Name: production, crystallization, and structure determination of the ikk-binding domain of nemo
Uploaded: 2019-12-28T12:00:00
Description: Watch this Scientific Journal Video about Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO at JoVE.com

We thank Prof. D. Madden, for many helpful discussions throughout this project. We thank Prof. D. Bolon for the gift of the plasmid containing the optimized GCN4 coiled coil. We thank Dr. B. Guo for NEMO plasmids. We thank Christina R. Arnoldy, Tamar Basiashvili and Amy E. Kennedy for demonstrating the procedure. We thank the BioMT Crystallography Core Facility and the departments of Chemistry and of Biochemistry &#38; Cell Biology at Dartmouth for the use of the crystallography equipment and the BioMT personnel for their support. This research used the AMX beamline of the National Synchrotron Light Source II, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract No. DE-SC0012704. We thank the staff at NSLS II for their support. This work was funded by NIH grants R03AR066130, R01GM133844, R35GM128663 and P20GM113132, and a Munck-Pfefferkorn Novel and Interactive grant.

1. Design of construct for crystallography
<ol>
	<li>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 crystallizat...

<ol>
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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 IKK&#946;, existed in a state of conformational exchange, not suitable for crystallization9,<sup...

The NF-&#954;B 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 cancer2,3,4,5 have been correlated to hyperactivation of the pathway, which has made modulation of NF-&#954;B activity a prime target for the development of new therapies6,7.
The canonical NF-&#954;B pathway in particular is distinguished from the non-canonical pathway, responsible for lymphorganogenesis and B-cell activation, by the former&#39;s dependence on the scaffolding protein NEMO (NF-&#954;B essential modulator8) for the assembly of the IKK-complex with the kinases IKK&#945; and IKK&#946;. The IKK complex is responsible for the phosphorylation of I&#954;B&#945; (inhibitor of &#954;B) that targets it for degradation, freeing the NF-&#954;B dimers to translocate to the nucleus for gene transcription1 and is therefore an attractive target for the development of inhibitors to modulate NF-&#954;B activity.
Our research focuses on the characterization of the protein-protein interaction between NEMO and IKK&#946;, targeting NEMO for the development of small molecules inhibitors of IKK complex formation. The minimal binding domain of NEMO, required to bind IKK&#946;, encompasses residues 44-111, and its structure has been determined in complex with a peptide corresponding to IKK&#946; sequence 701-7459. NEMO and IKK&#946; form a four-helix bundle where the NEMO dimer accommodates the two helices of IKK&#946;(701-745) in an elongated open groove with an extended interaction interface. IKK&#946;(734-742), also known as the NEMO-binding domain (NBD), defines the most important hot-spot for binding, where the two essential tryptophans (739,741) bury deeply within the NEMO pocket. The details of the complex structure can aid in the structure-based design and optimization of small molecule inhibitors targeting NEMO. At the same time, it is difficult that binding of a small molecule or peptide would recreate in NEMO the full conformational change (i.e., extensive opening of the NEMO coiled-coil dimer) caused by binding of the long IKK&#946;(701-745), as observed in the crystal, and the structure of unbound NEMO or NEMO bound to a small molecule inhibitor may represent a better target for structure-based drug design and inhibitor optimization.
