a structural guide to proteins of the nf- b signaling...

A Structural Guide to Proteins of the NF-kBSignaling Module

Tom Huxford1 and Gourisankar Ghosh2

1Department of Chemistry and Biochemistry, San Diego State University, 5500 Campanile Drive, San Diego,California 92182-1030

2Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla,California 92093-0375

Correspondence: [email protected]

The prosurvival transcription factor NF-kB specifically binds promoter DNA to activate targetgene expression. NF-kB is regulated through interactions with IkB inhibitor proteins. Activeproteolysis of these IkB proteins is, in turn, under the control of the IkB kinase complex (IKK).Together, these three molecules form the NF-kB signaling module. Studies aimed at charac-terizing the molecular mechanisms of NF-kB, IkB, and IKK in terms of their three-dimen-sional structures have lead to a greater understanding of this vital transcription factor system.

NF-kB is a master transcription factor thatresponds to diverse cell stimuli by activat-

ing the expression of stress response genes.Multiple signals, including cytokines, growthfactors, engagement of the T-cell receptor, andbacterial and viral products, induce NF-kBtranscriptional activity (Hayden and Ghosh2008). A point of convergence for the myriadof NF-kB inducing signals is the IkB kinasecomplex (IKK). Active IKK in turn controlstranscription factor NF-kB by regulating pro-teolysis of the IkB inhibitor protein (Fig. 1).This nexus of three factors, IKK, IkB, andNF-kB, forms the NF-kB signaling module—amolecular relay switch mechanism that is con-served across diverse species (Ghosh et al.1998; Hoffmann et al. 2006). In this article,we introduce the human NF-kB, IkB, andIKK proteins, and discuss how they function

from the perspective of their three-dimensionalstructures.

NF-kB

Introduction to NF-kB

NF-kB was discovered in the laboratory ofDavid Baltimore as a nuclear activity with bind-ing specificity toward a ten-base-pair DNAsequence 50-GGGACTTTCC-30 present withinthe enhancer of the immunoglobin k lightchain gene in mature antibody-producing Bcells (Sen and Baltimore 1986). The biochemi-cally purified activity was found to be composedof 50 and 65 kilodalton (kDa) subunits. Cloningof the p50 subunit revealed significant amino-acid sequence homology between its amino-terminal 300 amino acids and the oncogene

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from the reticuloendotheliosis virus of turkeysand shared by v-Rel and its cellular proto-oncogene c-Rel. This portion of conservedamino acid sequence was termed the Rel ho-mology region (RHR). As is discussed later,the mRNA responsible for producing thep50 subunit was found to encode a longer pre-cursor protein of 105 kDa in size that possessesthe entire p50 amino-acid sequence at itsamino-terminal end and its own inhibitorwithin its carboxy-terminal region. Once thecDNA encoding the p65 subunit (also knownas RelA) was sequenced, it was also found tocontain an amino-terminal RHR. Twoadditional NF-kB family subunits, RelB andp52 (the processed product of a longer100-kDa precursor), were also discovered toharbor the conserved RHR within their amino-terminal regions. These five polypeptides, p50,p65/RelA, c-Rel, p52, and RelB, constitute theentire family of NF-kB subunits encoded bythe human genome (Fig. 2A).

Rel Homology Region Structure

The first glimpse at the structure of the RHR wasafforded by the successful determination of twox-ray crystal structures of the NF-kB p50:p50homodimer in complex with related kB DNA(Ghosh et al. 1995; Muller et al. 1995). Thestructures uncovered a symmetrical protein:DNA complex structure reminiscent of a but-terfly with double-stranded DNA comprisingthe “body” and two-protein subunit “wings”(Fig. 2B,C). These structures revealed a com-pletely novel DNA binding motif in which oneentire 300 amino acid RHR from each p50subunit in the dimer are involved in contactingone whole turn along the major groove ofdouble-stranded DNA (Baltimore and Beg1995; Muller et al. 1996).

As revealed by the NF-kB p50:DNA com-plex structures, the RHR consists of two foldeddomains. The amino-terminal domain (orRel-N) is approximately 160–210 amino acidsin length and exhibits a variant of the immu-noglobulin fold. The carboxy-terminal dimer-ization domain (referred to as Rel-C) spansroughly 100 amino acids and also adopts animmunoglobulin-like fold. The five RHR-containing NF-kB subunits assemble to formvarious homo- and heterodimer combinationsto form active NF-kB transcription factors.The two domains are joined by a short flexiblelinker approximately 10 amino acids in length.A carboxy-terminal flexible region in whichis embedded the nuclear localization signalterminates the conserved RHR portion ofthe NF-kB subunits. Besides dimerization, thisRHR is responsible for sequence-specific DNAbinding, nuclear localization, and interactionwith IkB proteins.

Outside of the conserved RHR, three NF-kBsubunits, p65/RelA, c-Rel, and RelB, contain atranscription activation domain (TAD) attheir extreme carboxy-terminal ends. Thisregion, which is poorly understood in proteinstructural terms, is responsible for the increasein target gene expression that results frominduction of NF-kB and, consequently, NF-kBdimers that possess at least one of these sub-units function as activators of transcription.

Inducingsignal

InactiveNF-kBCytoplasm

Nucleus

IKK

IkBproteolysis

IkB

b

ag

Active NF-kB

Transcription

Figure 1. The NF-kB signaling module. NF-kB existsin the cytoplasm of resting cells by virtue of itsnoncovalent association with an IkB inhibitorprotein. The IkB kinase (IKK) responds to diversestimuli by catalyzing the phosphorylation-dependent26 S proteasome-mediated degradation of complex-associated IkB. Active NF-kB accumulates in thenucleus where it binds with DNA sequence specificityin the promoter regions of target genes and activatestheir transcription.

