Structure-informed characterization of a TLR2-binding scFv reveals a CDR-centered recognition model.

Toll-like receptor 2 (TLR2) is a cell-surface pattern-recognition receptor that contributes to inflammatory signaling and represents an accessible target for extracellular protein binders. Here, we integrated phage-display screening, biophysical characterization, comparative protein-protein docking, in silico perturbation, and cellular assays to characterize scFv33, a TLR2-binding single-chain variable fragment. scFv33 showed nanomolar binding to recombinant TLR2 and preferential recognition over the tested receptor comparator. Independent docking workflows converged on a CDR-centered recognition model in which heavy- and light-chain CDRs form a distributed network of charged, polar, and aromatic contacts across the TLR2 leucine-rich-repeat surface. Targeted computational perturbation further supported the sensitivity of this recognition model to changes on both the binder and receptor sides. In Pam3CSK4-stimulated microglial cells, scFv33 attenuated pro-inflammatory cytokine transcription. Together, these findings establish scFv33 as a functional TLR2-directed antibody fragment and provide an experimentally testable framework for structure-informed optimization of innate immune receptor binders.