survey and summary molecular recognition by the kix domain

SURVEY AND SUMMARY

Molecular recognition by the KIX domain and itsrole in gene regulationJitendra K Thakur Archana Yadav and Gitanjali Yadav

National Institute of Plant Genome Research (NIPGR) Aruna Asaf Ali Marg New Delhi 110067 India

Received September 19 2013 Revised and Accepted October 24 2013

ABSTRACT

The kinase-inducible domain interacting (KIX)domain is a highly conserved independentlyfolding three-helix bundle that serves as a dockingsite for transcription factors whereupon promoteractivation and target specificity are achievedduring gene regulation This docking event is a har-binger of an intricate multi-protein assembly at thetranscriptional apparatus and is regulated in a highlyprecise manner in view of the critical role it plays inmultiple cellular processes KIX domains have beencharacterized in transcriptional coactivators suchas p300CREB-binding protein and mediator ofRNA polymerase II transcription subunit 15 andeven recQ protein-like 5 helicases in various organ-isms Their targets are often intrinsically disorderedregions within the transactivation domains of tran-scription factors that attain stable secondary struc-ture only upon complexation with KIX In this articlewe review the KIX domain in terms of its sequenceand structure and present the various implicationsof its ability to act as a transcriptional switch themechanistic basis of molecular recognition by KIXits binding specificity target promiscuity combina-torial potential and unique mode of regulation viaallostery We also discuss the possible roles of KIXdomains in plants and hope that this review willaccelerate scientific interest in KIX and pave theway for novel avenues of research on this criticaldomain

INTRODUCTION

Transcriptional activation is a key process in the regula-tion of gene expression in living organisms and more soin eukaryotes where the transcriptional apparatus is ahuge modular complex Its regulation in eukaryotes isaccomplished through an intricate network of specific

interactions at the DNAndashprotein interface and theproteinndashprotein interface Transcriptional activatorsachieve gene specificity by binding specific regulatoryelements on DNA via functionally independent DNA-binding domains In addition transcription factorsmediate recruitment of other factors subsequent chroma-tin modification and pre-initiation complex formationby docking one or more of their transactivation domainsto conserved sites on many cognate proteins like forexample transcriptional coactivators that are importantbinding partners for activation domains (ADs) Thesecoactivators thus serve as bridging molecules betweentranscription factors and the transcription machinery oras enzymes that introduce chemical modifications onDNA One such coactivator is the cAMP-responseelement-binding protein (CREB)-binding protein (CBP)a large multi-domain protein containing stretches ofintrinsically disordered linker regions and a number ofstructured protein-binding domains that interact with alarge variety of transcription factors One of thedomains of CBP is a vital independently folding andhighly conserved three-helix bundle region termed as thekinase-inducible domain interacting (KIX) domain It wasdiscovered by Parker et al (1) in 1996 in the mouse CBPas the specific and minimal region that was sufficient tobind and interact with phosphorylated CREB and thenactivate transcription Since its initial identification wasbased on its interaction with the phosphorylated kinase-inducible domain (KID) region of CREB protein it wasgiven the name lsquoKID-interacting domainrsquo or lsquoKIXrsquo (1)Following its discovery in CBP KIX domain has beenidentified in the human activator recruited cofactor 105-kDa component (ARC105) and yeast Gal11p both ofwhich are mediator of RNA polymerase II (RNA PolII) transcription subunit 15 (MED15) subunit ofmediator complex involved in regulation of transcriptionof specific genes (2ndash4) As shown in Figure 1 KIX is nowknown to be one of the most important molecular recog-nition sites for proteinndashprotein interaction during generegulation playing a significant role in assembly ofproteins to the transcriptional apparatus in yeast and

To whom correspondence should be addressed Tel +91 11 26735103 Fax +91 11 26741658 Email gynipgracinCorrespondence may also be addressed to Jitendra K Thakur Tel +91 11 26735221 Fax +91 11 26741658 Email jthakurnipgracin

2112ndash2125 Nucleic Acids Research 2014 Vol 42 No 4 Published online 18 November 2013doi101093nargkt1147

The Author(s) 2013 Published by Oxford University PressThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (httpcreativecommonsorglicensesby30) whichpermits unrestricted reuse distribution and reproduction in any medium provided the original work is properly cited