T. Huxford and G. Ghosh

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RelB also contains a predicted leucine zippermotif amino-terminal to its RHR. The NF-kB subunits p50 and p52 lack transactivationdomains. Rather, their extreme carboxy-terminal ends are rich in glycine. Consequently,NF-kB dimers consisting exclusively of p50and p52 subunits are capable of nuclear locali-zation and DNA binding but fail to activatetarget gene expression and, in fact, functionboth in vitro and in vivo as repressors of tran-scription (Franzoso et al. 1992).

NF-kB Dimerization

Assembly of individual NF-kB subunits intodimers capable of sequence-specific DNA

binding and activating target gene expressionis mediated entirely by the dimerizationdomain. In theory, a total of 15 unique homo-and heterodimers are possible from combinato-rial dimerization of the five NF-kB subunits(Hoffmann et al. 2006). Of these, 12 havebeen identified in vivo. Three that are notknown to exist are the RelB:RelB, RelB:c-Rel,and p52:c-Rel. However, of these three, onlyRelB homodimer fails to exist in vivo, and thestatus of the other two dimers is uncertain;they could exist under specialized conditionssuch as the p65/RelA:RelB heterodimer(Marienfeld et al. 2003).

The homo- and heterotropic interactionsbetween NF-kB family subunits follow the

Rel Homology Region (RHR)Class I

p50

Class IIp65

c-Rel

ReIB

C

kBDNA

NN

NN

C C

p50RHR

p50

D256 N200

F213p65

G G G 5´

Y269

R56

R58 H66

Y59

p65RHR

C

p52

A

C D

F

E

B

NTD DimD 433L G

NTD DimD 447L

NTD DimD TAD 551 NL

NTD DimD TAD 619 Amino-terminaldomain

Dimerizationdomain

C

Linker

L

NTDLZ DimD L TAD 557

G

Figure 2. The NF-kB family. (A) The human genome encodes five polypeptides that assemble in various dimercombinations to form active NF-kB transcription factors. Each of the subunits contains the Rel homology region(RHR) near its amino terminus. The RHR consists of two folded domains, the amino-terminal domain (NTD)and the dimerization domain (DimD), that are joined by a short flexible linker and a carboxy-terminal flexibleregion that contains the nuclear localization signal (L). Three of the subunits, p65, c-Rel, and RelB, also contain atranscription activation domain (TAD) at their carboxy-terminal ends. RelB contains a predicted leucine zippermotif (LZ) amino-terminal to its RHR. The NF-kB subunits p50 and p52 lack transactivation domains and haveglycine-rich regions (G). (B) A ribbon diagram representation of the RHR from p50 in its DNA-boundconformation. (C) The NF-kB p50:p65/RelA heterodimer bound to kB DNA. (D) Another view of thecomplex. (E) The NF-kB p50:p65/RelA heterodimer dimerization domains with key amino acid side chainslabeled. (F) kB DNA from the NF-kB:DNA complex with key base-contacting amino acid residues labeled.

Proteins of the NF-kB Signaling Module

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central dogma of all protein–protein com-plexes: A set of amino acid residues from eachpolypeptide participate in direct contacts withone another, forming the interface, whereas adifferent set of residues present outside of theinterface indirectly affects the stability of theinterface by modulating the local environment.One of the most stable NF-kB dimer interfacesis created by the p50:p65/RelA heterodimer(Fig. 2D). The p50:p65/RelA heterodimerassembles with a significantly greater stabilitythan either of the respective p50:p50 or p65/RelA:p65/RelA homodimers (Huang et al.1997). Differences in the amino acid sequencesof p50 and p65/RelA at two positions partiallyexplain the variation in dimerization stabilityobserved in the three dimers. The positions ofTyr-269 and Asp-256 in p50 are occupied byPhe-213 and Asn-200, respectively, in p65.Within the heterodimer, the Asp and Asn ap-proach one another symmetrically at the inter-face and stabilize the heterodimer throughformation of a highly stable hydrogen bond.In contrast, in the homodimers, the juxtaposi-tion of Asp-Asp and Asn-Asn at the interfaceare detrimental to dimer stability. Similarly,the hydroxyl group on Tyr-269 of p50 hydrogenbonds at the dimer interface, contributing tostabilization of both the p50:p50 homodimeras well as the p50:p65/RelA heterodimer. Thesubstitution of Phe at this position serves toweaken the p65/RelA:p65/RelA homodimerrelative to the other two. Taken together, theseobservations help to explain why the p65/RelA:p65/RelA homodimer forms with lowerstability than does the p50:p50 homodimerand why the p50:p65/RelA heterodimer ismore stable than both homodimers.

However, direct contact between comple-mentary amino acids at the interface fails tocompletely explain the observed trends ofNF-kB subunit dimerization. Mutation of non-interfacial residues contained within the dimer-ization domain has been shown to modulatedimerization. For example, changing p65/RelACys-216 to Ala affects homodimer formation(Ganchi et al. 1993). The role of noninterfacialamino acid residues in dimerization is moststrikingly illustrated in the case of RelB. All

interfacial residues in RelB are either identicalor homologous to those of other NF-kB sub-units. And yet, RelB assembles into a completelyunique domain-swapped homodimer (Huanget al. 2005). Domain swapping occurs as a con-sequence of the destabilization of the foldedRelB dimerization domain, suggesting thatdomain stability is an important determinantof protein–protein interaction. In cells, de-creased folding stability in both the amino-terminal and dimerization domains contributesto its degradation by the proteasome, whichexplains why the RelB homodimer does notexist in vivo (Marienfeld et al. 2001).

