Name: a guide to production, crystallization, and structure determination of human ikk1/&#945;
Uploaded: 2018-11-02T13:00:00
Description: Watch this Scientific Journal Video about A Guide to Production, Crystallization, and Structure Determination of Human IKK1/Î± at JoVE.com

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>
	<li>Xia, Y., Shen, S., Verma, I. M. <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,+an+active+player+in+human+cancers.">NF-kappaB, an active player in human cancers.</a> Cancer immunology research. 2 (9), 823-830 (2014).</li><li>Grivennikov, S. I., Greten, F. R., Karin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunity,+inflammation,+and+cancer.">Immunity, inflammation, and cancer.</a> Cell. 140 (6), 883-899 (2010).</li><li>Ben-Neriah, Y., Karin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+meets+cancer,+with+NF-kappaB+as+the+matchmaker.">Inflammation meets cancer, with NF-kappaB as the matchmaker.</a> Nature immunology. 12 (8), 715-723 (2011).</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 reports. 15 (1), 46-61 (2014).</li><li>Karin, M., Ben-Neriah, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+meets+ubiquitination:+the+control+of+NF-[kappa]B+activity.">Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity.</a> Annu Rev Immunol. 18, 621-663 (2000).</li><li>Ghosh, S., Karin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Missing+pieces+in+the+NF-kappaB+puzzle.">Missing pieces in the NF-kappaB puzzle.</a> Cell. 109, S81-S96 (2002).</li><li>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=The+noncanonical+NF-kappaB+pathway.">The noncanonical NF-kappaB pathway.</a> Immunol Rev. 246 (1), 125-140 (2012).</li><li>Bonizzi, G., Karin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+two+NF-kappaB+activation+pathways+and+their+role+in+innate+and+adaptive+immunity.">The two NF-kappaB activation pathways and their role in innate and adaptive immunity.</a> Trends Immunol. 25 (6), 280-288 (2004).</li><li>DiDonato, J. A., Hayakawa, M., Rothwarf, D. M., Zandi, E., Karin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cytokine-responsive+IkappaB+kinase+that+activates+the+transcription+factor+NF-kappaB.">A cytokine-responsive IkappaB kinase that activates the transcription factor NF-kappaB.</a> Nature. 388 (6642), 548-554 (1997).</li><li>Werner, S. L., Barken, D., Hoffmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulus+specificity+of+gene+expression+programs+determined+by+temporal+control+of+IKK+activity.">Stimulus specificity of gene expression programs determined by temporal control of IKK activity.</a> Science. 309 (5742), 1857-1861 (2005).</li><li>Zandi, E., Rothwarf, D. M., Delhase, M., Hayakawa, M., Karin, M. <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+(IKK)+contains+two+kinase+subunits,+IKKalpha+and+IKKbeta,+necessary+for+IkappaB+phosphorylation+and+NF-kappaB+activation.">The IkappaB kinase complex (IKK) contains two kinase subunits, IKKalpha and IKKbeta, necessary for IkappaB phosphorylation and NF-kappaB activation.</a> Cell. 91 (2), 243-252 (1997).</li><li>Rothwarf, D. M., Zandi, E., Natoli, G., Karin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IKK-gamma+is+an+essential+regulatory+subunit+of+the+IkappaB+kinase+complex.">IKK-gamma is an essential regulatory subunit of the IkappaB kinase complex.</a> Nature. 395 (6699), 297-300 (1998).</li><li>Yamaoka, 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=Complementation+cloning+of+NEMO,+a+component+of+the+IkappaB+kinase+complex+essential+for+NF-kappaB+activation.">Complementation cloning of NEMO, a component of the IkappaB kinase complex essential for NF-kappaB activation.</a> Cell. 93 (7), 1231-1240 (1998).</li><li>Li, Z. W., 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+IKKbeta+subunit+of+IkappaB+kinase+(IKK)+is+essential+for+nuclear+factor+kappaB+activation+and+prevention+of+apoptosis.">The IKKbeta subunit of IkappaB kinase (IKK) is essential for nuclear factor kappaB activation and prevention of apoptosis.</a> J Exp Med. 189 (11), 1839-1845 (1999).</li><li>Hayden, M. S., Ghosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shared+principles+in+NF-kappaB+signaling.">Shared principles in NF-kappaB signaling.</a> Cell. 132 (3), 344-362 (2008).</li><li>Sun, S. C., Chang, J. H., Jin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+nuclear+factor-kappaB+in+autoimmunity.">Regulation of nuclear factor-kappaB in autoimmunity.</a> Trends Immunol. 34 (6), 282-289 (2013).