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mammals (125) KIX has been shown to interact with arange of transcription factors other than CREB such asc-Myb (6) mixed lineage leukemia protein (MLL) (7)breast cancer 1 (BRCA1) (8) c-Jun (9) p53 (10) signaltransducers and activators of transcription (STAT1) (11)and sterol responsive element-binding protein (SREBP)(12) of humans (Figure 1AndashC) to Gal4p (13) Gcn4 (14)Pdr13 (2) and Oaf1 (3) of yeast (Figure 1C) cubitusinterruptus (15) of Drosophila and human t-lymphotropicvirus type 1 (HTLV-1) Tax (16) human immunodeficiencyvirus type 1 (HIV-1) Tat (17) and E2 (18) of viruses(Figure 1B) Direct involvement of KIX domain hasbeen demonstrated in long term storage of hippocam-pus-dependent memory in mammalian brains (19) lipidhomeostasis in mammals (4) multi-drug resistance (2)and fatty acid metabolism (3) in yeast and salicylic acidmediated defense response in Arabidopsis (20) Importanceof KIX domain in determination of seed size of rice hasbeen postulated by presence of single nucleotide poly-morphisms (SNPs) differentiating the genotypes of longgrain from that of short grain (21) KIX domain hasrecently been reported from human recQ protein-like 5(RECQL5) helicase as an RNA Pol II-binding domain(Figure 1D) which is in contrast to the regular role ofKIX domains of CBP ARC105 and Gal11p that bind

to ADs of transcription activators (2ndash422) In this casethe KIX domain of RECQL5 interacts with Rpb1 jawdomain of RNA Pol II to repress transcription (22)Thus the proteins that possess KIX domain namelyCBP mediator subunit MED15 and most recentlyhelicase RecQL5 are mainly transcriptional regulatorsand the major role of KIX is to act as a docking site fortranscription factors and for stabilization of proteinndashprotein interactions (1252324) Solution structures ofseveral KIX domains in mouse humans and yeast areavailable in the protein data bank (PDB) both inligand-bound complexes and in the unbound formsAccording to the Pfam database KIX domain alsoshows conservation in the plant kingdom and othergroups such as arthropods and nematodes although thestructure of KIX in these groups is not yet characterizedDespite elucidation of its crucial role in transcription

several gaps remain in our knowledge of the KIX domainspecifically in terms of its ability to recognize and assist infolding of intrinsically disordered proteins its role in sec-ondary structure determination its varying bindingmodes mechanism of allostery high structural plasticityand even some of its cellular functions Furthermore verylittle is known about KIX domain proteins in plantsalthough a recent association analysis of SNP loci in

Figure 1 Cartoon depicting the varied interactions and overall roles played by KIX in (A) the general transcriptional apparatus (B) regulation viaCBP and its coactivators (C) the mediator complex and (D) the RecQL5 helicase

Nucleic Acids Research 2014 Vol 42 No 4 2113


23 rice genotypes identified three non-synonymous SNPloci in plant KIX domains that showed strong associationwith important agronomic traits like grain size (21) Thusit is particularly relevant to review the KIX domain acrossvarious life forms and integrate the contributions of recentstudies in context of earlier reports to gain insights intothe precise nature of its interactions and functions and itspotential for combinatorial targeting and allosteric regu-lation in diverse processes such as cell cycle multi-drugresistance and fatty acid metabolism In this review weexplore the sequence structure binding modes dockingplasticity and mechanistic aspects of KIX and its role information and stabilization of vital multi-proteincomplexes during transcriptional activation We hopethat the present review will open up multiple newavenues for further research on this important domain