Post-translational modification may alsoplay a role in modulating dimerization propen-sity. One study has shown that RelB forms adimer with a p65/RelA that is phosphorylatedat position Ser-276 (Jacque et al. 2005). Thisserine is located in a loop within the dimeriza-tion domain, projected away from the dimerinterface. It is unclear as to how phosphory-lation of this serine positively impacts p65/RelA:RelB heterodimer formation. One expla-nation could be that the increased negativecharge may indirectly modulate dimer-formingresidues of both p65/RelA and RelB.

NF-kB Recognition of kB DNA

X-ray structures of NF-kB in complex withkB DNA revealed a new mode of DNA recog-nition wherein a dimer composed of the RHRof two NF-kB subunits intimately contactsdouble-stranded DNA within the major groovethrough one complete turn (Ghosh et al. 1995;Muller et al. 1995). NF-kB employs both itsamino-terminal and dimerization domains toencircle its target DNA. DNA contacts aremediated by amino acids emanating fromloops that connectb-strand elements of second-ary protein structure. The p50:p50 homodimerstructures revealed that this NF-kB subunitemploys its amino acids His-66, Arg-58, andArg-56 to contact three guanine nucleotidebases at the extreme 50 ends of its consensuskB DNA. However, X-ray analyses of NF-kBp65/RelA:p65/RelA homodimers bound to kBDNA revealed that, when bound to a canonical





10-base-pair kB DNA, one p65/RelA subunitcontacts DNA in an analogous manner asobserved in the p50 homodimer structures,whereas the second p65/RelA subunit signifi-cantly repositions its entire amino-terminaldomain to mediate interactions with the DNAbackbone (Chen et al. 1998b). Such a bindingmode, which is afforded by flexibility in theshort linker region that connects the twostructured domains of the RHR, preservesbinding affinity at the cost of fewer contactsto DNA bases. A close inspection of thehomodimer:DNA complex structures leads tothe suggestion that a homodimer of p50 mightoptimally bind to an 11-base-pair sequencecomposed of two 50-GGGPuN half sites bracket-ing a central A:T base pair. In contrast, theNF-kB p65/RelA homodimer optimally recog-nizes a nine-base-pair target sequence contain-ing two 50-GGPuN half sites and a central A:Tbase pair. Determination of a second X-raystructure of NF-kB p65/RelA homodimer incomplex with the kB DNA sequence from thepromoter of IL-8 (50-GGAA T TTCC-30) con-firmed this hypothesis (Chen et al. 2000).

The lessons learned from structural analysesof p50 and p65/RelA homodimers bound todiverse kB DNA sequences suggested that thecanonical NF-kB p50:p65/RelA heterodimermight recognize its 10-base-pair target se-quence with the p50 subunit binding specifi-cally to a 50-GGGPyN half site, whereas thep65/RelA subunit binds to a 50-GGPyN halfsite separated from the p50 site by one A:Tbase pair. X-ray structure determination of ap50:p65/RelA RHR heterodimer bound to kBDNA from the original k light chain gene pro-moter, which is identical to a kB sequencethat is present in the promoter of HIVgenome (50-GGGAC T TTCC-30), served toconfirm this speculation (Chen et al. 1998a).Additional crystal structures of NF-kB p50:p65/RelA heterodimer bound to different kBDNA further support the basic rules of DNAhalf-site recognition developed from thehomodimer:DNA structures (Berkowitz et al.2002; Escalante et al. 2002).

X-ray crystal structures of several additionalNF-kB:DNA complexes have now been

determined (Cramer et al. 1997; Cramer et al.1999; Moorthy et al. 2007; Panne et al. 2007;Fusco et al. 2009). Taken together, these struc-tures suggest a model for how NF-kB dimersrecognizekB DNA that contain significant devi-ations from the consensus sequence. Changesof kB DNA sequence can be of two types. Inthe first, alteration occurs within the G:Cbase pairs that occupy positions on the out-side of the kB DNA and that are directly con-tacted by the amino-terminal domain. Anexample is the altered kB sequence 50-GGGACT TTTC-30 (change from immunoglobin kBsequence is underlined). Because of the lossof an important C:G base pair, the NF-kBp65/RelA subunit conformation when boundto TTCC-30 is drastically different than toTTTC-30. The flexible linker region andmodular domain architecture within the RHRallows the amino-terminal domain to repositionitself and bind the DNA backbone to accommo-date such variations in kB DNA sequence. Asecond type of kB DNA sequence alterationinvolves changes within the central five basepairs. Of these, the central A:T is not directlycontacted by the protein and the others arerecognized nonspecifically through van derWaals contacts by p50 Tyr-59 (Tyr-36 in p65/RelA). The third base pair from the 50-end orits symmetric pair are less sensitive to change,as illustrated by a comparison of immunoglobinkB and IFN-b kB sequences (50-GGGAC TTTCC-30 and 50-GGGAA T TTCC-30, respec-tively). The C:G to A:T base-pair change doesnot affect DNA recognition by the protein butmay influence stability of binding because ofthe more rigid DNA structure of IFN-b kBsites. In this second case, overall conformationsof the protein:DNA complexes are similar, butbinding affinity could differ significantly.

Molecular dynamics simulations have re-vealed intriguing structural transitions of a 20-base-pair DNA containing the kB site of IL-2promoter (AGAA A TTCC). The central A:Tbase pair (underlined) undergoes cross-standstacking and the central A:T base pair flips outof the DNA axis (Mura and McCammon2008). This dynamic behavior of the DNAsuggests a highly complex mechanism of DNA





recognition by the NF-kB dimers, in whichDNA sequence variations may play a significantrole in the recognition process. In general, onecan confidently say that the sequences at thecenter of a kB sequence may profoundly affectthe binding affinity and specificity.

