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

Irvine KL, Hopkins LJ, Gangloff M, Bryant CE - Vet. Res. (2013)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB30ES, UK. ceb27@cam.ac.uk.

ABSTRACT
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.

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Species-differences of the ligand binding pockets of TLR2/1. Crystal structures of human TLR2 and TLR1 [PDB code 2Z7X], and a model of human TLR6, were viewed in PyMol and non-conserved residues between the human and horse highlighted. (A) Conserved TLR2 ligand-binding residues (cyan) form the majority of contacts, whereas non-conserved (magenta) are at the periphery of the pocket. The TLR1 pocket is lined with conserved residues (yellow); only G313 and V339 (lilac; S317 and I343 in the horse) are non-conserved. (B) L318 (green) and F319 (blue), which interact with Pam2CSK4 (red), are conserved in the horse (I318 and F319). The region surrounding L318 and F319 is conserved apart from F317 (Y317 in the horse; cyan), which lies immediately lateral to L318. F343 and F365 (magenta), which block the TLR6 pocket, are also shown.
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Figure 4: Species-differences of the ligand binding pockets of TLR2/1. Crystal structures of human TLR2 and TLR1 [PDB code 2Z7X], and a model of human TLR6, were viewed in PyMol and non-conserved residues between the human and horse highlighted. (A) Conserved TLR2 ligand-binding residues (cyan) form the majority of contacts, whereas non-conserved (magenta) are at the periphery of the pocket. The TLR1 pocket is lined with conserved residues (yellow); only G313 and V339 (lilac; S317 and I343 in the horse) are non-conserved. (B) L318 (green) and F319 (blue), which interact with Pam2CSK4 (red), are conserved in the horse (I318 and F319). The region surrounding L318 and F319 is conserved apart from F317 (Y317 in the horse; cyan), which lies immediately lateral to L318. F343 and F365 (magenta), which block the TLR6 pocket, are also shown.

Mentions: Residues within human TLR2/1 responsible for ligand binding and dimerisation have been identified [2]. We compared the equine and human receptors to determine whether non-conservative changes between the two species’ sequences are located in areas of the proteins that could affect ligand association, and explain species specificity at TLR2/1. Human-equine non-conserved residues are spread diffusely across both receptor ectodomains (data not shown). The binding pockets of TLR2 and TLR1 are lined predominantly with human-equine conserved residues, whereas the regions of the two receptors flanking the ligand peptide group are virtually all non-conserved (Additional file 5). Seven of the twenty four residues of human TLR2 that interact with Pam3CSK4 in the crystal structure are non-conserved between human and equine, but these lie mainly on the periphery of the ligand binding pocket (Figure 4A). All but three of the TLR dimerisation residues of TLR2 are conserved between the two species, and all of the residues that interact with both the ligand and form the dimerisation interface are also conserved (not shown). Two of the eighteen residues in human TLR1 that interact with Pam3CSK4 are non-conserved between the two species (Figure 4A), and all but four of the main dimerisation interface residues are conserved (not shown). Of the two non-conserved residues, only V339 (I343 in the equine) contacts the amide acyl chain of Pam3CSK4. In summary, nine ligand-binding and seven dimerisation residues differ between the equine and human TLR2/1 dimers, which could potentially explain the small species differences in ligand recognition at this receptor pair.


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

Irvine KL, Hopkins LJ, Gangloff M, Bryant CE - Vet. Res. (2013)

Species-differences of the ligand binding pockets of TLR2/1. Crystal structures of human TLR2 and TLR1 [PDB code 2Z7X], and a model of human TLR6, were viewed in PyMol and non-conserved residues between the human and horse highlighted. (A) Conserved TLR2 ligand-binding residues (cyan) form the majority of contacts, whereas non-conserved (magenta) are at the periphery of the pocket. The TLR1 pocket is lined with conserved residues (yellow); only G313 and V339 (lilac; S317 and I343 in the horse) are non-conserved. (B) L318 (green) and F319 (blue), which interact with Pam2CSK4 (red), are conserved in the horse (I318 and F319). The region surrounding L318 and F319 is conserved apart from F317 (Y317 in the horse; cyan), which lies immediately lateral to L318. F343 and F365 (magenta), which block the TLR6 pocket, are also shown.
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Figure 4: Species-differences of the ligand binding pockets of TLR2/1. Crystal structures of human TLR2 and TLR1 [PDB code 2Z7X], and a model of human TLR6, were viewed in PyMol and non-conserved residues between the human and horse highlighted. (A) Conserved TLR2 ligand-binding residues (cyan) form the majority of contacts, whereas non-conserved (magenta) are at the periphery of the pocket. The TLR1 pocket is lined with conserved residues (yellow); only G313 and V339 (lilac; S317 and I343 in the horse) are non-conserved. (B) L318 (green) and F319 (blue), which interact with Pam2CSK4 (red), are conserved in the horse (I318 and F319). The region surrounding L318 and F319 is conserved apart from F317 (Y317 in the horse; cyan), which lies immediately lateral to L318. F343 and F365 (magenta), which block the TLR6 pocket, are also shown.
Mentions: Residues within human TLR2/1 responsible for ligand binding and dimerisation have been identified [2]. We compared the equine and human receptors to determine whether non-conservative changes between the two species’ sequences are located in areas of the proteins that could affect ligand association, and explain species specificity at TLR2/1. Human-equine non-conserved residues are spread diffusely across both receptor ectodomains (data not shown). The binding pockets of TLR2 and TLR1 are lined predominantly with human-equine conserved residues, whereas the regions of the two receptors flanking the ligand peptide group are virtually all non-conserved (Additional file 5). Seven of the twenty four residues of human TLR2 that interact with Pam3CSK4 in the crystal structure are non-conserved between human and equine, but these lie mainly on the periphery of the ligand binding pocket (Figure 4A). All but three of the TLR dimerisation residues of TLR2 are conserved between the two species, and all of the residues that interact with both the ligand and form the dimerisation interface are also conserved (not shown). Two of the eighteen residues in human TLR1 that interact with Pam3CSK4 are non-conserved between the two species (Figure 4A), and all but four of the main dimerisation interface residues are conserved (not shown). Of the two non-conserved residues, only V339 (I343 in the equine) contacts the amide acyl chain of Pam3CSK4. In summary, nine ligand-binding and seven dimerisation residues differ between the equine and human TLR2/1 dimers, which could potentially explain the small species differences in ligand recognition at this receptor pair.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB30ES, UK. ceb27@cam.ac.uk.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus