Structural studies of Toll like receptor signaling adaptors.

Signaling downstream of the Toll like receptors (TLRs) involves Toll-IL-1R homology (TIR) domains as well as recruitment of critical signaling adaptors MyD88 and TIRAP/Mal. Studies in human patients, dominant negative mutants and knockout mice have shown that mutations in Arg196, the BB loop, DD loop, “Poc” site Ile179 are critical for MyD88 TIR domain signaling and appropriate responses to pathogen stimuli. We previously reported the structure of MyD88 TIR domain that showed a unique conformation for its BB loop, as well as a crystallographic lattice that reveals interactions between the BB-DD and BB-EE loops. We now report additional crystal forms which retain BB-DD and BB-EE interactions as well as extend the resolution to 1.4 Å. A yeast two-hybrid analysis of the domain interface residues observed from the crystal structure lattice shows that key residues including the human Arg196Cys mutation are critically important for homotypic TIR domain binding. Additionally, we report NMR solution studies showing binding between MyD88 and bacterial TIR domain protein TcpC, which has been demonstrated to negatively regulate MyD88-dependent TLR adaptor signaling. Finally, we report the X-ray structure of a second critical TIR adaptor protein, TIRAP/Mal. We observe key residues and loops important for TIRAP/Mal adaptor function. Structural insights from these studies may aid our understanding of the molecular mechanisms by which TLRs and TIR domain adaptors interact and signal.