The Effects of Trifluoroethanol on the Helical Structure of SIKE

Abstract

Suppressor of IKKe (SIKE)inhibits TBK1 mediated phosphorylation of interferon regulatory factor 3 (IRF3) and misregulation of TBK1 is involved in both autoimmune disorders and cancer. Our work (Marion et al, J.Biol.Chem 288, 18612–23, 2013) demonstrated that TBK1 can phosphorylate SIKE in up to 6 positions, altering its ability to inhibit TBK1. Little however is known concerning the three dimensional structure of SIKE. SIKE (his tagged) has been expressed and purified using Nickel NTA affinity chromatography and shown to be homogenous by MALDI‐tof mass spectrometry. To examine the structure of SIKE we have performed two dimensional proton NMR experiments and Circular Dichroism spectroscopy and compared the results with both secondary and tertiary structure predictions. 2D NOESY spectra obtained using several mixing times in general revealed little stable structure in the molecule as detected in the NH‐NH cross peak region, suggesting the protein may contain significant disordered regions. CD spectra suggest significant alpha helix content and the possibility of coiled coil regions of structure. Addition of Trifluoroethanol, known to stabilize alpha helical structures, led to increased CD signal at 222nm. Using a range of TFE concentrations optimal stabilization of helical structure in SIKE allowed comparison with helical secondary structure predications.Support or Funding InformationThis work was supported by NIH grant R21A1 107447 to JKB