Using Biophysical Characterization to Explore Suppressor of IKKepsilon Structure

The innate immune system is the first line of defense against pathogens. Suppressor of IKKepsilon (SIKE) is a downstream component of the antiviral TLR3 innate immune pathway, although its function in this pathway has not yet been determined. In order to derive information about SIKE function, we undertook characterization of SIKE structure to determine a structure‐function relationship. Using PHYRE2, a SIKE model of the human isoform was predicted that consisted of a V‐shaped coiled coil fold. The secondary structure of SIKE was assessed via circular dichroism. Spectra were consistent with the predicted alpha helical model. Thermal melts (15–80°C) showed a linear, rather than sigmoidal, unfolding transition consistent with computational predictions (GlobPlot, DisEMBL, PONDR), which suggested SIKE contained 35% disordered regions. Addition of up to 25% trifluoroethanol to stabilize helical structure increased the helical content of SIKE by 35%. Fluorescence polarization and size exclusion chromatography assessed the solution size of SIKE (23 kDa) with respect to globular standards. SEC revealed a concentration dependent (0.9 – 3.6 mg/mL) association of SIKE into three distinct species of 79, 185 and 1,325 kDa. Fluorescence polarization of each species compared to standards of lysozyme (14 kDa), albumin (66 kDa) and glutamate dehydrogenase (355 kDa) were consistent with the SEC results. Limited proteolysis coupled to tandem mass spectrometry as well as crosslinking assessed by SDS‐PAGE mapped accessible SIKE sequences and association between SIKE monomers, respectively. Limited proteolysis at 1, 5, and 10 min identified trypsin cleavage sites consistent with a dimeric model of SIKE. Crosslinking with bis(sulfosuccinimidyl)suberate (BS3) identified predominantly a dimer species as well as a smaller proportion of a tetrameric species. In the cleft of the V‐shaped coiled coil, 3DLigandSite proposed a zinc binding site. To confirm an interaction between SIKE and divalent cations, SIKE‐ANS titration and fluorescence‐based thermal shift assays were completed +/− Mg, Mn, Ca, and Zn. In each case, a subset of ions perturbed SIKE structure, demonstrating a divalent cation interaction. In conclusion, using various biophysical techniques, we have found that SIKE's structure has regions of disorder bookended by alpha helices; SIKE monomers associate primarily into a dimeric species; divalent metals contribute to SIKE stabilization, and the homology model presented is consistent with these biophysical characterizations. Support or Funding Information Work supported by NIH grant R21AI107447 and USD SURE program.