Molecular mechanisms for the subversion of MyD88 signaling by TcpC from virulent uropathogenic Escherichia coli

Abstract

The Toll/IL-1 receptor (TIR) domains are crucial signaling modules during innate immune responses involving the Toll-like receptors (TLRs) and IL-1 receptor (IL-1R). Myeloid differential factor 88 (MyD88) is a central TIR domain-containing adapter molecule responsible for nearly all TLR-mediated signaling and is targeted by a TIR domain-containing protein C (TcpC) from virulent uropathogenic Escherichia coli, a common human pathogen. The mechanism of such molecular antagonism has remained elusive. We present the crystal structure of the MyD88 TIR domain with distinct loop conformations that underscore the functional specialization of the adapter, receptor, and microbial TIR domains. Our structural analyses shed light on the genetic mutations at these loops as well as the Poc site. We demonstrate that TcpC directly associates with MyD88 and TLR4 through its predicted DD and BB loops to impair the TLR-induced cytokine induction. Furthermore, NMR titration experiments identify the unique CD, DE, and EE loops from MyD88 at the TcpC-interacting surface, suggesting that TcpC specifically engages these MyD88 structural elements for immune suppression. These findings thus provide a molecular basis for the subversion of TLR signaling by the uropathogenic E. coli virulence factor TcpC and furnish a framework for the design of novel therapeutic agents that modulate immune activation.

Fig. 1.

Comparison of the TIR domain structures. (A) Cartoon representation of the MyD88 TIR domain crystal structure, labeled with key secondary structure elements. The five parallel β-strands are colored orange and the rest of the structure in cyan. (B) Superposition of structures for the TIR domains from MyD88 (cyan), TLR2 (lime), TLR1 (pale green), and Paracoccus PdTLP (magenta). Regions of major structural differences are marked.

Fig. 2.

Poc site residue anchors the BB loop. (A) Cartoon representation of the interactions between the MyD88 TIR domain Poc site residue I179 and its BB loop residues. (B) Root mean square fluctuations of the Cα atoms for the wild-type (blue) and I179N mutant (red) MyD88 TIR domains in a 5-ns MD simulation.

Fig. 3.

Suppression of TLR signaling by the TcpC TIR domain and its peptides. (A) Pull-down assays of the cellular lysates from the Myc-MyD88 transfected HEK293 cells were performed with the TIR TcpC Strep-Tactin Macroprep beads (+) or empty Strep-Tactin Macroprep beads (-). The beads were washed with 500 mM NaCl, and the remaining proteins were detected by Western blot using an anti-Myc antibody after elution. (B) The same assay was performed as in A using lysates from the Flag-TLR4 transfected cells. The beads were washed with two different NaCl concentrations as indicated before elution. (C and D) BMDMs were stimulated with TLR ligands in the presence of titrated amounts of the TcpC BB loop peptide (0.0026, 0.026, 0.26, or 2.6 µM, C) or the TcpC DD loop peptide (0.0025, 0.025, 0.25, or 2.5 µM, D), and the TNF-α in the culture supernatants was analyzed 3 h after stimulation. BB and DD indicate TNF-α release induced by the highest dose of each peptide in the absence of a TLR ligand. Error bars represent SD of three individual experiments. (E and F) The same pull-down assays were performed as in A and B, except that BB or DD loop peptide bearing a Strep-tag was bound to Strep-Tactin Macroprep beads. Empty Strep-Tactin Macroprep beads (-) were used as negative control. After washing with a buffer containing 500 mM NaCl, bound TLR4 (E) or MyD88 (F) were detected by Western blot after elution.

Fig. 4.

Identification of the TcpC-binding site at the MyD88 TIR domain. (A) NMR titration of the ¹⁵N-labeled MyD88 TIR domain with TcpC. The ¹H-¹⁵N chemical shift changes were plotted in the presence of TcpC (black) and buffer (red). The dashed line marks the threshold value (mean + 1.5 SD). (B) Mapping of the TcpC-binding site on the surface of the MyD88 TIR domain. Residues with significant chemical shift changes in A are colored blue and labeled. (C) HEK293 cells were transfected with MyD88-encoding plasmids including the wild-type, C203S, C280S, and C203S plus C280S mutants, as well as titrated amounts of TIR-TcpC ΔTAT, as indicated. Luciferase activities were determined 48 h after transfection. Error bars represent SD of three individual experiments.