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.

Divalent cations influence the dimerization mode of murine S100A9 protein by modulating its disulfide bond pattern

“…More recently, Harms and coworkers proposed a biphasic zinc binding mode for the hS100A9 homodimer (unpublished results). In their model, both the His20/Asp30/His91/His95 tetrad and the C-terminal histidines may contribute to zinc chelation, possibly through two distinct binding sites (Loes et al, 2019).…”

Zinc is an important inter-kingdom signal between the host and microbe

“…; initiate intracellular inflammatory signal transduction; and play an important role in the regulation of immune and inflammatory responses [7,78]. Although S100A9 activates TLR4 to induce inflammation that does not depend on zinc ions, it still acts as a zinc chelator and results in decreased intracellular free zinc levels in the cell [80]. The apoptosis-inducing activity of S100A8/S100A9 is also dependent on both receptormediated and zinc exclusion-modulated pathways [81,82].…”