IkB

Introduction to IkB

Almost immediately on detecting NF-kB inimmune cells, researchers discovered that alatent kB DNA binding activity was present inthe cytoplasm of all resting cells and that thispool of NF-kB could be activated by treatmentof cell lysates with the weak detergent deoxy-cholate (Baeuerle and Baltimore 1988a). Thissuggested that noncovalent interaction with aninhibitor protein was responsible for maintain-ing NF-kB in an inactive state. Purification ofthe inhibitor activity led to the cloning of theinhibitory proteins IkBa and IkBb (Baeuerleand Baltimore 1988b; Thompson et al. 1995).A third IkB gene was later identified by sequencehomology in an EST database and was calledIkB1 (Li and Nabel 1997; Simeonidis et al.

1997; Whiteside et al. 1997). Subsequent exper-iments demonstrated that it also exhibits NF-kBinhibitory activity.

Classical IkB Sequence and Structure

Both IkBa and IkBb, as well as the more recentlydiscovered IkB1, contain a central ankyrin re-peat domain (ARD) that contains six ankyrinrepeats (Fig. 3A). The ankyrin repeat (ANK) isa roughly 33-amino-acid consensus aminoacid sequence that appears in multiple copiesin numerous proteins (Fig. 3B) (Sedgwick andSmerdon 1999). Ankyrin repeats are part of agreater superfamily of helical repeat motifs,which include HEAT repeats, armadillo re-peats, and leucine-rich repeats, and are commonto proteins involved in protein–protein inter-actions (Groves and Barford 1999). At theiramino-terminal ends, classical IkB proteinscontain a sequence of amino acids that do notadopt a folded structure in solution (Jaffrayet al. 1995). Contained within this signalresponse region are the conserved serine sitesof phosphorylation by IKK. Roughly 10 aminoacids amino-terminal to this pair of serines

K

K

K

S

S

S

E ANK

ANK

ANK

ANKG

G

L

L

DimD

DimD

NTD

NTD

ANK ANK ANK ANK ANK

ANK ANK ANK ANK ANK ANK

ANK DeaD S

DeaD SANK

ANK ANK ANK ANK ANK ANK ANK



ANK

ANK ANK ANK ANK ANK

ANK ANK ANK ANK

ANK ANK ANK ANK AN PEST

PEST

327

446

629

898

969

361

356

317

1N

2

3

4

5

6C

PEST region

Ankyrinrepeatdomain

IkBa

p50

p65

NF-kB precursors

p105

p100

Nuclear IkB

Classical IkB

IkBa

IkBb

IkBe

A B C

D

PEST

Figure 3. The family of human IkB proteins. (A) IkB proteins are classified as in text. Classical IkB proteinspossess ankyrin repeats (ANK) flanked by an amino-terminal signal response region and carboxy terminalPEST region. The signal response regions contain sites of phosphorylation by IKK (S), ubiquitination (K),and nuclear export (E). The NF-kB precursors serve as IkB proteins as well as the source of the mature p50and p52 NF-kB subunits. (B) Ribbon diagram of the IkBa structure from the NF-kB:IkBa complex crystalstructure. Individual ankyrin repeats are numbered, ANK 4 is colored magenta, and the PEST region islabeled. (C) Ribbon diagram of the NF-kB:IkBa complex. (D) Another view of the complex.





reside conserved lysine amino acid sites of poly-ubiquitination. This amino-terminal region ofIkBa also contains a functional nuclear exportsequence (Johnson et al. 1999; Huang et al.2000). It is not masked on binding to NF-kBand contributes to the observed cytoplasmiclocalization of NF-kB:IkBa complexes. Nei-ther IkBb nor IkB1 possess this inherentnuclear export potential. Consequently, stableNF-kB:IkBb complexes reside stably either inthe cytoplasm or the nucleus, whereas IkB1appears to function as a negative feedback regu-lator of cytoplasmic NF-kB (Malek et al. 2001;Tam and Sen 2001; Kearns et al. 2006). At theircarboxy-terminal ends, the three classical IkBproteins contain a short sequence rich in theamino acids proline, glutamic acid, serine,and threonine. This so-called PEST region iscommon to many proteins that, like IkB, dis-play rapid turnover in cells (Rogers et al. 1986;Pando and Verma 2000). The PEST region ofIkBa, however, is also required for its abilityto disrupt preformed NF-kB:DNA complexes(Ernst et al. 1995).

IkB Interactions with NF-kB

The X-ray structure of IkBa in complex with theNF-kB p50:p65/RelA heterodimer was deter-mined independently by two separate labora-tories in 1998 (Huxford et al. 1998; Jacobs andHarrison 1998). Both groups relied on a simi-lar strategy of removing the signal responseregion of IkBa and the amino-terminaldomain of the p50 subunit to stabilize the con-formationally dynamic complex for cocrystalli-zation (Huxford et al. 2000). The structurereveals how IkBa uses its entire ankyrin repeat-containing domain as well as its carboxy-terminal PESTsequence to mediate an extensiveprotein–protein interface of roughly 4300 A2

(Fig. 3C,D). The carboxy-terminal 30 aminoacids from the NF-kB p65/RelA subunitRHR, which were disordered in NF-kB:DNAcomplex structures, adopt an ordered helicalstructure that contacts the first two ankyrinrepeats and forms significant hydrophobiccontacts with the amino-terminal face of theIkBa ankyrin repeat stack. This interaction

masks the p65/RelA nuclear localization signal.Ankyrin repeats three through five participatein multiple van der Waals contacts with onesurface of the p50:p65/RelA heterodimer di-merization domains. The sixth ankyrin repeatand PEST region of IkBa present a vast acidicpatch, which opposes the largely positivelycharged DNA binding surfaces of the p65/RelA amino-terminal domain. As a conse-quence of this electrostatic interaction, thep65/RelA amino-terminal domain occupies aposition relative to the dimerization domainthat is rotated roughly 1808 and translated40 A when compared with its DNA bound struc-tures. The transition of p65/RelA to the confor-mation observed in the NF-kB:IkB complexdoes not disrupt the amino-terminal domainstructure and is afforded entirely by the flexiblelinker region that connects the amino-terminaland dimerization domains. The structure of asimilar construct of IkBb bound to the di-merization domain from the NF-kB p65/RelA:p65/RelA homodimer suggests that IkBbuses a similar strategy in binding to NF-kB,although it relies less on interactions with thep65/RelA amino-terminal domain for com-plex stability (Malek et al. 2003).

