DNA binding to proteolytically activated TLR9 is sequence‐independent and enhanced by DNA curvature

Toll‐like receptor 9 (TLR9) recognizes microbial DNA in endolysosomal compartments. The ectodomain of TLR9 must be proteolytically cleaved by endosomal proteases to produce the active receptor capable of inducing an innate immune signal. We show that the cleaved TLR9 ectodomain is a monomer in solution and that DNA ligands with phosphodiester backbones induce TLR9 dimerization in a sequence‐independent manner. Ligands with phosphorothioate (PS) backbones induce the formation of large TLR9–DNA aggregates, possibly due to the propensity of PS ligands to self‐associate. DNA curvature‐inducing proteins including high‐mobility group box 1 and histones H2A and H2B significantly enhance TLR9 binding, suggesting that TLR9 preferentially recognizes curved DNA backbones. Our work sheds light on the molecular mechanism of TLR9 activation by endogenous protein–nucleic acid complexes, which are associated with autoimmune diseases including systemic lupus erythematosus.