</li><li>Claudio, E., Brown, K., Park, S., Wang, H., Siebenlist, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAFF-induced+NEMO-independent+processing+of+NF-kappa+B2+in+maturing+B+cells.">BAFF-induced NEMO-independent processing of NF-kappa B2 in maturing B cells.</a> Nat Immunol. 3 (10), 958-965 (2002).</li><li>Senftleben, U., 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=Activation+by+IKKalpha+of+a+second,+evolutionary+conserved,+NF-kappa+B+signaling+pathway.">Activation by IKKalpha of a second, evolutionary conserved, NF-kappa B signaling pathway.</a> Science. 293 (5534), 1495-1499 (2001).</li><li>Coope, H. 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. A., Lee, S. C., Barry, G. F., Olins, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+infectious+recombinant+baculoviruses+by+site-specific+transposon-mediated+insertion+of+foreign+genes+into+a+baculovirus+genome+propagated+in+Escherichia+coli.">Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli.</a> J Virol. 67 (8), 4566-4579 (1993).</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=Structural+Basis+for+the+Activation+of+IKK1/alpha.">Structural Basis for the Activation of IKK1/alpha.</a> Cell reports. 17 (8), 1907-1914 (2016).</li><li>Otwinowski, Z. a. M., W, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+X-ray+Diffraction+Data+Collected+in+Oscillation+Mode.">Processing of X-ray Diffraction Data Collected in Oscillation Mode.</a> Methods in Enzymology. 276 (Macromolecular Crystallography, part A), 307-326 (1997).</li><li>Liu, 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=Crystal+structure+of+a+human+IkappaB+kinase+beta+asymmetric+dimer.">Crystal structure of a human IkappaB kinase beta asymmetric dimer.</a> J Biol Chem. 288 (31), 22758-22767 (2013).</li><li>Emsley, P., Lohkamp, B., Scott, W. G., Cowtan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+and+development+of+Coot.">Features and development of Coot.</a> Acta crystallographica. Section D, Biological crystallography. 66 (Pt 4), 486-501 (2010).</li><li>McCoy, A. 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=Phaser+crystallographic+software.">Phaser crystallographic software.</a> Journal of applied crystallography. 40 (Pt 4), 658-674 (2007).</li><li>Vagin, A. T., Teplyakov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MOLREP:+an+automated+program+for+molecular+replacement.">MOLREP: an automated program for molecular replacement.</a> J. Appl. Cryst. 30, 1022-1025 (1997).</li><li>Brunger, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Version+1.2+of+the+Crystallography+and+NMR+system.">Version 1.2 of the Crystallography and NMR system.</a> Nature protocols. 2 (11), 2728-2733 (2007).</li><li>Brunger, A. T., 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=Crystallography+&amp;+NMR+system:+A+new+software+suite+for+macromolecular+structure+determination.">Crystallography &amp; NMR system: A new software suite for macromolecular structure determination.</a> Acta crystallographica. Section D, Biological. 54 (Pt 5), 905-921 (1998).</li><li>McRee, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XtalView:+a+visual+protein+crystallographic+software+system+for+X11/Xview.">XtalView: a visual protein crystallographic software system for X11/Xview.</a> J. Mol Graph. 10, 44-47 (1992).</li><li>Schroder, G. F., Levitt, M., Brunger, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+biomolecular+crystallography+with+low-resolution+data.">Super-resolution biomolecular crystallography with low-resolution data.</a> Nature. 464 (7292), 1218-1222 (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 biology. 11 (6), e1001581 (2013).</li><li>Christopher, J. A., 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+discovery+of+2-amino-3,5-diarylbenzamide+inhibitors+of+IKK-alpha+and+IKK-beta+kinases.">The discovery of 2-amino-3,5-diarylbenzamide inhibitors of IKK-alpha and IKK-beta kinases.</a> Bioorganic &amp; medicinal chemistry letters. 17 (14), 3972-3977 (2007).</li><li>Karplus, P. A., Diederichs, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linking+crystallographic+model+and+data+quality.">Linking crystallographic model and data quality.</a> Science. 336 (6084), 1030-1033 (2012).</li><li>Chen, V. B., 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=MolProbity:+all-atom+structure+validation+for+macromolecular+crystallography.">MolProbity: all-atom structure validation for macromolecular crystallography.</a> Acta crystallographica. Section D, Biological crystallography. 66 (Pt 1), 12-21 (2010).</li></ol>