IkBa Dynamics

Protein dynamics, or the rates with which aprotein exchanges between quasi-stable foldedstates, is an extremely important aspect of pro-tein structure and function. Several indepen-dent lines of investigation, including thermaland chemical denaturation, computer simu-lations, NMR spectroscopy, and failed crystal-lization attempts, have led to the conclusionthat the IkBa protein exhibits a high degree ofstructural dynamics in solution (Huxford et al.2000; Pando and Verma 2000; Croy et al. 2004;Bergqvist et al. 2006). This runs counter tothe data that have emerged from protein engi-neering studies that clearly show that ankyrinrepeat proteins designed after consensus se-quences or those that appear in nature are ex-tremely stably folded (Binz et al. 2003; Binzet al. 2004). IkBa has, therefore, evolved as aninherently unstable ankyrin repeat-containing





protein. The consequences of this are twofold.First of all, free IkBa is easily degraded in cells.This signal-independent degradation involvesthe 20S proteasome and the carboxy-terminalPEST of IkBa (Mathes et al. 2008). Moreover,recent computational modeling of the NF-kBpathway through a systems biology approachhas confirmed that regulation of NF-kB acti-vation can be controlled by small changes inthe rate of degradation of a constitutivelyexpressed free cytoplasmic IkBa (O’Dea et al.2007). On binding to NF-kB, the dynamic IkBaankyrin repeat fold becomes stable and thePEST region protein turnover signal sequenceis adopted as a DNA-inhibitory functionalelement (Sue et al. 2008). Degradation ofIkBa is shifted from the steady state to a signal-dependent pathway that requires phosphory-lation within the flexible amino-terminal signalresponse region. It is likely that the inherentfolding instability of IkBa contributes to itbeing targeted by the proteasome, whereasNF-kB, which is composed of two stably foldeddomains and with which IkBa is associated ina complex at subnanomolar dissociation bind-ing constant, remains intact.

Nonclassical IkB Proteins

Through the efforts of investigators attemptingto understand regulation of NF-kB, a morediverse family of IkB proteins has emerged.Proteins of the IkB family are all linked by thefact that they contain ankyrin repeats andinteract with NF-kB subunits to affect geneexpression. The classical IkB proteins, IkBa,IkBb, and IkB1, have been described. A secondclass of IkB proteins is represented by theNF-kB precursor proteins p105 and p100(Basak et al. 2007). These two proteins actboth as precursors of NF-kB p50 and p52 sub-units, respectively, and as inhibitors of NF-kB(Fig. 3A). The NF-kB precursor proteins areresponsible for inhibiting nearly half of theNF-kB in resting cells. However, unlike classicalIkB proteins that inhibit NF-kB by forming 1:1complexes, these NF-kB precursors participatein large multiprotein assemblies, wherein morethan one NF-kB dimer can bind to multiple

p100 and/or p105 subunits. The assembly ofp100 and p105 into largercomplexes is mediatedby an oligomerization domain located immedi-ately amino-terminal to their ankyrin-repeatdomains. This assembly is heterogeneous, i.e.,several different NF-kB subunits can be inhib-ited in a single inhibitory complex (Savinovaet al. 2009). Therefore, stimulus-specific degra-dation of p100 or p105 can in principle releasedifferent NF-kB dimers. Together, these dimersexhibit a much broader spectrum of generegulatory activities (Savinova et al. 2009, Shihet al. 2009). The large heterogeneous NF-kBinhibitory complex assemblies have beendubbed NF-kBsomes to distinguish them fromthe smaller NF-kB inhibitory complexesformed by IkBa, -b, and -1.

Together with IkBa and IkB1, theNF-kB precursors are targets of NF-kB-driventranscription. Newly synthesized IkB serveto block NF-kB activity postinduction. Thisnegative feedback regulation of NF-kB byIkB proteins is critical for control of inflam-mation and other diseases (Hoffmann et al.2002).

Nuclear IkB Proteins

A third, entirely different class of IkB is rep-resented by the proteins Bcl-3, IkBz/MAIL,and IkBNS (Fig. 3A). Bcl-3 was cloned as aconsequence to its proximity to a breakpointmutation in some leukemias (Ohno et al.1990). IkBz was discovered in a screen ofgenes that displayed increased expression afterinduction of immune cells with bacterial lipo-polysaccharide (LPS) or the inflammatory cyto-kine interleukin-1 (IL-1) (Kitamura et al. 2000;Haruta et al. 2001; Yamazaki et al. 2001). IkBNSwas identified as a gene that is expressed in Tcells during negative selection (Fiorini et al.2002). Unlike classical IkB, these proteins donot contain amino-terminal signal-dependentphosphorylation sites or carboxy-terminalPEST regions. Their classification as “nuclearIkB” derives from the fact that they containankyrin repeats, bind NF-kB subunits, and con-centrate within the nucleus when expressed incells (Michel et al. 2001).





