Zinc-independent activation of Toll-like receptor 4 by S100A9

ABSTRACT The homodimer formed by the protein S100A9 induces inflammation through Toll-like receptor 4 (TLR4), playing critical roles in both healthy and pathological innate immune responses. The molecular mechanism by which S100A9 activates TLR4 remains unknown. Previously, the interaction between purified S100A9 and TLR4 was shown to depend on Zn 2+ ; however, the Zn 2+ binding site(s) on S100A9 were not identified. Here, we investigated the role of Zn 2+ binding in the pro-inflammatory activity of S100A9. We found that the S100A9 homodimer was prone to reversible, Zn 2+ -dependent aggregation in vitro . Using a combination of site-directed mutagenesis and Isothermal Titration Calorimetry (ITC), we identified multiple residues that contribute to Zn 2+ binding in S100A9. We then used mutagenesis to construct a version of S100A9 with no detectable Zn 2+ binding by either ITC or Inductively Coupled Plasma-Mass Spectrometry. This protein did not exhibit aggregation upon addition of saturating Zn 2+ . Further, despite the lack of Zn 2+ -binding, this protein was capable of activating TLR4 in a cell-based functional assay. We then modified the functional assay so the Zn 2+ concentration was exceedingly low relative to the concentration of S100A9 added. Again, S100A9 was able to activate TLR4. This reveals that, despite the ability of S100A9 to bind Zn 2+ , S100A9 does not require Zn 2+ to activate TLR4. Our work represents an important step in clarifying the nature of the interaction between S100A9 and TLR4.