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Engineering Stabilized Variants of the NEMO N‐terminal Domain Coiled‐Coil
Author(s) -
Zhou Li,
Whitty Adrian
Publication year - 2013
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.27.1_supplement.790.17
Subject(s) - circular dichroism , cysteine , chemistry , coiled coil , protein subunit , dimer , alanine , fluorescence anisotropy , biophysics , random coil , biochemistry , amino acid , biology , enzyme , organic chemistry , membrane , gene
NF‐κB essential modulator (NEMO) is a subunit of the Inhibitor of κB Kinase (IKK) complex that regulates NF‐κB signaling. Inhibiting the protein‐protein interaction between NEMO and its ligand IKKβ represents a potential approach to treating inflammatory diseases and certain types of cancer. Rushe. et. al. (Structure, 16:798, 2008) reported that the IKKα/β binding domain fragment NEMO44 111 was disordered in the absence of IKKβ. To understand the structure and stability of the NEMO N‐terminal domain we have prepared several variants of NEMO N‐terminal domain by mutating all cysteine residues to alanine, or retaining just Cys54, or retaining Cys54 plus an additional engineered cysteine at position 107. All variants were expressed as His‐SUMO fusions containing a TEV cleavage site, purified by Ni/NTA chromatography, and then cleaved with TEV protease. We evaluated the binding affinity of each construct for IKKβ in a fluorescence anisotropy binding assay, and their structure and stability by circular dichroism spectroscopy and thermal, and chemical denaturation, under reducing and nonreducing conditions. The results address whether the N‐terminal domain of NEMO intrinsically disordered, define the role of Cys54 in the stability of the NEMO dimer, and identify stabilized forms of NEMO N‐terminal domain for crystallography.