Each of the three nuclear IkB proteins arethemselves the products of NF-kB-dependentgenes (Eto et al. 2003; Ge et al. 2003; Hirotaniet al. 2005). They bind to NF-kB, but, whereasthe classical IkB proteins prefer dimers thatpossess at least one p65/RelA or c-Rel subunit,or nonclassical p105 and p100 binds all NF-kBsubunits, nuclear IkBs bind specifically tohomodimers of p50 (Hatada et al. 1992;Yamazaki et al. 2001; Trinh et al. 2008).Finally, association of nuclear IkB proteinswith nuclear NF-kB can have diverse butimportant consequences on gene expression(Franzoso et al. 1992; Muta et al. 2003;Hirotani et al. 2005; Motoyama et al. 2005;Riemann et al. 2005). In peritoneal macro-phages derived from mice lacking the geneencoding IkBz, for example, a completeinability to produce the NF-kB-dependentcytokine interleukin-6 (IL-6) in response toLPS treatment was observed (Yamamoto et al.2004). As IL-6 is a gene that is expressed in alater phase of NF-kB induction, it is apparentthat the early induction and nuclear accu-mulation of IkBz plays a vital role in theLPS-dependent expression of this pluripotentcytokine.

IKK

Introduction to IKK

In a thrilling conclusion to a search to identify anenzymatic activity that was capable of phosphor-ylating the two serine amino acids of the signalresponse region of IkBa, researchers from threelabs reported in 1997 the IkB Kinase complex(IKK) (DiDonato et al. 1997; Mercurio et al.1997; Regnier et al. 1997). This IKK was purifiedfrom cytokine-induced HeLa cells and exhibitedan apparent molecular mass of 700–900 kDa.Microsequencing revealed two related kinasedomain-containing subunits, referred to asIKKa and IKKb (or alternatively as IKK1 andIKK2, respectively) (DiDonato et al. 1997;Zandi et al. 1997). These exhibit molecularmasses of 85 and 87 kDa, respectively, anddisplay 50% identity at the amino-acid level.The IKKa subunit was recognized to be the

same protein that was originally cloned in 1995as CHUK, a kinase with homology to the helix-loop-helix transcription factors, and IKKb wassubsequently identified as a hit in a yeast two-hybrid screen with NIK, a kinase suspected tofunction upstream of IKK, as bait (Connellyand Marcu 1995; Regnier et al. 1997; Woroniczet al. 1997). A third subunit, known as IKKg(also known as NEMO), was also identified as a49 kDa member of the IKK complex (Rothwarfet al. 1998; Yamaoka et al. 1998; Li et al. 1999b;Mercurio et al. 1999).

The function of IKK1/IKKa in cells issomewhat exotic and continues to be elucidated(Hu et al. 2001; Senftleben et al. 2001; Anestet al. 2003; Yamamoto et al. 2003; Sil et al.2004; Lawrence et al. 2005). However, multiplestudies including mouse knockouts haverevealed that IKK2/IKKb is responsible forphosphorylating IkB in response to NF-kB-inducing signals (Li et al. 1999a; Li et al.1999c; Tanaka et al. 1999). Because of its roleas the primary inducer of the NF-kB transcrip-tion factor, IKK2/IKKb plays the critical rolein promoting inflammation and cell survivalin response to proinflammatory stimuli. TheNEMO/IKKg subunit does not possess anykinase domain or enzymatic activity andinstead it acts as an adapter subunit that linksthe catalytic subunits to receptor proximalsignaling molecules. Mouse knockout studiesclearly reveal that proper NF-kB signalingthrough IKK is not possible without thissubunit (Makris et al. 2000; Rudolph et al.2000; Schmidt-Supprian et al. 2000).

IKK Catalytic Subunit Domain Organization

The IKK1/IKKa and IKK2/IKKb subunitsexhibit an uncommon domain organization(Fig. 4A). Their first roughly 300 amino acidscontain a clearly recognizable kinase domain.This is followed by a short region that exhibitsdistant homology to ubiquitin (Ikeda et al.2007). A central region contains a leucinezipper motif followed by a region with slighthomology to the helix-loop-helix transcriptionfactors. This is followed by a serine-rich region.Finally, the carboxy-terminal element of IKKb





has been shown to interact directly withNEMO/IKKg (May et al. 2000; May et al.2002). A genome-wide analysis has identifieda small clade of proteins with domain organi-zation reminiscent of the catalytic IKK1/IKKaand IKK2/IKKb subunits (Manning et al.2002). This kinase subgroup includes the pro-teins IKK1 and TBK1/NAK. Both have beencharacterized as upstream modulators of IKKactivity and are, therefore, both structurallyand functionally related to the catalytic IKKsubunits (Tojima et al. 2000; Peters andManiatis 2001).

With the exception of the extreme carboxy-terminal NEMO/IKKg-interacting motif, thestructures of IKK1/IKKa and IKK2/IKKb areunknown. Although it is clear that the kinasedomain of IKK2/IKKb is necessary for catalyz-ing phospho-transfer, regions of the proteinoutside of this domain are necessary for direct-ing specificity to the amino-terminal serinesof IkBa. Deletion of the leucine zipper andhelix-loop-helix has been shown to yield amutant enzyme that, although it is capable ofcatalyzing phospho-transfer to IkBa, fails torecognize the amino-terminal serines requiredfor NF-kB activation in response to inflamma-tory signaling (Shaul et al. 2008).

