An oligomeric signaling platform formed by the Toll-like receptor signal transducers MyD88 and IRAK-4

Abstract

Toll-like receptors (TLRs) mediate responses to pathogen-associated molecules as part of the vertebrate innate immune response to infection. Receptor dimerization is coupled to downstream signal transduction by the recruitment of a post-receptor complex containing the adaptor protein MyD88 and the IRAK protein kinases. In this work, we show that the death domains of human MyD88 and IRAK-4 assemble into closed complexes having unusual stoichiometries of 7:4 and 8:4, the Myddosome. Formation of the Myddosome is likely to be a key event for TLR4 signaling in vivo as we show here that pathway activation requires that the receptors cluster into lipid rafts. Taken together, these findings indicate that TLR activation causes the formation of a highly oligomeric signaling platform analogous to the death-inducing signaling complex of the Fas receptor pathway.

FIGURE 1.

Solution properties of the MyD88 and IRAK-4 death. A, schematic representation of hMyD88 and IRAK-4 showing the DD, the ID, the TIR, and the kinase domain (KD). B, SDS-PAGE of purified proteins: IRAK-4 DD-(1–110) (lane 1); hMyD88 DD-ID-(1–152) (lane 2), and the hMyD88-IRAK-4 complex (lane 3). M, molecular mass marker. C, sedimentation velocity AUC profiles and the c(S) distributions for hMyD88 death domains (at 4 and 0.3 mg ml⁻¹⁾. D, sedimentation velocity AUC profiles and the c(S) distributions for hMyd88 at 0.8 mg ml⁻¹. When compared with C, additional peaks are present representing oligomers of 100 and 150 kDa. E, chemical cross-linking of MyD88 protein (see ”Experimental Procedures“). Lane 1, no treatment; lane 2, 0.02 mm bis(sulfosuccinimidyl) suberate; lane 3, 0.2 mm bis(sulfosuccinimidyl) suberate.

FIGURE 2.

hMyD88 and IRAK-4 death domains assemble into complexes with stoichiometries of 7:4 and 8:4. A, elution profile of the hMyD88-IRAK-4 complex on a Superdex S200 gel filtration column. hMyD88 and IRAK-4 death domains were mixed to give a slight molar excess of IRAK-4. Samples from peak 1 and peak 2 are shown in Fig. 1B, lanes 3 and 1, respectively. Calibration of the column gave molecular mass estimates of 175 kDa for peak 1 and 12 kDa for peak 2 (not shown). B, sedimentation velocity data and the c(S) distribution for the hMyD88-IRAK-4 DD complex. C, nano-electrospray ionization mass spectra of MyD88-IRAK-4 complexes acquired under conditions that preserve non-covalent interactions. Inset, mass spectrum acquired with minimal gas phase activation, showing IRAK-4 monomers at low m/z and a set of poorly resolved ion series at higher m/z. Main panel, mass spectra acquired with moderate gas phase collisional activation to remove solvent adducts. Two well resolved overlapping ion series are revealed, corresponding to complexes with MyD88-IRAK-4 stoichiometries of 7:4 (red triangles, observed mass = 165,800 ± 34 Da, expected mass = 165,725 Da) and 8:4 (yellow circles, observed mass = 182,372 ± 6 Da, expected mass = 182,284 Da). Further unresolved ions are evident in the noise. (The starred peak forms part of an ion series extending to higher m/z, arising from gas phase dissociation of complexes; see supplemental Fig. 1.)

FIGURE 3.

Structural analysis of the hMyd88-IRAK-4 complexes. A, x-ray scattering profiles of the hMyD88-IRAK-4 death domain oligomer. Also shown is the theoretical scattering profile for the PIDDosome core complex (gray line), calculated from the crystal structure of the PIDD-RAIDD death domain complex (PDB code 2of5). The distance distribution functions are shown as insets from which maximum particle dimensions 12.3 nm are obtained. Two orthogonal orientations of the ab initio models of the hMyD88-IRAK-4 complex are also shown. B, three-dimensional image reconstructions of the MyD88 complexes. Two views of the model are shown rotated through 90° (see also supplemental Fig. 2).

FIGURE 4.

Density-dependent activation of TLR4 by the antagonistic antibody HTA125. HEK293-TLR4-YFP cells were cultured on polystyrene plastic previously coated with increasing concentrations of anti-TLR4 monoclonal antibody (mAb) (HTA125) (A) or control anti-TLR2 mAB (TL2.1), or cells were treated with LPS (100 ng/ml), LPS (100 ng/ml) with MD-2 (conditioned supernatants), or IL-1β (100 ng/ml) (B). IL-8 secretion into supernatants was assessed by ELISA. C, HTA125 or TL2.1 was immobilized alone or together with increasing concentration of cholera toxin subunit B. HEK293-TLR4-YFP cells were subsequently plated, and IL-8 secretion was measured in supernatants by ELISA. HEK293-TLR4-YFP (D) or HEK293-TLR4-YFP-CD14 cells (E) were cultured on polystyrene previously coated with HTA125 alone or with increasing concentrations of anti-CD14 (26ic) monoclonal antibody. IL-8 was measured in supernatants by ELISA. Shown are means and S.D. (error bars) of triplicates of representative experiments of three (A, B, and C) or two (D and E) repeats. B, cholera toxin subunit B enhances the ability of HTA125 to activate the TLR4 pathway. C, HTA125 activates TLR4 independently of lipopolysaccharide. A control antibody against TLR2 is ineffective in this assay. Open squares, no treatment; triangles, 0.5 ng ml⁻¹ LPS; closed squares, 5 ng ml⁻¹; diamonds, 50 ng ml⁻¹.

FIGURE 5.

Potential assembly pathway for the Myddosome.