Wound inducible transcript 3.0 (WIT3.0) is a novel cytoskeleton protein that regulates fibroblast migration and initiates rapid wound closure. WIT3 shares ~50% sequence identity with an innate immune protein of unknown function, Suppressor of IKKepsilon (SIKE), that also associates with cytoskeletal proteins. We hypothesize that SIKE and WIT3 will have similar biochemical and biophysical characteristics that contribute to a shared function. To assess these similarities, we undertook characterization of WIT3 structure utilizing circular dichroism, fluorescence‐based thermal shift assays, ligand binding studies using an ANS reporter, and crosslinking with BS3. Using PHYRE2, a WIT3 model was predicted that consisted of an alpha helical coil with 30% disordered regions. The secondary structure of WIT3 was assessed via circular dichroism. Wavelength spectra is consistent with an alpha helical protein and thermal melt data showed a linear unfolding pattern with Tm of 37°C, suggesting a minimally stable protein. As part of the PHYRE2 model, 3DLigandSite proposed a zinc binding site. To assess an interaction between WIT3 and divalent cations, WIT3‐ANS titration and fluorescence‐based thermal shift assays were completed +/‐ Mg, Mn, Ca, Ni and Zn. An interaction between WIT3 and Zn was confirmed where, in the presence of zinc, WIT3's affinity for ANS was 2 fold higher than in the absence of zinc. The initial comparison suggests that WIT3 and SIKE share similar helical structure and similar FTS melt curve topography indicating exposed hydrophobic residues prior to denaturation, but different structural stability and a potential divalent cation binding site on WIT3. Together, these data suggest that WIT3 and SIKE share several biochemical characteristics that may allow these two proteins to have complimentary function.Support or Funding InformationStudies were funded in part by USD SURE.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.