NEMO/IKKg Domain Organization

Structural interest in NEMO/IKKg arises fromits lack of a kinase domain or, for that matter,homology to any other protein of known struc-ture. Secondary-structure prediction methodssuggest that NEMO/IKKg is a mostly helicalprotein with two signature coiled coil (CC)elements and a leucine zipper motif in themiddle are flanked by a helical dimerizationdomain near the amino-terminal end and aZn-finger motif at the carboxyl terminus.Three-dimensional structures of several frag-ments of the NEMO/IKKg subunit either asfree polypeptides or bound to ligands haverecently been elucidated. These NEMO/IKKgstructures allow one to envision that with theexception of the very ends, NEMO/IKKg con-sists of long helices that are punctuated byshort unstructured regions. These helical seg-ments wrap around each other forming a longcoiled-coil dimer with fraying of the mono-mers at each end. The carboxy-terminal endcontains a Zn-finger motif and the 40-residuelong amino terminus is likely to be flexible andunstructured.

The NEMO/IKKg fragment structuressuggest how IKKg might be involved in

KDIKKa

A B

DC

IKKb

IKKg

N C

N

N N

N

C C

C

C

C

N

CN

IKKgDi-Ub

Di-UbIKKg

CC1 CC2 L ZF

U L H S

KD U L H S

N

N

745

756

419

IKKgIKKb

F734

F734

Zn

W739

W739

W741

W741IKKg

IKKg

H413

C417

C400

C397

IKKb

Figure 4. Subunits of the human IKK complex. (A) Domain organization of IKK subunits. Catalytic subunitscontain a kinase domain (KD), ubiquitin-like domain (U), leucine zipper (L), helix-loop-helix (H),serine-rich (S), and NEMO-binding motif (N). The NEMO/IKKg subunit contains two predictedcoiled-coil motifs (CC1 and -2), a leucine zipper (L), and a carboxy-terminal zinc-finger (ZF). (B) Ribbondiagram of the IKK2/IKKb:NEMO/IKKg complex. Individual polypeptides are labeled as well as some ofthe conserved hydrophobic amino acid side chains from IKK2/IKKb. (C) Ribbon diagram of the NEMO/IKKg:di-ubiquitin complex. (D) The NEMO/IKKg carboxy-terminal zinc-finger motif structure.





cellular signaling. NEMO/IKKg was previouslythought to form a multimer with differentsegments shown to form dimers, trimers, ortetramers (Agou et al. 2002; Tegethoff et al.2003; Marienfeld et al. 2006; Drew et al. 2007;Herscovitch et al. 2008). With new structuralinformation, it now appears that previous con-clusions might not be accurate. It is not sur-prising as flexible coiled-coil motifs migratethrough the gel filtration beads differentlythan the stably folded globular proteins ofidentical mass.

Two Distinct IKK Activation Pathways

The IKK1/IKKa and IKK2/IKKb subunits areactivated through distinct signaling pathways(Senftleben et al. 2001). These pathways areactivated by two distinct sets of stimuli. Thecanonical pathway is triggered by LPS orinflammatory cytokines TNF-a or IL-1 andsignals through IKK2/IKKb to activate p65/RelA and c-Rel dimers through the degradationof IkBa, IkBb, IkB1, and p105. The noncanoni-cal pathway results from BAFF, LT-b, and CD40signaling through IKKa, leading to activation ofthe p52:RelB heterodimer through processingof p100 into p52 (Ghosh and Karin 2002).Activation of both pathways requires inter-actions of signaling molecules through specificpoly-ubiquitin moieties that are covalentlylinked to some of these molecules. In addition,upstream protein kinases are also essential foractivation of both kinases. The major distinc-tion between these two pathways is the involve-ment of a single upstream kinase, known asNF-kB inducing kinase (NIK), to activateIKKa, whereas multiple kinases can activateIKKb. IKKa and IKKb are both present in thesame particle in vivo. One puzzling question,however, is whether the different pathwaystarget these two distinct subunits in a singlecomplex or if there is a separate pool of IKKapresent in cells that is activated by the nonca-nonical pathway. For the canonical pathway,however, the IKKb subunit of the heterodimermust be activated. The role for IKKa in canoni-cal signaling is unclear. The presence of the IkBkinases in multiple signaling pathways suggests

that they are capable of participating in diversesignaling complexes throughout the cell.

IKK Complex Oligomerization

Although several combinations of the IKK1/IKKa, IKK2/IKKb, and NEMO/IKKg sub-units have been proposed to account for theoriginal 700–900 kDa complex, no successfulreconstitution from purified components hasresulted in an active complex of this size.Therefore, 10 years after isolation of the com-plex, even the oligomerization state of the IKKcomplex remains unclear. Given the presenceof multiple conserved elements that can poten-tially mediate homo- and heteromeric inter-actions between subunits, there exist manypossibilities. Early attempts to determine thearrangement of subunits in the complex led tothe model that, independent of its NEMO/IKKg scaffolding protein, IKK1/IKKa andIKK2/IKKb are capable of forming stablehomo- or heterodimers (Zandi et al. 1998).More recent work with human IKK2/IKKbpurified in milligram quantities from recombi-nant baculovirus-infected sf9 insect cells hasshown that the full length subunit purifies as atetramer. Removal of the carboxy-terminal100 amino acids containing the serine-richand NEMO/IKKg-binding regions results in adimeric enzyme, whereas removal of the entirecarboxy-terminal portion beginning at theleucine zipper renders the kinase monomeric.Interestingly, it was observed that althoughthis monomeric IKK2/IKKb kinase domainremains catalytically active, it fails to specificallyphosphorylate the signal response region ofIkBa and instead catalyzes phosphorylationof the PEST (Shaul et al. 2008). The presenceof multiple domains linked to flexibility in allthree subunits shows why it is difficult to struc-turally characterize functional IKK complexes.