DISCOVERY OF KIX AND SIGNIFICANCE OF THECREBndashCBP COMPLEX

The KIX was discovered as a stretch of 90 residues inmouse CBP that behaved as an inducible high affinitydocking site for phosphorylated Ser-133 of CREB KIDdomain resulting in CREBndashCBP complex formation (1)Although many other KIX domains have beencharacterized since then along with their transcriptionfactors this first reported interaction between CBP KIXand CREB KID of mouse resulting in the CREBndashCBPcomplex remains the most extensively studied and wellunderstood in molecular terms CREB is a well-knowntranscription factor which in response to cAMP regulatescellular gene expression by binding to cAMP responseelement (CRE) CRE (50-TGACGTCA-30) is a conservedregion present in promoters of many genes and isrecognized by the CREB dimer (25) As shown inFigure 1A protein kinases activate CREB byphosphorylating a particular conserved serine residue inits KID domain when external stimuli such as calciumgrowth factors protein hormones light etc triggers sig-naling cascades (126ndash28) As can be seen in this figureCBPndashCREB complexation occurs via KIDndashKIX inter-action and this leads to interaction of CBP with RNAPol II at the cysteine and histidine rich domain 3 (CH3)of CBP domain site thereby activating transcription (29)There have been suggestions that CBP interacts with tran-scription factor II B as well as with TATA-bindingprotein a component of transcription factor II D(3031) Although CBP and p300 are present in limitedamount in nucleus they are known to associate withlarge variety of proteins most of which are transcriptionfactors such as CREB STAT1 p53 c-Myb etc as shownin Figure 1BCBPp300 proteins perform crucial roles invarious cellular events because CBPp300 is a commontarget for many transcription factors which are involvedin cell growth differentiation proliferation repair andapoptosis (32ndash34) Furthermore CBP is a target ofmany viral oncoproteins such as HTLV-1 TAX adeno-virus early region 1A (E1A) and simian vacuolating virus40 T(SV40-T) antigen (Figure 1B) E1A is an antagonist top300CBP-associated factor (pCAF) which binds CBP

p300 during cell cycle The CBPp300-pCAF complexcan arrest cell cycle progression and E1A targets CBPp300 and drives cell into S phase to cause tumors (35)Thus CBP not only acts as a molecular bridge that stabil-izes the transcription apparatus but also tightly regulatesthe expression of specific genes when recruited by differenttranscription factors It does so by using distinct domainswithin its sequence namely receptor interaction domainKIX and transcription adaptor putative zinc finger all ofwhich enable it to interact with such a wide range of tran-scription factors CBPp300 also has an intrinsic histoneacetyltransferase (HAT) activity that allows it to activatetranscription through chromatin remodeling and alter-ation of DNA accessibility (Figure 1B) apart fromplaying a critical role in nuclear receptor (NR) signaling(3637)

ROLE OF KIX IN COMPLEXATION ANDSTRUCTURAL STABILIZATION

The intimate physical binding of CREB and CBP requirescertain domains from both sides This is where the role ofKIX domain comes into play with KIX being the specificsite in CBP that binds to phosphorylated KID (pKID)region of CREB as shown in Figure 1AThe CBP-KIXdomain sequence comprising 90 amino acids is highlyconserved in yeast and animals Figure 2A depicts thealignment of selected CBP-KIX domains At the time ofthe discovery of mouse CBP KIX domain circular dichro-ism studies predicted it to be rich in alpha helices andhydrophobic interactions were found to play a dominatingrole in complex formation (1) This was confirmed by thefirst nuclear magnetic resonance (NMR) structure of KIXrevealing it to be a small bundle of three mutually inter-acting alpha helices and two short 310 helices joined bysmall loops enclosing a hydrophobic region as shown inFigure 2B (38) As per convention the alpha helices ofKIX domain are denoted as a1 a2 and a3 while 310helices as G1 and G2 As can be seen in Figure 2Bhelices a1 and a3 helices are coplanar and also approxi-mately parallel to one another at an angle of 17whereas helices a1 and a2 are at an angle of 55 toone another Residues of a1 and a2 share contactsthroughout their lengths whereas residue contactsbetween a2 and a3 are very limited The first 310 helixG1 lies near N terminus of KIX domain whereas thesecond 310 helix G2 lies in the linker region between a1and a2 which is more flexible and longer than linkerregion between a2 and a3 (Figure 2B)

KIX plays a dominant role in secondary structure for-mation of pKID because the isolated KID region is largelydisordered and devoid of significant preformed helicityAfter binding to KIX the pKID adopts an ordered struc-ture of two distinct mutually perpendicular helices(namely aA and aB) linked by a turn that wrap arounda3 of KIX in a shallow hydrophobic pocket on KIXsurface (Figure 2B) This pKIDndashKIX interaction isstabilized both by hydrophobic and electrostatic inter-actions the former betweenaB of KID and the KIXhydrophobic pocket in the region of helices a1 and a3

