title: The C2 domain of Tollip, a Toll-like receptor signalling regulator, exhibits broad preference for phosphoinositides.
abstract: TLRs (Toll-like receptors) provide a mechanism for host defence immune responses. Activated TLRs lead to the recruitment of adaptor proteins to their cytosolic tails, which in turn promote the activation of IRAKs (interleukin-1 receptor-associated kinases). IRAKs act upon their transcription factor targets to influence the expression of genes involved in the immune response. Tollip (Toll-interacting protein) modulates IRAK function in the TLR signalling pathway. Tollip is multimodular, with a conserved C2 domain of unknown function. We found that the Tollip C2 domain preferentially interacts with phosphoinositides, most notably with PtdIns3P (phosphatidylinositol 3-phosphate) and PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate), in a Ca2+-independent manner. However, NMR analysis demonstrates that the Tollip C2 domain binds Ca2+, which may be required to target the membrane interface. NMR and lipid-protein overlay analyses suggest that PtdIns3P and PtdIns(4,5)P2 share interacting residues in the protein. Kinetic studies reveal that the C2 domain reversibly binds PtdIns3P and PtdIns(4,5)P2, with affinity values in the low micromolar range. Mutational analysis identifies key PtdIns3P- and PtdIns(4,5)P2-binding conserved basic residues in the protein. Our findings suggest that basic residues of the C2 domain mediate membrane targeting of Tollip by interaction with phosphoinositides, which contribute to the observed partition of the protein in different subcellular compartments.
authors.affiliation: Protein Signaling Domains Laboratory, Department of Biological Sciences, Virginia Tech, Blacksburg, VA 24061, USA.
authors.forename: Gayatri
authors.initials: G
authors.lastname: Ankem
authors.forename: Sharmistha
authors.initials: S
authors.lastname: Mitra
authors.forename: Furong
authors.initials: F
authors.lastname: Sun
authors.forename: Anna C
authors.initials: AC
authors.lastname: Moreno
authors.forename: Boonta
authors.initials: B
authors.lastname: Chutvirasakul
authors.forename: Hugo F
authors.initials: HF
authors.lastname: Azurmendi
authors.forename: Liwu
authors.initials: L
authors.lastname: Li
authors.forename: Daniel G S
authors.initials: DG
authors.lastname: Capelluto
mesh_terms: D002118:Calcium / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009154:Mutation; D010716:Phosphatidylinositols / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000378:metabolism
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D010716:Phosphatidylinositols; C410602:TOLLIP protein, human; D002118:Calcium