The Emerging Structure of NEMO/IKKg

A working structural model of the NEMO/IKKg subunit has begun to take shape with therecent successful determination of several X-rayand NMR structures of discrete functional





portions. The structure of an IKK2/IKKb poly-peptide bound the amino-terminal helicalregion (residues 44 to 111) of NEMO/IKKgrevealed the complex forms a parallel four-helixbundle where two IKK2/IKKb peptides associ-ate with the NEMO/IKKg dimer (Rushe et al.2008). The IKK2/IKKb peptides appear tofold on binding to the NEMO/IKKg-dimerscaffold, making several contacts (Fig. 4B). Theaffinity of interaction between NEMO/IKKgand IKK2/IKKb subcomplex is high (KD is inlow nanomolar range) and requires a large arrayof contacts. However, few of the hydrophobicresidues in IKK2/IKKb that are observed to linethe hydrophobic pocket formed by the NEMO/IKKg dimer are required for complex formation.Although no clearexperimental data is available,the IKK:IKKg complexes might exist as a 2:2complex, where a dimeric NEMO/IKKg bindsto an IKK dimer. The amino-terminal helicaldimerization domain of NEMO/IKKg interactswith both the IKK1/IKKa and IKK2/IKKbcarboxy-terminal peptides, forming a 1:1:2(IKK1/IKKa:IKK2/IKKb:NEMO/IKKg) IKKcomplex. This is possibly the basal state of theIKK complex in cells.

Several reports demonstrated an interactionbetween poly-ubiquitin chains and the CC2-LZ region of NEMO/IKKg (Lo et al. 2009).Although its was previously thought that thepoly-ubiquitin chain is linked through lysine63 and glycine 76 of ubiquitin, experimentshave now shown that linear ubiquitin chainsbind to NEMO/IKKg with 100-fold tighterbinding affinity than do K63-linked chains.The X-ray structure of the complex betweenthe CC2-LZ of NEMO/IKKg and a lineardi-ubiquitin motif has been recently elucidated(Rahighi et al. 2009). Two di-ubiquitin motifsare bound to two chains of the LZ motif(Fig. 4C). It is striking that this central regionof NEMO/IKKg also exhibits the elongatedcoiled-coil motif that was observed in the pre-viously determined IKK2/IKKb:NEMO/IKKgcomplex as well as in the X-ray structure of acomplex between the carboxy-terminal helicalregion of NEMO/IKKg and the viral proteinvFLIP from Kaposi’s sarcoma virus (Bagneriset al. 2008).

The extreme carboxy-terminal region ofNEMO/IKKg adopts a CCHC-type zinc-fingermotif. The solution structure of this region hasbeen determined by multidimensional NMRspectroscopy (Cordier et al. 2008). Its structureadopts the familiar fold of a zinc-finger motif(Fig. 4d). Furthermore, this motif has beencharacterized as a ubiquitin-binding motifrequired for NF-kB signaling in response toTNF-a (Cordier et al. 2009).

When taken together, the NEMO/IKKgsubstructures suggest that this subunit adoptsan elongated helical structure. Two longhelices bind one another through extendedcoiled-coil interactions to form the dockingsite for a pair of kinase subunits at the aminoterminus, binding sites for di-ubiquitin andother proteins throughout the central region,and a pair of zinc-finger ubiquitin bindingsites at the carboxyl terminus. This modulararrangement of docking sites for diverse pro-teins seems appropriate for a subunit that isthought to function principally as a scaffoldfor inducible signaling. Although significantstructural work on IKK complexes remains tobe carried out, this oddly elongated NEMO/IKKg model serves to explain some of thecomplications associated with early studiesthat relied primarily on size exclusion chro-matography for characterization of complexsize and subunit stoichiometry.

ACKNOWLEDGMENTS

T.H. is supported by American Cancer Societygrant RSG-08-287-01-GMC. G.G. is supportedby National Institutes of Health (NIH) grants(GM085490, AI064326, and NCI141722).

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2009; doi: 10.1101/cshperspect.a000075Cold Spring Harb Perspect Biol Tom Huxford and Gourisankar Ghosh

B Signaling ModuleκA Structural Guide to Proteins of the NF-

Subject Collection NF-kB

BκRegulators of NF-Use of Forward Genetics to Discover Novel

Tao Lu and George R. Stark

BκOncogenic Activation of NF-Louis M. Staudt

B ResponseκSelectivity of the NF-Ranjan Sen and Stephen T. Smale System

B SignalingκThe Regulatory Logic of the NF-

Ellen O'Dea and Alexander HoffmannB in the Nervous SystemκNF-

Barbara Kaltschmidt and Christian Kaltschmidt Development and FunctionB Pathway in LymphocyteκRoles of the NF-

Steve Gerondakis and Ulrich SiebenlistB: Regulation by UbiquitinationκSignaling to NF-

Ingrid E. Wertz and Vishva M. Dixit ActivationBκThe IKK Complex, a Central Regulator of NF-

Alain Israël

BκNF-Ubiquitination and Degradation of the Inhibitors of

Schueler-Furman, et al.Naama Kanarek, Nir London, Ora

B in the Nervous SystemκNF-Barbara Kaltschmidt and Christian Kaltschmidt

Signaling ModuleBκA Structural Guide to Proteins of the NF-

Tom Huxford and Gourisankar GhoshInflammation

B Pathway inκThe Nuclear Factor NF-

Toby LawrenceDrosophilaB in the Immune Response of κNF-

Charles Hetru and Jules A. Hoffmann and CancerB as a Critical Link Between InflammationκNF-

Michael Karin

Responses by Chromatin OrganizationB-dependent TranscriptionalκControl of NF-

Gioacchino NatoliProteins

BκSpecification of DNA Binding Activity of NF-

Fengyi Wan and Michael J. Lenardo

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