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Effects of Divalent Metal Ions on the Structure and Stability of SIKE
Author(s) -
Kim SeungHwan,
Bell Ellis,
Bell Jessica
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1110.2
Subject(s) - chemistry , divalent , metal ions in aqueous solution , metal , organic chemistry
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. A Three dimensional model of SIKE was made using the Phyre2 server using a variety of template molecules and ab initio modelling. The optimal model (with 97% of the sequence modelled at >90% confidence) was submitted to the 3DLigandSite server to predict potential binding sites. The model was predicted to contain a divalent metal ion binding site comprised of Aspartate 143, Leucine 173 and Glutamate 176 residues. His tagged SIKE was expressed, extracted using a Guanidine Hydrochloride solubilization procedure and purified using Nickel NTA affinity chromatography after renaturation on the resin. The purified protein was used in Fluorescence based Thermal Shift assays to in the presence and absence of a variety of divalent metal ions to screen for binding. Preliminary analysis of the results suggest that divalent metal ions including zinc do in fact affect the thermal stability of SIKE suggesting the presence of a metal binding site consistent with the optimal model. Support or Funding Information This work was supported by NIH grant R21A1 107447 to JKB