Name: a guide to production, crystallization, and structure determination of human ikk1/&#945;
Uploaded: 2018-11-02T13:00:00
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We thank the staff at the beamlines 19ID, 24ID, and 13ID at Advanced Photon Source, Lemont, IL, for support during data collection on various crystals. We are grateful to Dmitry Lyumkis, Salk Institute for fetching us the low resolution cryo-EM map at early stages of EM map/model building, which was used to build the initial IKK1 molecular replacement search model. The research leading to these results has received funding from NIH grants AI064326, CA141722, and GM071862 to GG. SP is currently a Wellcome Trust DBT India Intermediate Fellow.

1. Preparation of Recombinant Virus Suitable for Large Scale Expression of IKK1/&#945;
<ol>
	<li>P1 virus preparation32
	<ol>
		<li>Day 1: Plate Sf9 cells (~6 X 105) (passage number less than 10) in 2 mL of Sf900 III insect cell medium in each well of a 6-well plate and incubate at 27 &#176;C. Passage cells when they reach a density of 2 to 3 X 106 cells per mL in suspension by diluting into fresh media at a density of ~6 X 105.</li>
		<li>Day 2:...

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40+regulates+the+processing+of+NF-kappaB2+p100+to+p52.">CD40 regulates the processing of NF-kappaB2 p100 to p52.</a> EMBO J. 21 (20), 5375-5385 (2002).</li><li>Dejardin, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lymphotoxin-beta+receptor+induces+different+patterns+of+gene+expression+via+two+NF-kappaB+pathways.">The lymphotoxin-beta receptor induces different patterns of gene expression via two NF-kappaB pathways.</a> Immunity. 17 (4), 525-535 (2002).</li><li>Xiao, G., Fong, A., Sun, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+p100+processing+by+NF-kappaB-inducing+kinase+involves+docking+IkappaB+kinase+alpha+(IKKalpha)+to+p100+and+IKKalpha-mediated+phosphorylation.">Induction of p100 processing by NF-kappaB-inducing kinase involves docking IkappaB kinase alpha (IKKalpha) to p100 and IKKalpha-mediated phosphorylation.</a> The Journal of biological chemistry. 279 (29), 30099-30105 (2004).</li><li>Xiao, G., Harhaj, E. W., Sun, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF-kappaB-inducing+kinase+regulates+the+processing+of+NF-kappaB2+p100.">NF-kappaB-inducing kinase regulates the processing of NF-kappaB2 p100.</a> Molecular cell. 7 (2), 401-409 (2001).</li><li>Qing, G., Qu, Z., Xiao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stabilization+of+basally+translated+NF-kappaB-inducing+kinase+(NIK)+protein+functions+as+a+molecular+switch+of+processing+of+NF-kappaB2+p100.">Stabilization of basally translated NF-kappaB-inducing kinase (NIK) protein functions as a molecular switch of processing of NF-kappaB2 p100.</a> J Biol Chem. 280 (49), 40578-40582 (2005).</li><li>Zarnegar, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noncanonical+NF-kappaB+activation+requires+coordinated+assembly+of+a+regulatory+complex+of+the+adaptors+cIAP1,+cIAP2,+TRAF2+and+TRAF3+and+the+kinase+NIK.">Noncanonical NF-kappaB activation requires coordinated assembly of a regulatory complex of the adaptors cIAP1, cIAP2, TRAF2 and TRAF3 and the kinase NIK.</a> Nat Immunol. 9 (12), 1371-1378 (2008).</li><li>Vallabhapurapu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonredundant+and+complementary+functions+of+TRAF2+and+TRAF3+in+a+ubiquitination+cascade+that+activates+NIK-dependent+alternative+NF-kappaB+signaling.">Nonredundant and complementary functions of TRAF2 and TRAF3 in a ubiquitination cascade that activates NIK-dependent alternative NF-kappaB signaling.</a> Nat Immunol. 9 (12), 1364-1370 (2008).</li><li>Annunziata, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequent+engagement+of+the+classical+and+alternative+NF-kappaB+pathways+by+diverse+genetic+abnormalities+in+multiple+myeloma.">Frequent engagement of the classical and alternative NF-kappaB pathways by diverse genetic abnormalities in multiple myeloma.</a> Cancer cell. 12 (2), 115-130 (2007).</li><li>Keats, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promiscuous+mutations+activate+the+noncanonical+NF-kappaB+pathway+in+multiple+myeloma.">Promiscuous mutations activate the noncanonical NF-kappaB pathway in multiple myeloma.</a> Cancer cell. 12 (2), 131-144 (2007).</li><li>Staudt, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncogenic+activation+of+NF-kappaB.">Oncogenic activation of NF-kappaB.</a> Cold Spring Harbor perspectives in biology. 2 (6), a000109 (2010).</li><li>Polley, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+structural+basis+for+IkappaB+kinase+2+activation+via+oligomerization-dependent+trans+auto-phosphorylation.">A structural basis for IkappaB kinase 2 activation via oligomerization-dependent trans auto-phosphorylation.</a> PLoS Biol. 11 (6), e1001581 (2013).</li><li>Hinz, M., Scheidereit, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+IkappaB+kinase+complex+in+NF-kappaB+regulation+and+beyond.">The IkappaB kinase complex in NF-kappaB regulation and beyond.</a> EMBO Rep. 15 (1), 46-61 (2014).</li><li>Scheidereit, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IkappaB+kinase+complexes:+gateways+to+NF-kappaB+activation+and+transcription.">IkappaB kinase complexes: gateways to NF-kappaB activation and transcription.</a> Oncogene. 25 (51), 6685-6705 (2006).</li><li>Luckow, V. 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Production, crystallization and structure solution of two related IKK proteins 
We set out to determine the X-ray crystal structure of IKK1/&#945; with the notion that it would be a relatively straightforward exercise given our experience with IKK2/&#946; protein production, crystallization, and structure determination. However, we were highly surprised that these two related proteins behaved very differently regarding the ease of crystallization. Despite efforts from several high-profile laboratori...

