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

Summary

We describe protocols for the structure determination of the IKK-binding domain of NEMO by X-ray crystallography. The methods include protein expression, purification and characterization as well as strategies for successful crystal optimization and structure determination of the protein in its unbound form.

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.

Introduction

The NF-κB pathway is activated in response to a variety of stimuli, including cytokines, microbial products and stress, to regulate expression of target genes responsible for inflammatory and immune response, cell death or survival and proliferation¹. Pathologies including inflammatory and autoimmune diseases and cancer^2,3,4,5 have been correlated to hyperactivation of the pathway, which has made modulation of NF-κB activity a prime target for the development of new therapies^6,

Protocol

1. Design of construct for crystallography

1. Clone the sequence of NEMO-EEAA as in previous publication¹² in a vector for expression in E. coli using the T7 promoter, including a N-terminal hexa-histidine tag and a protease cleavage site.
   NOTE: In this protocol, we used a vector modified to include a N-terminal hexa-histidine tag and a Tobacco Etch Virus (TEV) cleavage site¹⁰. This vector facilitates cleavage of the His tag for protein crystallization and leaves only the short extension of GSW residues before the start of the desired protein sequence. The vector from which this was derived, and ....

Results

Cloning, expression and purification of the IKK-binding domain of NEMO.
The protocol followed in this study to obtain the final sequence of NEMO-EEAA (Figure 1A), which produced diffraction quality crystals, involved the expression and characterization of all the intermediate constructs, including the addition of the coiled-coil adaptors at N- and or C-terminus, the mutations C76A, C95S and the mutations E56A, E57A. Figure 1.......

Discussion

Crystallization attempts of NEMO in the unbound form were unsuccessful, including attempts using the full-length protein and several truncation constructs encompassing the IKK-binding domain. Our biophysical characterization of the IKK-binding domain of NEMO (residues 44-111) by circular dichroism, NMR spectroscopy and fluorescence anisotropy indicated that the construct, albeit able to bind IKKβ, existed in a state of conformational exchange, not suitable for crystallization^9,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
20% w/v γ-PGA (Na+ form, LM)	Molecular Dimensions	MD2-100-150	For fine screen crystallization of NEMO-EEAA
3.5 kDa MWCO Dialysis Membrane	Spectra/Por	132724	For dialysis removal of imidazole
Amicon Stirred Cell	Millipose Sigma	UFSC 05001	For protein concentration
Ammonium Chloride	Millipore Sigma	G8270	For minimal media labeling
Benzonase Nuclease	Millipore Sigma	9025-65-4	For digestion of nucleic acid
BL21-CodonPlus (DE3)-RIL Competent Cells	Agilent Technologies	Model: 230245	TEV expression
CryoPro	Hampton Research	HR2-073	Cryo-protectants kit
D-Glucose (Dextrose)	Millipore Sigma	A9434	For minimal media labeling
Difco Terrific Broth	ThermoFisher	DF043817	For culture growth
Dithiothreitol > 99%	Goldbio	DTT25	For reduction of disulfides
E. coli: Rosetta 2 (DE3)	Novagen	71400-3	Expression of unlabeled NEMO-EEAA
FORMULATOR	Formulatrix		Liquid handler/ screen builder
HCl – 1.0 M Solution	Hampton Research	HR2-581	For fine screen crystallization of NEMO-EEAA
HiLoad 16/600 Superdex 75 pg	GE Healthcare	28989333	For size exclusion purification
HisTrap HP 5 mL column	GE Healthcare	17524802	For purification of His-tagged NEMO-EEAA
HT 96 MIDAS	Molecular Dimensions	MD1-59	For sparse matrix screening of NEMO-EEAA
HT 96 Morpheous	Molecular Dimensions	MD1-46	For sparse matrix screening of NEMO-EEAA
Imidazole	ThermoFisher	288-32-4	For elution from His-trap column
Isopropyl-beta-D-thiogalactoside	Goldbio	I2481C5	For induction of cultures
MRC2 crystallization plate	Hampton Research	HR3-083	Crystallization plate
NT8 - Drop Setter	Formulatrix		Crystallization
pET-16b	Millipore Sigma	69662	For cloning of NEMO-EEAA
pET-45b	Millipore Sigma	71327	For cloning of NEMO-EEAA
Phenylmethylsulfonyl fluoride	ThermoFisher	36978	For inhibition of proteases
Polycarbonate Bottle for use in Ultracentrifuge Rotor Type 45 Ti	Beckmann Coulter	339160	Ultracentrifuge bottle
Polyethylene Glycol 20,000	Hampton Research	HR2-609	For fine screen crystallization of NEMO-EEAA
pRK793 (TEV)	Addgene	Plasmid 8827	For TEV production
QuikChange XL II	Agilent Technologies	200522	Site directed mutagenesis
Required Cap Assembly:	Beckmann Coulter	355623	Ultracenttrifuge bottle cap
ROCK IMAGER	Formulatrix		Crystallization Imager
Seed Bead Kit	Hampton Research	HR2-320	Seed generation
Sodium Chloride ≥ 99%	Millipore Sigma	S9888	For buffering of purification solutions
TCEP (Tris (2-Carboxyethyl) phosphine Hydrochloride)	Goldbio	TCEP1	Reducing agent
The Berkeley Screen	Rigaku	MD15-Berekely	For sparse matrix screening of NEMO-EEAA
The PGA Screen	Molecular Dimensions	MD1-50	For fine screen crystallization of NEMO-EEAA
Tris – 1.0 M Solution	Hampton Research	HR2-589	For fine screen crystallization of NEMO-EEAA
Ultrapure Tris Buffer (powder format)	Thermofisher	15504020	For buffering of purification solutions
Urea	ThermoFisher	29700	For denaturation of NEMO-EEAA