HMGB1 binds to and disrupts the hairpin structure of RNA15 and inhibits toll-like receptor activation

Toll-like receptor 13 (TLR13) is a critical innate immune sensor that recognizes a conserved RNA sequence, RNA15 (2054-2068, ACG GAA AGA CCC CGU), within bacterial 23S rRNA, thereby initiating a proinflammatory response. While the alarmin high mobility group box 1 (HMGB1) is known to modulate various TLR pathways, its influence on TLR13 signaling has remained unexplored. Here, we reveal that HMGB1 directly binds RNA15 with high affinity and profoundly disrupts its hairpin structure, which is essential for TLR13 recognition. Using a combination of fluorescence anisotropy, FRET assays, NMR spectroscopy, and enhanced-sampling molecular dynamics simulations, we demonstrate that HMGB1 binding remodels RNA15 into a stem-open conformation, making it thermodynamically unfavorable for receptor activation. Functionally, HMGB1 significantly inhibits RNA15-induced TLR13 activation, leading to a dose-dependent reduction in inflammatory mediators. These findings uncover a novel "ligand remodeling" mechanism, whereby HMGB1 acts as a negative regulator of TLR13 signaling by structurally altering the RNA ligand rather than directly blocking the receptor. This work provides new insights into host-pathogen interactions and suggests important implications for the design and immunogenicity of RNA-based therapeutics and vaccines.