Biophysical characterization of Suppressor of IKKepsilon structure, stability, and metal binding properties

Abstract

Suppressor of IKKepsilon (SIKE) is phosphorylated in the signaling cascade that initiates the anti‐viral innate immune response mediated by Toll‐like receptor 3. Although shown to be associated with cytoskeletal proteins, the function of SIKE in association with the cytoskeleton or as part of the innate immune response is not yet known. To gain insight into SIKE's function, structural and biophysical characterization were undertaken. Fluorescence‐based thermal shift assays (FTSA) indicated that the SIKE structure displayed hydrophobic residues prior to thermal denaturation. With increasing temperature, additional hydrophobic character was displayed giving rise to unique FTS curves showing three transitions, initial binding of fluor, unfolding, and quenching of fluorescence, presumably by protein aggregation. From modeling software, a divalent cation binding site was predicted. Using FTSA, the addition of zinc and calcium appear to alter the thermal stability of SIKE. Using ANS as a reporter group or direct tryptophan fluorescence of SIKE, the binding affinity of SIKE to various divalent cations, zinc, magnesium, manganese and calcium, was assessed. Fluorescence quenching experiments with neutral and charged quenchers were used to probe the local environment of the lone Trp residue of SIKE. The quenching coefficient for the Trp in SIKE determined from acrylamide studies was significantly lower than that of free Trp in solution indicating that the residue is fairly protected. Together, these studies provide biophysical parameters with which to interrogate and refine our predicted SIKE structure that is being employed in interpreting how SIKE interacts with cytoskeletal proteins to mediate host defense mechanisms.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.