Effect of Phosphorylation on SIKE Quaternary Structure

Abstract

Signaling pathways of the innate immune system are essential conduits to cellular defenses against pathogen yet dysregulation or mutation often results in various autoimmune diseases, cancers, and other ailments. Obtaining a complete understanding of native immune signal pathways will facilitate identification of the complex defense mechanisms and has the potential to yield molecular origins of disease states. In the Toll‐Like Receptor 3 (TLR3) immune pathway, Suppressor of IKKepsilon (SIKE) has undefined downstream function. Previous studies identified SIKE as a high affinity substrate of TANK Binding Kinase 1 in the TLR3 pathway, resulting in phosphorylation at six serine residues primarily clustered near SIKE’s C‐terminus. This phosphorylation enhanced the previously observed SIKE‐tubulin interaction. As tubulin functions in diverse cellular processes, including cellular proliferation, intracellular transportation, and cell motility, SIKE’s interaction may serve to connect these processes to the TLR3 immune response. To facilitate future work in understanding the effects of SIKE modification on tubulin function, this study aimed to understand how phosphorylation alters SIKE structure. To study the effects of phosphorylation on SIKE quaternary structure, Size Exclusion Chromatography (SEC) was performed on a phosphomimetic variant of SIKE (S6E SIKE) +/− bis(sulfosuccinimidyl)suberate crosslinking. SDS‐PAGE analyses of peak fractions revealed S6E SIKE existed in a monomer‐dimer distribution, whereas wildtype (WT) SIKE was dimer‐hexamer‐aggregate distribution. The S6E SIKE dimer eluted earlier than the WT SIKE dimer suggesting a more extended S6E structure. To map phosphorylation sites with respect to the dimer interface of a SIKE structural model, glutamates were substituted for serines at the phosphorylation sites using PyMOL’s mutagenesis wizard. The electrostatic surface potential was then assessed using the APBS electrostatics plugin. This model suggested the density of negative charge at the dimer interface might trigger a shift from dimer to monomer and support a more extended S6E structure. Despite significant shifts in quaternary structure of S6E SIKE, secondary structure was not appreciably altered as measured by Circular Dichroism (CD) spectra; however, unlike WT SIKE, S6E SIKE was not reversibly denatured, but refolded to a new, distinct state. Fluorescence‐based Thermal Shift (FTS) data revealed WT and S6E SIKE have exposed hydrophobic surfaces prior to denaturation, as noted by initial, significant SYPRO Orange fluorescence. Under all conditions, S6E SIKE was less stable than WT SIKE as assessed by Tm values. Thermal melts from pH 5 to 9 yielded pH‐dependent changes in Tm values that were consistent with a pKa of 5.7 governing thermal unfolding for both WT and S6E. Both proteins were significantly more stable at pH 5. In conclusion, our results suggest phosphorylation destabilizes SIKE quaternary structure without affecting secondary structure, consistent with an overall more extended structure. In terms of stability, the phosphomimetic SIKE does not thermally refold to its initial state and is less stable compared to WT, but retains hydrophobic character on its surface and a pH stability pattern similar to WT SIKE.