Transcriptional activities of the NF-&#954;B family of dimeric transcription factors are required for diverse cellular functions ranging from inflammation and immunity to survival and death. These activities are stringently controlled in cells and a loss of regulation leads to various pathological conditions, including autoimmune disorders, and cancer1,2,3. In the absence of a stimulus, the activities of NF-&#954;B are kept inhibited by I&#954;B (Inhibitor of -&#954;B) proteins4. The phosphorylation of specific Ser residues on I&#954;B proteins marks them for ubiquitination and subsequent proteasomal degradation or selective processing5. Two highly homologous Ser/Thr kinases, IKK2/&#946; and IKK1/&#945;, act as central regulators of NF-&#954;B activities by carrying out these phosphorylation events6,7.
Interactions between a ligand and a receptor transduces a signal through a series of mediators leading to the activation of NF-&#954;B factors. The NF-&#954;B signalling process can broadly be classified into two distinct pathways &#8211; canonical and non-canonical (alternative)8. The activity of IKK2/&#946; primarily regulates the NF-&#954;B signalling of the canonical pathway that is essential for inflammatory and innate immune responses9. A distinct feature of this pathway is a rapid and short-lived activation of IKK2/&#946;10 within a hitherto biochemically uncharacterized IKK complex &#8212; presumed to be composed of IKK1 and IKK2, as well as a regulatory component, NEMO (NF-&#954;B Essential Modulator)11,12,13. Between the two catalytic IKK subunits of the IKK complex, IKK2 is primarily responsible14 for the phosphorylation of specific residues of prototypical I&#954;Bs (&#945;, -&#946;, &#38; -&#947;) bound to NF-&#954;B, and also an atypical I&#954;B protein, NF-&#954;B1/p105, which is a precursor of the NF-&#954;B p50 subunit5. Phosphorylation induced ubiquitination and proteasomal degradation of I&#954;B (or processing of p105) leads to the release and activation of a specific set of NF-&#954;B dimers15. Aberrant NF-&#954;B activity due to mis-regulated function of IKK2 has been observed in many cancers as well as in autoimmune disorders2,3,16.
In contrast to IKK2/&#946;, activity of IKK1/&#945; regulates NF-&#954;B signalling of the non-canonical pathway, which is essential for development and immunity. IKK1 phosphorylates specific residues of NF-&#954;B2/p100 on its C-terminal I&#954;B&#948; segment, which leads to its processing and the generation of p52. The formation of transcriptionally active p52:RelB heterodimer initiates a slow and sustained response to developmental signals7,17,18,19,20. Interestingly, the generation of the central NF-&#954;B factor p52 of this pathway is critically dependent on another factor, NF-&#954;B Inducing Kinase (NIK)21,22, but not on IKK2 or NEMO. In resting cells, the level of NIK remains low due to its constant proteasome-dependent degradation23,24,25. Upon stimulation of cells by &#39;non-canonical&#39; ligands, and in certain malignant cells, NIK becomes stabilized to recruit and activate IKK1/&#945;. Kinase activities of both NIK and IKK1 are essential for efficient processing of p100 into p527. IKK1 and NIK phosphorylate three serines (Ser866, 870 and 872) of NF-&#954;B2/p100 on its C-terminal I&#954;B&#948; segment leading to its processing and the generation of p52. Aberrant activation of the non-canonical pathway has been implicated in many malignancies including multiple myeloma26,27,28.
Several highly efficient and specific inhibitors for IKK2/&#946; are known, although none so far have turned out to be an effective drug. In contrast, IKK1/&#945;-specific inhibitors are sparse. This may stem partly from our lack of structural and biochemical information on IKK1/&#945;, which limits our understanding of the mechanistic basis of activation of NF-&#954;B by IKK1 in cells, and rational drug design. The X-ray structures of IKK2/&#946; provided us with insights into the activation mechanism of IKK2/&#946;29; however, these structures could not reveal how different upstream stimuli trigger activation of IKK1/&#945; or IKK2/&#946; to regulate distinct sets of NF-&#954;B activities 30,31. To understand the mechanistic basis underlying the distinct signalling function of IKK1/&#945;, and to establish a platform for rational drug design, we focused on determining the structure of IKK1/&#945;.

