Structural Basis of Ligand Recognition by the Tollip C2 and CUE Domains

Abstract

Toll-like receptors (TLRs) provide a mechanism of host defense responses by activating the innate and adaptative immune responses. Subsequent downstream events result in the recruitment of one or more adaptor proteins, a process mediated by the cytosolic tail of TLRs. These protein-protein interactions promote the activation of the interleukin-1 receptor-associated kinases (IRAKs) 1, 2, M, and 4 that act upon their transcription factor targets to influence the expression of genes involved in the innate immune response. The Toll-interacting protein (Tollip) controls IRAK function in the TLR signaling pathway. Tollip presents an N-terminal Tom1-binding domain, a central C2 domain, and a C-terminal coupling of ubiquitin to endoplasmic reticulum degradation (CUE) domain. We found that the Tollip C2 domain preferentially interacts with phosphoinositides including phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in a calcium-independent manner. NMR and lipid-protein overlay analyses suggest that PI3P and PI(4,5)P2 share the same binding site in the protein. Kinetic analysis reveals that the Tollip C2 domain reversibly binds PI3P and PI(4,5)P2 with affinities in the low micromolar range. Mutational analysis identifies key phosphoinositide-binding basic residues in the Tollip C2 domain located in a flexible region nearby the beta-groove. The CUE domain binds ubiquitin, although the biological consequences of the association as well the molecular basis of the interaction are unknown. Using NMR spectroscopy, we have identified the Tollip CUE domain residues that recognize ubiquitin as well as the ubiquitin residues that bind to the Tollip CUE domain. Structural and kinetical analyses suggest that a dimeric Tollip CUE domain forms a complex with ubiquitin in conserved binding pockets with nanomolar affinity. Overall, our findings will provide the basis to understand how Tollip is intracellularly partitioned in a ligand-dependent manner and how these interactions modulate TLR signaling.