Full length NEMO and smaller truncation constructs encompassing the IKK-binding domain have proven intractable for structure determination in the unbound form via X-ray crystallography and nuclear magnetic resonance (NMR) methods10, which prompted us to design an improved version of the IKK-binding domain of NEMO. Indeed, NEMO (44-111) in the unbound form is only partially folded and undergoes conformational exchange and we therefore set to stabilize its dimeric structure, coiled-coil fold and stability, while preserving binding affinity for IKK&#946;. By appending three heptads of ideal dimeric coiled-coil sequences11 at the N-and C-termini of the protein, and a series of four point mutations, we generated NEMO-EEAA, a construct fully dimeric and folded in a coiled coil, which rescued IKK-binding affinity to the nanomolar range as observed for full length NEMO12. As an additional advantage, we hoped the coiled-coil adaptors (based on the GCN4 sequence) would facilitate crystallization and eventually aid in the X-ray structure determination via molecular replacement. Coiled-coil adaptors have been similarly utilized to both increase stability, improve solution behavior and facilitate crystallization for trimeric coiled coils and antibody fragments13,14. NEMO-EEAA is easily expressed and purified from Escherichia. coli cells with a cleavable Histidine tag, is soluble, folded in a stable dimeric coiled coil and is easily crystallized, with diffraction to 1.9 &#197;. The presence of the ordered coiled-coil regions of GCN4 could additionally aid in phasing the data from crystals of NEMO-EEAA by molecular replacement using the known structure of GCN415.
Given the results obtained with apo-NEMO-EEAA, we believe the protocols described here could also be applied to the crystallization of NEMO-EEAA in the presence of small peptides (like the NBD peptide) or small molecule inhibitors, with the goal of understanding the requirements for NEMO inhibition and structure-based optimization of initial lead inhibitors to high affinity. Given the plasticity and dynamic nature of many coiled-coil domains16, the use of the coiled-coil adaptors could find more general applicability in aiding structural determination.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>20% w/v &#947;-PGA (Na+ form, LM)</td>
			<td>Molecular Dimensions</td>
			<td>MD2-100-150</td>
			<td>For fine screen crystallization of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>3.5 kDa MWCO Dialysis Membrane</td>
			<td>Spectra/Por</td>
			<td>132724</td>
			<td>For dialysis removal of imidazole</td>
		</tr>
		<tr>
			<td>Amicon Stirred Cell</td>
			<td>Millipose Sigma</td>
			<td>UFSC 05001</td>
			<td>For protein concentration</td>
		</tr>
		<tr>
			<td>Ammonium Chloride</td>
			<td>Millipore Sigma</td>
			<td>G8270</td>
			<td>For minimal media labeling</td>
		</tr>
		<tr>
			<td>Benzonase Nuclease</td>
			<td>Millipore Sigma</td>
			<td>9025-65-4</td>
			<td>For digestion of nucleic acid</td>
		</tr>
		<tr>
			<td>BL21-CodonPlus (DE3)-RIL Competent Cells</td>
			<td>Agilent Technologies</td>
			<td>Model: 230245</td>
			<td>TEV expression</td>
		</tr>
		<tr>
			<td>CryoPro</td>
			<td>Hampton Research</td>
			<td>HR2-073</td>
			<td>Cryo-protectants kit</td>
		</tr>
		<tr>
			<td>D-Glucose (Dextrose)</td>
			<td>Millipore Sigma</td>
			<td>A9434</td>
			<td>For minimal media labeling</td>
		</tr>
		<tr>
			<td>Difco Terrific Broth</td>
			<td>ThermoFisher</td>
			<td>DF043817</td>
			<td>For culture growth</td>
		</tr>
		<tr>
			<td>Dithiothreitol &#62; 99%</td>
			<td>Goldbio</td>
			<td>DTT25</td>
			<td>For reduction of disulfides</td>
		</tr>
		<tr>
			<td>E. coli: Rosetta 2 (DE3)</td>
			<td>Novagen</td>
			<td>71400-3</td>
			<td>Expression of unlabeled NEMO-EEAA</td>
		</tr>
		<tr>
			<td>FORMULATOR</td>
			<td>Formulatrix</td>
			<td></td>
			<td>Liquid handler/ screen builder</td>
		</tr>
		<tr>
			<td>HCl &#8211; 1.0 M Solution</td>
			<td>Hampton Research</td>
			<td>HR2-581</td>
			<td>For fine screen crystallization of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>HiLoad 16/600 Superdex 75 pg</td>
			<td>GE Healthcare</td>
			<td>28989333</td>
			<td>For size exclusion purification</td>
		</tr>
		<tr>
			<td>HisTrap HP 5 mL column</td>
			<td>GE Healthcare</td>
			<td>17524802</td>
			<td>For purification of His-tagged NEMO-EEAA</td>
		</tr>
		<tr>
			<td>HT 96 MIDAS</td>
			<td>Molecular Dimensions</td>