<table>
	<tbody>
		<tr>
			<td>Cellfectin/Cellfectin II</td>
			<td>Thermo Fisher Scientific</td>
			<td>10362100</td>
			<td>Cellfectin is now discontinued, replaced by Cellfectin II</td>
		</tr>
		<tr>
			<td>Sf900 III Insect cell medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>12658-027</td>
			<td></td>
		</tr>
		<tr>
			<td>SF9 cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>12659017</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-IKK1 antibody</td>
			<td>Novus Biologicals</td>
			<td>NB100-56704</td>
			<td>Previously sold by Imgenex</td>
		</tr>
		<tr>
			<td>anti-PentaHis antibody</td>
			<td>Qiagen</td>
			<td>34460</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td>Nitrocellulose can also be used</td>
		</tr>
		<tr>
			<td>Ni-NTA agarose</td>
			<td>Qiagen</td>
			<td>30210</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford assay reagent</td>
			<td>BioRad</td>
			<td>500001</td>
			<td></td>
		</tr>
		<tr>
			<td>Superdex 200 column</td>
			<td>GE Healthcare</td>
			<td>28989335</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon concentrator</td>
			<td>Millipore</td>
			<td>UFC801008, UFC803008, UFC201024, UFC203024</td>
			<td></td>
		</tr>
		<tr>
			<td>Compound A</td>
			<td>Bayer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calbiochem IKK-inhibitor XII</td>
			<td>Calbiochem</td>
			<td>401491</td>
			<td></td>
		</tr>
		<tr>
			<td>Staurosporine</td>
			<td>SIGMA</td>
			<td>S4400</td>
			<td></td>
		</tr>
		<tr>
			<td>MLN120B</td>
			<td>Millenium</td>
			<td>Gift item</td>
			<td></td>
		</tr>
		<tr>
			<td>AMPPNP</td>
			<td>SIGMA</td>
			<td>A2647</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran sulfate</td>
			<td>SIGMA</td>
			<td>51227, 42867, 31404,</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran sulfate</td>
			<td>Alfa Aesar</td>
			<td>J62101</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG</td>
			<td>SIGMA</td>
			<td>93593, 81210, 88276, 95904, 81255, 89510, 92897, 81285, 95172</td>
			<td>Some of them are new Cat # on SIGMA catalogue. What we had was originally from Fluka that had different Cat #.</td>
		</tr>
		<tr>
			<td>Crystallization Screens</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal Screen I and II (Crystal Screen HT)</td>
			<td>Hampton Research</td>
			<td>HR2-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Index HT</td>
			<td>Hampton Research</td>
			<td>HR2-134</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG/Ion and PEG/Ion2 (PEG/Ion HT)</td>
			<td>Hampton Research</td>
			<td>HR2-139</td>
			<td></td>
		</tr>
		<tr>
			<td>PEGRX 1 and PEGRx 2 (PEGRx HT)</td>
			<td>Hampton Research</td>
			<td>HR2-086</td>
			<td></td>
		</tr>
		<tr>
			<td>SaltRx 1 and SaltRx 2 (SaltRx HT)</td>
			<td>Hampton Research</td>
			<td>HR2-136</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal mounts</td>
			<td>Hampton Research</td>
			<td>HR8-188, 190, 192, 194</td>
			<td></td>
		</tr>
		<tr>
			<td>Synchrotron</td>
			<td>The Advanced Photon Source (APS) at the U.S. Department of Energy&#8217;s Argonne National Laboratory</td>
			<td>Beamline 19 ID</td>
			<td>The Advanced Photon Source (APS) at the U.S. Department of Energy&#8217;s Argonne National Laboratory provides ultra-bright, high-energy storage ring-generated X-ray beams for research in almost all scientific disciplines.</td>
		</tr>
	</tbody>
</table>

