Insights into human TLR9 structural dynamics and recognition of pathogen-associated CpG DNA using molecular dynamics simulation

Human toll-like receptor 9 (TLR9) is a key pattern-recognition receptor that detects unmethylated CpG motifs in pathogen single-stranded (ss) DNA. Although structural information for non-human TLR9 ectodomains exists, the conformational dynamics of human TLR9 remain poorly understood. Here, we modeled the TLR9 ectodomain and performed all-atom simulations of apo TLR9, CpG-bound TLR9, and CpG-immunomodulator (IM) co-bound TLR9. We show that the apo ectodomain formed a stable pre-assembled homodimer and CpG ssDNA was observed to bind with a 2:2 stoichiometry, bridging both monomers and inducing localized rearrangements within the leucine-rich repeat region. CpG ssDNA binding enhanced Z-loop flexibility by ∼2 folds in the TLR9_CpG and TLR9_CpG_IM complexes. Furthermore, CpG ssDNA and IM engagement reorganized inter-monomer contacts and revealed previously unrecognized functional residues like H612, H641, T642, and K690, that supports cooperative ligand stabilization. Together, these simulations identify candidate structural transitions and putative allosteric nodes in human TLR9 for future experimental studies.