			<td>MD1-59</td>
			<td>For sparse matrix screening of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>HT 96 Morpheous</td>
			<td>Molecular Dimensions</td>
			<td>MD1-46</td>
			<td>For sparse matrix screening of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>ThermoFisher</td>
			<td>288-32-4</td>
			<td>For elution from His-trap column</td>
		</tr>
		<tr>
			<td>Isopropyl-beta-D-thiogalactoside</td>
			<td>Goldbio</td>
			<td>I2481C5</td>
			<td>For induction of cultures</td>
		</tr>
		<tr>
			<td>MRC2 crystallization plate</td>
			<td>Hampton Research</td>
			<td>HR3-083</td>
			<td>Crystallization plate</td>
		</tr>
		<tr>
			<td>NT8 - Drop Setter</td>
			<td>Formulatrix</td>
			<td></td>
			<td>Crystallization</td>
		</tr>
		<tr>
			<td>pET-16b</td>
			<td>Millipore Sigma</td>
			<td>69662</td>
			<td>For cloning of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>pET-45b</td>
			<td>Millipore Sigma</td>
			<td>71327</td>
			<td>For cloning of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonyl fluoride</td>
			<td>ThermoFisher</td>
			<td>36978</td>
			<td>For inhibition of proteases</td>
		</tr>
		<tr>
			<td>Polycarbonate Bottle for use in Ultracentrifuge Rotor Type 45 Ti</td>
			<td>Beckmann Coulter</td>
			<td>339160</td>
			<td>Ultracentrifuge bottle</td>
		</tr>
		<tr>
			<td>Polyethylene Glycol 20,000</td>
			<td>Hampton Research</td>
			<td>HR2-609</td>
			<td>For fine screen crystallization of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>pRK793 (TEV)</td>
			<td>Addgene</td>
			<td>Plasmid 8827</td>
			<td>For TEV production</td>
		</tr>
		<tr>
			<td>QuikChange XL II</td>
			<td>Agilent Technologies</td>
			<td>200522</td>
			<td>Site directed mutagenesis</td>
		</tr>
		<tr>
			<td>Required Cap Assembly:</td>
			<td>Beckmann Coulter</td>
			<td>355623</td>
			<td>Ultracenttrifuge bottle cap</td>
		</tr>
		<tr>
			<td>ROCK IMAGER</td>
			<td>Formulatrix</td>
			<td></td>
			<td>Crystallization Imager</td>
		</tr>
		<tr>
			<td>Seed Bead Kit</td>
			<td>Hampton Research</td>
			<td>HR2-320</td>
			<td>Seed generation</td>
		</tr>
		<tr>
			<td>Sodium Chloride &#8805; 99%</td>
			<td>Millipore Sigma</td>
			<td>S9888</td>
			<td>For buffering of purification solutions</td>
		</tr>
		<tr>
			<td>TCEP (Tris (2-Carboxyethyl) phosphine Hydrochloride)</td>
			<td>Goldbio</td>
			<td>TCEP1</td>
			<td>Reducing agent</td>
		</tr>
		<tr>
			<td>The Berkeley Screen</td>
			<td>Rigaku</td>
			<td>MD15-Berekely</td>
			<td>For sparse matrix screening of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>The PGA Screen</td>
			<td>Molecular Dimensions</td>
			<td>MD1-50</td>
			<td>For fine screen crystallization of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>Tris &#8211; 1.0 M Solution</td>
			<td>Hampton Research</td>
			<td>HR2-589</td>
			<td>For fine screen crystallization of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>Ultrapure Tris Buffer (powder format)</td>
			<td>Thermofisher</td>
			<td>15504020</td>
			<td>For buffering of purification solutions</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>ThermoFisher</td>
			<td>29700</td>
			<td>For denaturation of NEMO-EEAA</td>
		</tr>
	</tbody>
</table>

production, crystallization, and structure determination of the ikk-binding domain of nemo

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and IKK&#946;. Upon activation, the IKK complex phosphorylates the I&#954;B molecules leading to NF-&#954;B nuclear translocation and activation of target genes. Inhibition of the NEMO/IKK interaction is an attractive therapeutic paradigm for the modulation of NF-&#954;B 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.

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</stron...

Watch this Scientific Journal Video about Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO at JoVE.com

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

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.

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and ...

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