Predicting and validating a model of Suppressor of IKKepsilon through biophysical characterization.

Suppressor of IKKepsilon (SIKE) is a 207 residue protein that is implicated in the TLR3-TANK binding kinase 1-mediated response to viral infection. SIKE's function in this pathway is unknown, but SIKE forms interactions with two distinct cytoskeletal proteins, α-actinin and tubulin, and SIKE knockout reduces cell migration. As structure informs function and in the absence of solved structural homologs, our studies were directed toward creating a structural model of SIKE through biochemical and biophysical characterization to probe and interrogate SIKE function. CD revealed a primarily (73%) helical structure of minimal stability (<Tm >=32°C), but reversibly denatured. Limited proteolysis (LP) and chemical modification identified the N-terminal 2/3 of the protein as dynamic and accessible while size exclusion chromatography confirmed three homo-oligomeric species. SEC coupled to chemical crosslinking characterized the primary species as dimeric, a secondary hexameric species, and a higher order aggregate/polymer. Fluorescence polarization using intrinsic tryptophan fluorescence contextualized the anisotropy value for the SIKE dimer (MW 51.8kDa) between proteins of known structure, bovine serum albumin (66kDa) and glutamate dehydrogenase (332kDa). Radii of gyration for BSA and GDH provided exclusionary values for SIKE tertiary and dimeric quaternary models that otherwise conformed to secondary structure, LP, and modification data. Dimeric quaternary models were further culled using acrylamide quenching data of SIKE's single tryptophan that showed a single, protected environment. The low cooperativity of folding, regions of dynamic and potentially disordered structure advance the hypothesis that SIKE forms a conformational ensemble of native states that accommodate SIKE's interactions with multiple, distinct protein binding partners. This article is protected by copyright. All rights reserved.