a guide to production, crystallization, and structure determination of human ikk1/&#945;

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its precursor p100. p52 functions as a homodimer or heterodimer with another NF-&#954;B subunit, RelB. These dimers in turn regulate the expression of hundreds of genes involved in inflammation, cell survival, and cell cycle. IKK1/&#945; primarily remains associated with IKK2/&#946; and NEMO as a ternary complex. However, a small pool of it is also observed as a low molecular weight complex(es). It is unknown if the p100 processing activity is triggered by activation of IKK1/&#945; within the larger or the smaller complex pool. Constitutive activity of IKK1/&#945; has been detected in several cancers and inflammatory diseases. To understand the mechanism of activation of IKK1/&#945;, and enable its use as a drug target, we expressed recombinant IKK1/&#945; in different host systems, such as E. coli, insect, and mammalian cells. We succeeded in expressing soluble IKK1/&#945; in baculovirus infected insect cells, obtaining mg quantities of highly pure protein, crystallizing it in the presence of inhibitors, and determining its X-ray crystal structure. Here, we describe the detailed steps to produce the recombinant protein, its crystallization, and its X-ray crystal structure determination.

Cloning and expression of different constructs of IKK1/&#945; 
Full length human IKK1/&#945; was cloned into the baculovirus expression vector pFastBacHTa within its EcoRI and NotI restriction sites to obtain an N-terminal hexa-Histidine tagged IKK1. The tag could be removed by TEV protease digestion. Since full length IKK1/&#945; contains flexible regions on both ends, and flexible regions usually render a protein difficult to crystallize, we cloned various truncate...

Watch this Scientific Journal Video about A Guide to Production, Crystallization, and Structure Determination of Human IKK1/ÃŽÂ± at JoVE.com

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â±

I&#954;B Kinase 1/&#945; (IKK1/&#945; CHUK) is a Ser/Thr protein kinase that is involved in a myriad of cellular activities primarily through activation of NF-&#954;B transcription factors. Here, we describe the main steps necessary for the production and crystal structure determination of this protein.

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/&#945;

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its ...

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