2114 Nucleic Acids Research 2014 Vol 42 No 4


and the latter between specific charged residues on eachdomain It is interesting that both aA and aB of pKID aremainly stabilized by KIX domainrsquos interacting surface andintra pKID interactions do not play any important role inits stabilization (38) The core globular KIX domain isstabilized by a cationndashpi interaction between aromaticring of Tyr-640 and positively charged guanidiniumgroup of a highly conserved Arg-600 (Figure 2B) It hasbeen shown that replacement of this arginine by glutaminecan impair KIX binding even though Arg-600 does notmake direct contacts with KID of CREB (1)Interestingly since the cationndashpi interaction is consideredcrucial for specifying the folded KIX structure (Figure 3)disruption of this interaction by arginine methylation mayserve as a regulatory mechanism of inhibition of KIXfunction and is discussed in the penultimate section

Apart from the cationndashpi interaction residues that liebeyond the globular KIX core have been shown to influ-ence its secondary structure and stability In particular astretch of six residues (KRRSRL) observed at the Cterminus of an extended a3 helix in some KIX domains(see alignment in Figure 2A) has been shown to be im-portant for stabilization Since these residues are not partof the hydrophobic core of KIX they have been proposedto contribute indirectly to helix stabilization via electro-static interactions with the helix dipole based on chemicalshift perturbation mapping (39)

The phosphorylation event is also critical for stability ofthe pKIDndashKIX complex since the phosphorylated serineresidue in CREB is directly involved in the binding event

with CBPp300 In human CREB phosphorylation site ofKID is Ser-118 whereas it is 133 in rat and mouse CREBpSer-133 is essential for complex formation as its mutationdiminishes KIDndashKIX affinity This residue is present inthe same helix (aB) of KID that makes direct contactswith the KIX domain The phosphate group of pSer-133forms a hydrogen bond with Tyr-658 and a salt bridgewith Lys-662 of KIX domain (3840) This coupledbinding and folding event introduces a large entropybarrier which is compensated by the electrostatic contri-bution of the phosphate group to the binding free energythereby making this phosphorylation event a criticalthermodynamic switch for the inducible formation of theCREBndashCBP complex (41) Furthermore such foldingafter binding facilitates selectivity and speed of inter-action and also lets the CREB exclusively interact withCBP among other coactivators (1842) Two critical pointsregarding KIX may be noted here first KIX binding isnot always phosphorylation dependent as in the case ofc-Myb an oncoprotein essential for cellular proliferationsof immature hematopoietic cells that constitutively inter-acts with CBP at its KIX domain (43) Since interactionbetween c-Myb and KIX is not phosphorylation depend-ent contributions to hydrogen bond and salt bridgeformation from Tyr-658 and Lys-662 of KIX were notobserved Interestingly this may account in large partfor the 50-folds lower affinity of interaction betweenc-Myb and CBP as compared to that between CREB-KID and CBP detected by fluorescence anisotropy andITC experiments (4445) Second not all protein kinases

Figure 2 The CBP-KIX domain sequence and structure (A) Alignment of selected KIX domain sequences showing high conservation of allsecondary structural elements (B) Opposing views of the KIX domain structure (gray) in complex with pKID (purple) depicting the helicalnature of both proteins in cartoon representation (PDB ID 2LXT) Secondary structural elements are marked in gray and purple for KIX andpKID respectively The highly hydrophobic surface that binds KID is shown in shaded red representation The cationndashpi interaction between thearomatic ring of Tyr-640 and guanidinium group of Arg-300 that stabilizes the KIX domain structure is depicted in yellow



can result in KIDndashKIX complexation making this inter-action stimulus specific For example Ca2+calmodulin-dependent protein kinase II is incapable of stimulatingCREB activity although it can phosphorylate Ser-133 ofKID This is because certain protein kinases additionallyphosphorylate Ser-142 in aB of pKID and phosphoryl-ation at Ser-142 has an inhibitory effect on complexation(46) Phosphorylated Ser-142 causes juxtaposition of abulky group near Tyr-650 which in turn weakens theinteraction between aB of pKID and hydrophobicgroove of KIX thereby blocking formation of KIXndashKID and further implications are discussed in a latersection (4046)