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/Î± at JoVE.com

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

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

The goal of this procedure is to provide guidance for the production, crystallization and structural determination of human IKK1-alpha in order to understand the mechanistic basis of its signaling function and to establish platforms for rational drug design. This method should help researchers determine the structure of IKK1, which would provide answers to key queries in the field of immune signaling. This technique describes a streamlined path to obtaining crystals of IKK proteins, which are rather refractory to crystallization and to provide various critical information in overcoming bottlenecks in determining the structures.Generally, individuals new to this method will struggle, because generating milligram quantities of soluble, well-behaved protein, requires its expression in insect cells, and crystallization can be done under only a highly narrow window of conditions. The molecular replacement procedure used in determining the structure of IKK1 can also be very tricky, especially due to poor deflection property of the crystals, not at the site of the asymmetric unit containing many IKK1 molecules in the absence of appropriate search models. Demonstrating the procedures will be Kyle Shumate and Sonjiala Hotchkiss, students from my laboratory.On day one, plate Sf9 cells in two milliliters of Sf903 insect cell medium, in each well of a six-well plate and incubate at 27 degrees Celsius. Passage the cells when they reach a density of two to three times 10 to the six cells per milliliter in suspension by diluting it to fresh media, at a density of approximately six times 10 to the five. On day two, dilute eight microliters of Sf9 transfection reagent in 100 microliters of SF-903 or Grace's Insect Medium.Then vortex the mixture briefly. In a separate tube, dilute one microgram of kit-purified recombinant bacmid in 100 microliters of the same medium. Combine the diluted DNA and transfection reagent.After incubating at room temperature for 15 to 30 minutes, remove the medium from the well. Then add one milliliter of fresh medium. Next, add the diluted DNA transfection reagent mixture dropwise onto the cells, adjusting the drop size such that it does not dislodge adherent cells.After six hours, add 1.5 milliliters of fresh medium on top of the cells. Incubate the cells at 27 degrees Celsius, checking the wells periodically every 12 hours for signs of viral infection. On day five, remove the medium containing cells, 60 to 72 hours post-infection.After transferring the medium into tubes, centrifuge for five minutes at 500 times g and four degrees Celsius. When finished, save the supernatant, which is the P1 virus stack. On day one, resuspend the previously prepared His-IKK1 cell pellet in 40 milliliters of lysis buffer.Place the cell suspension on ice and lyse the cells by sonication at 60%to 70%duty cycles and five to 10 pulses of 30-second duration at an interval greater than one minute. Clarify the lysate by centrifugation at greater than or equal to 28, 000 g for 45 minutes at four degrees Celsius. After preparing a Nickel-NTA Agarose resin, according to the text protocol, elute the His-tagged IKK1 alpha protein under gravity flow using 20 milliliters of elution buffer.Collect one to 1.5 milliliter fractions. After combining the fractions containing the protein, digest the sample with TEV protease overnight at four degrees Celsius. On the morning of day two, incubate the protein with one millimolar of ATP, in the presence of magnesium chloride, beta-glycerophosphate, sodium fluoride and sodium orthovanadate for one hour at 27 degrees Celsius.Following incubation, filter the protein solution through a 0.45 micron filter. Then load approximately six milliliters of the sample on to a 120 milliliter preparative size-exclusion column, attached to an automated liquid chromatography system. After equilibration, run the size-exclusion chromatography at a flow rate of one milliliter per minute and collect two-milliliter fractions.During the run, monitor elution at 280 and 254 nanometers. After pulling the pure fractions, concentrate in a 30-kilodalton, molecular weight cut-off centrifugal concentrator, following the manufacturer's instructions. Then dispense 25-microliter aliquots of the concentrated protein and flash-freeze in liquid nitrogen.Using a robot, pipette 80 to 100 microliters of a crystallization reagent into the reservoir of a 96-well plate. Using a crystallization robot, dispense and mix 0.2 to 0.25 microliters of IKK1 and its inhibitor complex with the same volume of reservoir solution. Immediately after setting the drops, seal each plate with optically clear films to avoid evaporation.Incubate one plate at 18 degrees Celsius