Figure 1.
(A) Overview of the TLR4 signalling pathway. Both the NF-κB and the interferon pathways are induced by stimulation with lipopolysaccharide. Adapted from [2], [3]. (B) Mechanism of signal transduction by TLR4. The curved ectodomains (ECD) are illustrated in light blue and the co-receptor protein MD-2 in grey. The TIR domains are shown in yellow and red respectively. M = membrane, L = LPS. (i) Prior to activation, receptor molecules are able to diffuse in the membrane and may form transient dimers. The ectodomains are rigidly connected to the cytoplasmic TIRs by the transmembrane helix. (ii) Receptor dimerization following activation by LPS binding to MD2. By analogy with Drosophila Toll (see [45]), which is activated by a dimeric protein ligand, the receptor complexes are likely to be symmetrical. Conformational rearrangements constrain the TIR domains to interact through equivalent surfaces forming a symmetrical dimer. (iii) The dimerized TIRs provide a new molecular surface that can bind to the ‘bridging adaptor’ molecules TRAM and Mal with high affinity. Interaction with the downstream adaptors TRIF and MyD88 leads to NFκB and IRF3 mediated signalling respectively.
Figure 2.
Structure based sequence alignments of TIR domains.
The program JOY was used to annotate the alignments for TLR1, TLR2 TLR4, TLR10, Mal and TRAM. Numbers on top of amino acid sequences are alignment positions. The key to JOY annotations is as follows (a graphical version is viewable as Table S1); solvent inaccessible – UPPER CASE; solvent accessible – lower case; α-helix – dark grey shaded; β-strand – mid-grey shaded; 310 helix – light grey shaded; hydrogen bond to main chain amide – bold; hydrogen bond to main chain carbonyl – underline; hydrogen bond to other sidechain – tilde; disulphide bond – cedilla; positive φ--->φ - italic; cis-peptide – breve.
Figure 3.
Structural modelling of the TLR4 TIR domain homodimer, Mal and TRAM.
The BB loops of the two TLR4 protomers are coloured blue and yellow respectively. For Mal and TRAM they are coloured green (A) TLR4 top view. (B) TLR4 side view. (C) Mal. (D) TRAM.
Table 1.
Interacting residues in the dimer interface of TLR4.
Figure 4.
Docking studies predict that the adaptors bind at the side of the TLR4 homodimer interface.
The TLR4 protomers, represented as ribbon diagrams are in green and cyan. Docked Mal and TRAM are represented as stick models and the 50 best docking solutions generated by GRAMM for either Mal (A) or TRAM (B) have been superimposed upon one another. (C) High resolution complex of TLR4 dimer (green and cyan), Mal (pink) and TRAM (yellow). The position of each BB loop is labelled.
Table 2.
Potential structural impact of TLR4 TIR mutations.
Table 3.
Summary of effects of mutation on NFκB and IFN-β activation.
Table 4.
Residues that produce strong interactions (ΔASA>40 Å2) in the interface of the TLR4 dimer-Mal complex.
Table 5.
Residues that produce strong interactions (ΔASA>40 Å2) in the interface of the TLR4 dimer-TRAM complex.
Figure 5.
Modelling suggests a molecular explanation for the caspase 1 dependence of Mal and the malfunctional human polymorphism Ser180Leu.
The models are shown as van der Waal surface representations. (A) Side view showing the position of the BB loop (green) and the phosphorylated tyrosine, Tyr86. In the complex this part of Mal forms the interface with TLR4. The position of the α-E helix (red) which is cleaved out by caspase 1 is shown on the opposite surface to the BB loop. (B) Back view of Mal (rotated 90° to the right relative to (A)). (C), (D) Mal with the α-E helix removed highlighting the deep groove created and the exposed position of the otherwise buried Ser180 residue (yellow).