CONSERVATION OF KIX IN THE MEDIATORSUBUNIT MED15

Apart from CBPp300 homologous KIX domains havealso been identified and characterized in MED15 asubunit of the mediator complex found in all eukaryotesFunctional and structural studies have revealed significantsimilarity between KIX domains of MED15 proteins ofdiverse organisms at sequence structure and functionallevels The KIX domain in MED15 (also called ARC105)a subunit in the mediatorARC complex of humans wasoriginally identified as a SREBP1a-binding domain (512)SREBP proteins belong to a family of transcriptionfactors involved in cholesterol and lipid homeostasis inmammals and they bind to a region on ARC105 withsignificant sequence and structural similarity with theCBP KIX domain (4) SREBP1a has been shown to act

as a common substrate for both CBP KIX and ARC105KIX although it is not the same with some other tran-scriptional activators such as CREB and c-Myb which donot bind significantly with ARC105 KIX domainimplying an unexpected specificity among KIX domains(4) This specificity can potentially be ascribed to theabsence of residues Tyr-658 and Lys-662 in MED15since both are present in a3 of CBP KIX and have beenimplicated in the interaction with protein kinaseA-phosphorylated CREB and c-Myb ADs (4045) Thusit is the residues in helix a3 of KIX that provide activatorbinding specificity and the lack of conservation of theseresidues in ARC105 KIX is the reason why CREB and c-Myb are unable to effectively bind to ARC105 KIXdomain (4)

Gal11pMED15 is a subunit of mediator complex inyeast that also harbors KIX domain initially discoveredas Pdr1p-binding domain with a specific role in xeno-biotic-dependent regulation of multidrug resistance inyeast and pathogenic fungi (2) Family members ofPdr13p in Saccharomyces cerevisiae and Candidaglabrata recognize the presence of xenobiotics by directlybinding to them and then binding to KIX domain ofGal11p (247) KIX domain of Gal11pMED15 interactswith ADs of several transcription factors such as Gal4pPdr1Pdr3p Oaf1p Gcn4p etc (231314) NMR studiesindicated substantial structural similarity of this domainto human ARC105MED15 KIX domain and there wasevidence of a similar hydrophobic core in both proteinsforming the binding interface for various transcriptionfactors This resemblance to human MED15 KIXdomain was found to extend in functional terms as wellsince Gal11p KIX domain could interact with humanSREBP1a but not with CREB and c-Myb activators(2) Gal11p KIX domain also plays an important role inthe regulation of lipid metabolism in fungi where fattyacids such as oleic acid (OA) lauric acid (LA) myristicacid (MA) and palmitic acid (PA) are able to directly bindto transcription factor Oaf1p and activate it to induceexpression of Fatty Acid oxidation 2 (FOX2) and FOX3genes that are involved in metabolism (3) Here too theGal11p KIX domain was found to directly interact withOaf1p at a hydrophobic groove formed between three ahelices (with a2 participating in the majority of inter-actions) resulting in recruitment of the transcriptionalmachinery This activation however was specific toOaf1 interaction with OA LA PA and MA but notstearic acid for instance or other molecules like peroxi-some proliferators or non-steroidal anti-inflammatorydrugs that could bind to Oaf1p but could not stimulateOaf1ndashKIX interaction or consequently transcriptional ac-tivation suggesting that specific conformational changesare facilitated in Oaf1 by cognate ligands that stimulateKIXndashOaf1 interaction (3)

KIX DOMAIN IN RECQL5 AND ITS ROLE INSTRUCTURAL MIMICRY

The most recent KIX domain has been reportedfrom RecQL5 one of the five human DNA helicases

Figure 3 3D view of the ternary complex of KIX domain (PDB ID2LXT) depicting two distinct spatially separated binding surfacesEach binding surface is colored by hydrophobicity (graded scale ofred) and lies on opposite sides of the KIX domain represented incartoon with rainbow colors The two binding partners MLL(magenta) and KID (purple) are also shown at their respective siteson KIX The TAD motifs in both ligands are depicted as lines Thecationndashpi interaction is shown in yellow surface representation



