Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

Abstract

NEMO is a scaffolding protein which plays an essential role in the NF-κB pathway by assembling the IKK-complex with the kinases IKKα and IKKβ. Upon activation, the IKK complex phosphorylates the IκB molecules leading to NF-κB nuclear translocation and activation of target genes. Inhibition of the NEMO / IKK interaction is an attractive therapeutic paradigm for the modulation of NF-κB pathway activity, making NEMO a target for inhibitors design and discovery. To facilitate the process of discovery and optimization of NEMO inhibitors, we engineered an improved construct of the IKK-binding domain of NEMO that would allow for structure determination of the protein in the apo form and while bound to small molecular weight inhibitors. Here we present the strategy utilized for the design, expression and structural characterization of the IKK-binding domain of NEMO. The protein is expressed in E. coli cells, solubilized under denaturing conditions and purified through three chromatographic steps. We discuss the protocols for obtaining crystals for structure determination and describe data acquisition and analysis strategies. The protocols will find wide applicability to the structure determination of complexes of NEMO and small molecule inhibitors.

The structure of a NEMO construct engineered for screening reveals novel determinants of inhibition

“…As zipNEMO still binds IKKβ approximately 40-fold better than zipNEMO(50-113), it is likely that the longer construct provides additional stabilization. It is notable that even a very short NEMO sequence can be stabilized so that to achieve full-length like binding affinity, as in the adapter-stabilized NEMO(50-113), K D = 12 nM 28,29 . The biophysical and structural data indicate an additive, if not synergic, stabilization of the structure as more mutations were introduced.…”

“…Disulfide stabilized constructs 27 and coiled-coil adaptors stabilized construcs 16,28 induce a dimeric coiled-coil fold and improve IKKβ-binding activity, in vitro and in cells. We recently reported the X-ray structure of the unbound IKK-binding domain of NEMO utilizing the latter construct 16,29 . The coiled-coil adaptors engineered NEMO construct crystallizes easily and enables structure-based drug discovery efforts through NEMO-inhibitor complexes but is not suitable for NMR-based screening assays due to the elongated size of the extended coiled coil, which causes rapid NMR signal relaxation.…”