with residues lining the entrance to MD-2's hydrophobic pocket. Crucially, dendrimer glucosamine interferes with the electrostatic binding of: (i) the 4'phosphate on the di-glucosamine of LPS to Ser118 on MD-2; (ii) LPS to Lys91 on MD-2; (iii) the subsequent binding of TLR4 to Tyr102 on MD-2. This is followed by additional co-operative interactions between several of the dendrimer glucosamine's carboxylic acid branches and MD-2. Collectively, these interactions block the entry of the lipid chains of LPS into MD-2's hydrophobic pocket, and also prevent TLR4-MD-2-LPS complex formation. Our studies have therefore defined the first nonlipid-based synthetic MD-2 antagonist using both animal model-based studies of pro-inflammatory cytokine responses and molecular modelling studies of a whole dendrimer with its target protein. Using this approach, it should now be possible to computationally design additional macromolecular dendrimer based antagonists for other Toll Like Receptors. They could be useful for treating a spectrum of infectious, inflammatory and malignant diseases.

Project description: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.

Dataset Information

Three-dimensional mapping of differential amino acids of human, murine, canine and equine TLR4/MD-2 receptor complexes conferring endotoxic activation by lipid A, antagonism by Eritoran and species-dependent activities of Lipid IVA in the mammalian LPS sensor system.

Publications

Three-dimensional mapping of differential amino acids of human, murine, canine and equine TLR4/MD-2 receptor complexes conferring endotoxic activation by lipid A, antagonism by Eritoran and species-dependent activities of Lipid IVA in the mammalian LPS sensor system.

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