employed for opening the replication fork and this reportled to the discovery of a vital function played by KIXdomain in DNA repair during replication (23) TheRecQL5 helicase is known to be essential for reducingtumor risk and genome stabilization during transcriptionreplication recombination and DNA repair It is the onlyhuman RecQ helicase that can interact directly with RNAPol II (48) Computational modeling revealed a KIX likeregion in RecQL5 that resembled the KIX domains inCBP p300 and MED15 in terms of sequence and thecommon three-helix bundle structure with a conservedhydrophobic core as well Mutations in this region ofRecQL5 led to disruption of interaction betweenRecQL5 and RNA Pol II which in turn led to inhibitionof RNA Pol II during initiation and elongation events oftranscription suggesting a crucial and specific role of KIXin transcriptional regulation as shown in Figure 1D (23)Later it was shown that RecQL5 helicase could recognizedouble strand breaks during DNA damage and stallfurther transcription by interacting with and inhibitingRNA Pol II through its KIX domain thereby enablingcell survival during DNA damage (49) A very recentstructural investigation into the mechanistic basis of thisinhibition revealed a surprising structural overlap betweenRecQL5 KIX domain and domain II of elongation factorTFIIS suggesting a dual mode of transcriptional repres-sion wherein the KIX domain enables RecQL5 to struc-turally mimic the Pol IIndashTFIIS interaction therebyoccluding the TFIIS-binding site on Pol II which inturn prevents transcription from resuming The RecQL5KIX domain and domain II of yeast and metazoan TFIISorthologs show sequence conservation and the authorsemployed three-dimensional (3D) crystallography to illus-trate structural mimicry wherein the KIX domain assumesa TFIIS like structure and binds to Pol II in a region thatoverlaps with its known binding site for TFIIS (22)A large number of cellular signals necessitate short-termsuspension of the transcriptional machinery and thismechanism of repression via blocking of TFIIS normalfunction might operate as a general mode of responseand regulation in the cell

KIX PROMISCUITY MULTIPLE BINDING SITES

For several years post-discovery the KIX domain wasassumed to have a single binding site despite observationsthat certain viral transcriptional activators like HTLV1-Tax and p53 could interact with the CREB-bound formof KIX (50ndash52) These interactions were believed to bemutually exclusive and the initial solution structures ofKIX in complex with KID and c-Myb also supportedthe single-site notion since both KID and c-Myb wereobserved to share a common binding site despite the factthat KID and c-Myb are quite distinct from each other incritical aspects of KIX mediated interaction These includethe facts that (i) the KIX-binding region of c-Myb has noobvious sequence similarity with the equivalent region onKID (ii) c-Myb binds CBP in a phosphorylation inde-pendent manner unlike KID (45) and (iii) the affinity ofKIX for pKID is 20-fold higher than that for c-Myb (53)

Furthermore initial CD- and NMR-based studies hadsuggested that despite both being intrinsically unstruc-tured or disordered proteins (IDPs) c-Myb is already inpartial (25) helical conformation in unbound stateunlike KID which has only 1 helicity when unbound(4445)In 2002 two independent groups working with MLL

and c-Jun coactivators discovered a secondary bindingsite in KIX through NMR chemical shift mapping a sitethat was remote and distinct from the one employed byKIDc-Myb (754) MLL is crucial for production ofnormal blood cells and is a homolog of the Drosophilatrithorax (Trx) protein involved in maintenance ofhomeobox (Hox) gene expression during early embryo-genesis while c-Jun is a human transcriptional coactivatorand a mediator of proteinndashprotein interactions involvedin diverse cellular process including differentiation andapoptosis The first discovery was the outcome of an in-vestigation into whether MLL and c-Myb proteins werecompeting for CBP binding or instead interacting co-operatively with CBP based on the facts that (i) MLLis a binding partner for both c-Myb and pKID (ii) bothMLL and c-Myb were known to express at the same timeduring development of hematopoietic precursor cells (iii)both play a pivotal role in blood cell proliferation andfinally (iv) both could bind KIX domain (755ndash58) Gotoet al (7) found evidence for cooperativity as occupationof one binding site on KIX decreased the entropic penaltyassociated with the second binding event and whilelocalizing the MLL-binding site on KIX spectral differ-ences indicated binding to a remote and new siteSimilarly in case of c-Jun the chemical shift perturbationsarising from conformational changes of aromatic ringsinduced by pJun were found to be smaller than thoseinduced by pKID revealing higher aromaticity invicinity of the pKIDndashKIX complex (Tyr-640 Tyr-649Trp-591 and Tyr-658 of KIX and Tyr-134 of pKID) incontrast to a single aromatic side chain Tyr-631 of KIX inthe vicinity of the c-Jun-binding site revealing two distinctinteraction surfaces (54) These initial reports showed thatboth ligands (c-Myb and c-JunMLL) could bind simul-taneously and cooperatively to the CBP KIX domain intheir corresponding hydrophobic grooves making ternarycomplexes providing a role for KIX in transcriptionalsynergy Figure 3 depicts the ternary complex of KIXwith KID and MLL and it can be seen that the KIDc-Myb site consists of a hydrophobic groove formed bya1 and a3 helices of KIX whereas the second spatiallyseparated MLL site is on the opposite surface of KIXand consists of a groove formed at the interface betweenG2 a1 and a3 helices of KIX domain Later studies firmlyestablished the presence of two independent bindingsurfaces on CBP KIX domain designated as c-Myb andMLL sites (5559) MLL site has since been shown to bindmany other proteins including c-Jun HTLV-1oncoprotein TAX and HIV-1 transactivator of transcrip-tion (TAT) (1760) Table 1 provides a list of various tran-scription factors that bind to these two binding sitesKIX thus provides two distinct binding surfaces for

