Myeloid differentiating factor 88 (MyD88) and MyD88 adaptor-like (Mal) are adaptor molecules critically involved in the Toll-like receptor (TLR) 4 signaling pathway. While Mal has been proposed to serve as a membrane-sorting adaptor, MyD88 mediates signal transduction from activated TLR4 to downstream components. The Toll/Interleukin-1 receptor (TIR) domain of MyD88 is responsible for sorting and signaling via direct or indirect TIR−TIR interactions between Mal and TLR4. However, the molecular mechanisms involved in multiple interactions of the TIR domain remain unclear. The present study describes the solution structure of the MyD88 TIR domain. Reporter gene assays revealed that 3 discrete surface sites in the TIR domain of MyD88 are important for TLR4 signaling. Two of these sites were shown to mediate direct binding to the TIR domain of Mal. Interestingly, Mal-TIR, but not MyD88-TIR, directly binds to the cytosolic TIR domain of TLR4. These observations suggested that the heteromeric assembly of TIR domains of the receptor and adaptors constitutes the initial step of TLR4 intracellular signal transduction.

Protein–phosphoinositide interaction participates in targeting proteins to membranes where they function correctly and is often modulated by phosphorylation of lipids. Here we show that protein phosphorylation of p47 ^phox , a cytoplasmic activator of the microbicidal phagocyte oxidase (phox), elicits interaction of p47 ^phox with phosphoinositides. Although the isolated phox homology (PX) domain of p47 ^phox can interact directly with phosphoinositides, the lipid-binding activity of this protein is normally suppressed by intramolecular interaction of the PX domain with the C-terminal Src homology 3 (SH3) domain, and hence the wild-type full-length p47 ^phox is incapable of binding to the lipids. The W263R substitution in this SH3 domain, abrogating the interaction with the PX domain, leads to a binding of p47 ^phox to phosphoinositides. The findings indicate that disruption of the intramolecular interaction renders the PX domain accessible to the lipids. This conformational change is likely induced by phosphorylation of p47 ^phox , because protein kinase C treatment of the wild-type p47 ^phox but not of a mutant protein with the S303/304/328A substitution culminates in an interaction with phosphoinositides. Furthermore, although the wild-type p47 ^phox translocates upon cell stimulation to membranes to activate the oxidase, neither the kinase-insensitive p47 ^phox nor lipid-binding-defective proteins, one lacking the PX domain and the other carrying the R90K substitution in this domain, migrates. Thus the protein phosphorylation-driven conformational change of p47 ^phox enables its PX domain to bind to phosphoinositides, the interaction of which plays a crucial role in recruitment of p47 ^phox from the cytoplasm to membranes and subsequent activation of the phagocyte oxidase.

Ubiquitination, a modification in which single or multiple ubiquitin molecules are attached to a protein, serves as a signalling function that controls a wide variety of cellular processes. To date, two major forms of polyubiquitin chain have been functionally characterized, in which the isopeptide bond linkages involve Lys48 or Lys63. Lys48‐linked polyubiquitin tagging is mostly used to target proteins for degradation by the proteasome, whereas Lys63‐linked polyubiquitination has been linked to numerous cellular events that do not rely on degradative signalling via the proteasome. Apparently linkage‐specific conformations of polyubiquitin chains are important for these cellular functions, but the structural bases distinguishing Lys48‐ and Lys63‐linked chains remain elusive. Here, we report NMR and small‐angle X‐ray scattering (SAXS) studies on the intersubunit interfaces and conformations of Lys63‐ and Lys48‐linked di‐ and tetraubiquitin chains. Our results indicate that, in marked contrast to Lys48‐linked chains, Lys63‐linked chains are elongated molecules with no stable non‐covalent intersubunit interfaces and thus adopt a radically different conformation from that of Lys48‐linked chains.