Modulation of Toll-like receptor 1 intracellular domain structure and activity by Zn2+ ions

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.

Human and Bacterial Toll-Interleukin Receptor Domains Exhibit Distinct Dynamic Features and Functions

“…While complexes of the TLR1-TIR and TLR2-TIR domains have eluded structural determination, our mass spectrometry data suggests that these domains form heterogenous disulfide-mediated heterodimers and homodimers, likely hampering such structural studies. This is consistent with a recent report on TLR1 signaling that indicates that the conserved C673 is important for its pro-inflammatory activity [ 43 ].…”

“…Dynamically, in contrast to the bacterial TIR domains that undergo a global exchange, which leads to their extreme line-broadening, the human TIR domains probed here exhibited a range in terms of chemical exchange. Specifically, most of the resonances for TLR1-TIR observed here that is consistent with a recent study reporting exchange on the fast timescale for this domain [ 43 ]. In contrast, both TLR2-TIR and IL-1R8-TIR exhibit slower exchange processes.…”

“…All mammalian TIR domains were also supplemented with 1 mM DTT, which was added upon refolding in denaturation buffer, refold buffer, and NMR buffer. We note that 15 N-HSQC spectra of TLR1-TIR and TLR2-TIR match those previously reported [ 9 , 43 ].…”

“…Thus, our data suggests that TLR2-TIR is largely monomeric and that the lack of observable resonances is likely due to an inherent dynamic that leads to severe line-broadening for the majority of the domain. In other words, unlike the apparent extremes of bacterial TIR domains, which are completely line-broadened ( Figure 3 A,B), and TLR1-TIR, which results in the majority of its resonances being partially line-broadened [ 43 ], TLR2-TIR lies in between, with over half of its resonances being line-broadened due to an inherent exchange.…”

“…However, as the IL-1R8-TIR domain gives rise to an asymmetrical elution profile unlike that of the distinct dimer/monomer peaks of the TLR1 and TLR2 TIR domains ( Figure 8 A, middle and bottom), this does suggest that weak self-association may persist. We next sought to determine whether the IL-1R8-TIR domain lacks resonances due to a dynamic exchange, as found for TLR2-TIR above and recently found for TLR1-TIR [ 43 ]. In contrast to the other mammalian TIR domains, the spectrum of the IL-1R8-TIR domain of residues 160–310 does not appear to be significantly line-broadened and comprised a sufficient number of resonances that could account for the entire protein ( Figure 8 B).…”