and the other plate at four degrees Celsius in a cold room. Use a stereo microscope with a polarizer to check for appearance of crystal in each drop, every day, for the first seven days and then at longer intervals. After preparing IKK1 and its inhibitor complex as previously described, transfer the well solutions to each well of a 24-well plate.Place one to 1.5 microliters of well solution on a clean glass coverslip. Add an equal volume of IKK1 and its inhibitor complex to the glass coverslip and mix gently by pipetting up and down three to four times. After applying grease on the rings of each well of the plate, turn the glass coverslip over and place on the respective well with forceps.Then seal the well by pressing down on the glass coverslip. After setting up all the drops in the 24-well plate, incubate in the cold room, in the dark. Occasionally check the drops under a microscope for appearance and growth of crystals.Once crystal growth is complete, gently remove the glass coverslip containing the crystal and place it onto a solid surface with the crystal drop facing upwards. Gently add 10 microliters of cryo A solution on top of the drop and gently mix by pipetting so that the crystal is not touched. Using a microscope, slowly remove some liquid from the crystal and keep about five to eight microliters of the solution.Gently add 2.5 to four microliters of cryo B solution onto the drop and gently mix by pipetting. Cover the glass coverslip with a small Petrie dish to avoid direct airflow over the crystal. After waiting five minutes, carefully pick a single crystal from the drop in an appropriate cryoloop mounted on a proper base.Flash-freeze the crystal in liquid nitrogen. Then store the crystal containing cryoloop in a puck immersed in liquid nitrogen in a Dewar flask until ready for X-ray diffraction at the synchrotron. After extensive trials with several different IKK1 variants, crystals were obtained with one truncated construct, which displayed suitable X-ray properties only in the presence of the IKK inhibitor XII.The combined effect of low-resolution X-ray data with weak intensities, a large number of IKK1 molecules in the asymmetric unit and conformational variation of the IKK1 monomeric-dimeric model, compared to known IKK2 models, made it difficult to obtain a molecular replacement solution, IKK1, and determine its structure. Different IKK2 structures indicated different inter-monomer orientation within its dimers so that the distance between the alpha carbons of P578 in the two kinase domains, in four different dimer models, varied between 39 and 61 angstroms. Obtaining a useful search model was possible because of the low-resolution cryoelectron microscopy map and a high-accuracy model of IKK1 domains that could be generated based on a high-resolution IKK2 structure.The initial model indicated an orientation of kinase domain rotated 24 degrees relative to that of an IKK2 monomer and an N-terminal opening of 58 angstroms. Using one of the dimers with the 52-angstrom opening as a model, six dimers in the asymmetric unit were located. Once mastered, this technique can be executed in a couple of months, if it is performed properly.While attempting this procedure, it's important to remember that obtainment of a soluble, active and well-behaved protein is the first critical step. After watching this video, you should have a good general understanding of all the steps that need to be followed in painstaking detail, to be successful in crystallizing and determining the structure of IKK1 protein. Learning this procedure, there's still structures of other IKK family proteins that can also be determined in order to answer additional questions about kinase-mitogen signaling.

Research

JoVE Journal

Biochemistry

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are ...

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a ...

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

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 ...

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are ...

Efficient and Scalable Production of Full-length Human Huntingtin Variants in Mammalian Cells using a Transient Expression System

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with a mass of approximately 350 ...

High-Throughput Screening for Protein Crystallization

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography

Author Spotlight: High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography  

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into ...

A Fibrinolytic Enzyme-Based Method to Study Cerebral Thrombus Cell Components

A Comprehensive Approach to Analyze the Cell Components of Cerebral Blood Clots

Author Spotlight: A Novel Method for Comprehensive Cell Component Analysis of Cerebral Blood Clots 

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of ...