Abstract
Toll-like receptors (TLRs) play an important role in the innate immune response. While a lot is known about the structures of their extracellular parts, many questions are still left unanswered, when the structural basis of TLR activation is analyzed for the TLR intracellular domains. Here we report the structure and dynamics of TLR1 toll-interleukin like (TIR) cytoplasmic domain in crystal and in solution. We found that the TLR1-TIR domain is capable of specific binding of Zn with nanomolar affinity. Interactions with Zn are mediated by cysteine residues 667 and 686 and C667 is essential for the Zn binding. Potential structures of the TLR1-TIR/Zn complex were predicted in silico. Using the functional assays for the heterodimeric TLR1/2 receptor, we found that both Zn addition and Zn depletion affect the activity of TLR1, and C667A mutation disrupts the receptor activity. Analysis of C667 position in the TLR1 structure and possible effects of C667A mutation, suggests that zinc-binding ability of TLR1-TIR domain is critical for the receptor activation.
Highlights
Toll-like receptors (TLRs) play an important role in the innate immune response
The TLR1-toll-interleukin like (TIR) was synthesized in Escherichia coli and was kept in the aqueous buffer, containing a potent reducing agent, tris(2-chloroethyl) phosphate (TCEP), to avoid the disulfide formation during all the purification stages
We investigated the hydrodynamic properties of the TLR1-TIR by dynamic light scattering (DLS) and Nuclear magnetic resonance (NMR) in the presence and in the absence of Zn2+ ions (Fig. 3d and Supplementary Fig. 7)
Summary
Toll-like receptors (TLRs) play an important role in the innate immune response. While a lot is known about the structures of their extracellular parts, many questions are still left unanswered, when the structural basis of TLR activation is analyzed for the TLR intracellular domains. The molecular mechanism of TLR activation is believed to be known: ligand binding induces the receptor dimerization, which, in turn, triggers the interaction of TIR domains with intracellular adaptor proteins, namely myeloid differentiation primary response 88 protein (MyD88), TIR domain containing adaptor protein (TIRAP), tumor necrosis factor receptor-associated factors, etc.[6]. Either heterodimerization itself or the specific conformation of the dimer, induced by the ligand binding, are believed to cause the interaction between the TIR domains of TLR2 and MyD88, and assembly of a signaling complex, referred to as myddosome[9,10]. TLR extracellular domains are studied rather well—much X-ray data are available, including several structures of dimeric domains in complex with various ligands[11,12,13,14,15,16,17,18,19]. To fill these “blank spots,” we initiated the investigation of TLR1-TIR domain structure and dynamics in crystal and in solution, focusing on the factors that can influence the interaction between the TIR domains, including the presence of metal ions
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