Equilibrium Binding Model for CpG DNA-Dependent Dimerization of Toll-like Receptor 9 Ectodomain.

Stephanie Reikine,Stephen H Mclaughlin,Yorgo Modis

doi:10.1021/acs.biochem.0c00466

Stephanie Reikine, Stephen H Mclaughlin + Show 1 more

Open Access

https://doi.org/10.1021/acs.biochem.0c00466

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Abstract

Microbial nucleic acids in the extracellular milieu are recognized in vertebrates by Toll-like receptors (TLRs), one of the most important families of innate immune receptors. TLR9 recognizes single-stranded unmethylated CpG DNA in endosomes. DNA binding induces TLR9 dimerization and activation of a potent inflammatory response. To provide insights on how DNA ligands induce TLR9 dimerization, we developed a detailed theoretical framework for equilibrium ligand binding, modeling the binding of the ssDNA at the two main sites on the TLR9 ectodomain. Light scattering and fluorescence anisotropy assays performed with recombinant TLR9 ectodomain and a panel of agonistic and antagonistic DNA ligands provide data that restrain the binding parameters, identify the likely ligand binding intermediates, and suggest cooperative modes of binding. This work brings us one step closer to establishing a rigorous biochemical understanding of how TLRs are activated by their ligands.

Highlights

Microbial nucleic acids in the extracellular milieu are recognized in vertebrates by Toll-like receptors (TLRs), one of the most important families of innate immune receptors
The crystal structures of TLR9 ectodomain fragments from mouse, horse, and cow have been determined without ligand, bound to antagonistic ligands 4084 and iSUPER, and bound to 1668-12mer,[10] a truncated version of the activating oligonucleotide ligand 1668.11 These structures provided the structural basis for the CpG specificity of TLR9 ligand recognition
An additional binding site in TLR9 was recently identified in the central region of the ectodomain, with specificity for short single-stranded DNA (ssDNA) oligonucleotides containing the motif 5′-xCx,[13] which function as auxiliary ligands to enhance signaling.[14]

Summary

Introduction

Microbial nucleic acids in the extracellular milieu are recognized in vertebrates by Toll-like receptors (TLRs), one of the most important families of innate immune receptors. A major family of innate immune receptors is the Toll-like receptors (TLRs).[2] TLR3, TLR7, TLR8, and TLR9 are found in endosomes and recognize nucleic acid PAMPs.[3−7] TLR9 recognizes single-stranded DNA (ssDNA) containing unmethylated CG nucleotide sequence motifs (CpGs).[7] CpGs are more prevalent in bacteria and viruses than in the mammalian genome, in which most CG sequences are methylated.[8,9]. A reductionist approach to determine the minimal sequence requirements for an oligonucleotide to maximally activate TLR9 identified a length of between 23 and 29 nucleotides as the optimal length for mouse TLR9 agonists, with a 5′-TCC motif and CpG motif located 5−7 nucleotides from the 5′ end.[17,18] It remains unclear why extending the length of the ligand beyond the 12 nucleotides observed in the TLR9:1668-12mer structure enhances signaling. Article than a 2:2 model) or of the Hill equation.[10,13,19,20] it is known that the ligand-saturated state of a signaling-competent

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