Mapping of a microbial protein domain involved in binding and activation of the TLR2/TLR1 heterodimer

J Immunol. 2009 Mar 1;182(5):2978-85. doi: 10.4049/jimmunol.0803737.

Abstract

The pentameric B subunit of type IIb Escherichia coli enterotoxin (LT-IIb-B(5)), a doughnut-shaped oligomeric protein from enterotoxigenic E. coli, activates the TLR2/TLR1 heterodimer (TLR2/1). We investigated the molecular basis of the LT-IIb-B(5) interaction with TLR2/1 to define the structure-function relationship of LT-IIb-B(5) and, moreover, to gain an insight into how TLR2/1 recognizes large, nonacylated protein ligands that cannot fit within its lipid-binding pockets, as previously shown for the Pam(3)CysSerLys(4) (Pam(3)CSK(4)) lipopeptide. We first identified four critical residues in the upper region of the LT-IIb-B(5) pore. Corresponding point mutants (M69E, A70D, L73E, S74D) were defective in binding TLR2 or TLR1 and could not activate APCs, despite retaining full ganglioside-binding capacity. Point mutations in the TLR2/1 dimer interface, as determined in the crystallographic structure of the TLR2/1-Pam(3)CSK(4) complex, resulted in diminished activation by both Pam(3)CSK(4) and LT-IIb-B(5). Docking analysis of the LT-IIb-B(5) interaction with this apparently predominant activation conformation of TLR2/1 revealed that LT-IIb-B(5) might primarily contact the convex surface of the TLR2 central domain. Although the TLR1/LT-IIb-B(5) interface is relatively smaller, the leucine-rich repeat motifs 9-12 in the central domain of TLR1 were found to be critical for cooperative TLR2-induced cell activation by LT-IIb-B(5). Moreover, the putative LT-IIb-B(5) binding site overlaps partially with that of Pam(3)CSK(4); consistent with this, Pam(3)CSK(4) suppressed TLR2 binding of LT-IIb-B(5), albeit not as potently as self-competitive inhibition. We identified the upper pore region of LT-IIb-B(5) as a TLR2/1 interactive domain, which contacts the heterodimeric receptor at a site that is distinct from, although it overlaps with, that of Pam(3)CSK(4).

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Bacterial Toxins / chemistry*
  • Bacterial Toxins / genetics
  • Bacterial Toxins / metabolism*
  • Cell Line, Tumor
  • Cells, Cultured
  • Crystallography, X-Ray
  • Dimerization
  • Enterotoxigenic Escherichia coli / chemistry*
  • Enterotoxigenic Escherichia coli / genetics
  • Enterotoxigenic Escherichia coli / metabolism
  • Enterotoxins / chemistry*
  • Enterotoxins / genetics
  • Enterotoxins / metabolism*
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Humans
  • Lipopeptides / chemistry
  • Lipopeptides / genetics
  • Lipopeptides / metabolism
  • Mice
  • Mice, Inbred BALB C
  • Peptide Mapping* / methods
  • Point Mutation
  • Protein Binding / genetics
  • Protein Binding / immunology
  • Protein Subunits / chemistry
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • Toll-Like Receptor 1 / antagonists & inhibitors
  • Toll-Like Receptor 1 / chemistry
  • Toll-Like Receptor 1 / metabolism*
  • Toll-Like Receptor 2 / antagonists & inhibitors
  • Toll-Like Receptor 2 / chemistry
  • Toll-Like Receptor 2 / metabolism*

Substances

  • Bacterial Toxins
  • Enterotoxins
  • Escherichia coli Proteins
  • Lipopeptides
  • Protein Subunits
  • TLR2 protein, human
  • Toll-Like Receptor 1
  • Toll-Like Receptor 2
  • heat-labile enterotoxin, E coli