interactions with transcriptional ADs and it is interestingto note that both binding sites interact with peptides that



contain the characteristic amphipathic j-x-x-j-j motif(j bulky hydrophobic residue and x any residue) Thisconserved TAD motif does not provide any binding sitespecificity to transcription factors as it is observed in theamphipathic a helices of transactivation domains of manytranscription factors such as CREB-KID MLL c-Junc-Myb p53 E2A and Forkhead box class O 3a(FOXO3a) (64) This similarity of TAD motif recognitionbetween the two sites extends in terms of function as welland combined with binding cooperativity it providesnew insights into the combinatorial recruitment of CBPby multiple ADs The simultaneous binding patterncombined with limiting quantities of CBP in cells suggestKIX to be a potential link between diverse transcriptionfactors contributing significantly to the synergy of geneexpression and resulting in enhanced recruitment ofCBP to target promotersInterestingly as mentioned in Table 1 some transcrip-

tion factors like p53 and FOXO3a have the ability to bindboth the sites of KIX domain by means of two tandemADs within their sequences and both transactivationdomains can simultaneously acquire the KID and MLLsites of KIX (6162) FOXO3a is very important since itactivates transcription of genes involved in cell differenti-ation DNA repair apoptosis and stress response It is anintrinsically disordered protein with three conservedregions (CR1ndashCR3) of which the CR3 and the C-terminal segment of conserved region 2 (CR2C) are bothTADs that can each occupy both the c-Myb and MLLsites through multiple promiscuous and dynamic inter-actions resulting in two stable conformations Similarlythe tumor suppressor p53 which causes cell arrest andapoptosis during DNA damage also has two transactiva-tion domains AD1 and AD2 and KIX acts as a sensor ofphosphorylation state of these two TADs therebymediating cell cycle regulation In contrast to FOXO3athe tandem p53 TADs can both bind in two differentorientations to each of the two KIX sites thereby support-ing eight distinct binding modes Studies have shown thatunlike other transcription factors AD1 and AD2 transac-tivation domains of p53 have similar affinity to bind bothpKIDc-Myb and MLL sites of KIX domain while AD1binds preferentially to MLL site (65) The binding is alsophosphorylation dependent like that of pKID althoughphosphorylated AD1 binds preferentially to MLL siteof KIX domain (61) Overall the MLL site exhibits

remarkable plasticity being able to accommodate avariety of peptides in various orientations without anysubstantial structural rearrangement (62)

It is noteworthy that despite its small size and a rela-tively simple helical bundle fold KIX has two distinctsites both with the ability to bind different IDP systemsFor a long time it was not possible to exactly unveil themechanism of this binding since dissecting the mechanismof recognition between IDPs and their physiologicalpartners can be extremely complex and requires extensivekinetic characterization However such a study hasrecently been conducted despite the inherent complexityof the process resulting in the establishment of a uniquelsquobinding-precedes-foldingrsquo mechanism for KIX binding toc-Myb The authors clearly demonstrate that the transac-tivation domain of c-Myb recognizes KIX domain in itsunstructured conformation and binds to it via a salt bridgebetween Arg-61 and Glu-306 subsequently followed by arapid folding step driven by h-bond interactions at theopposite side of the interface (66) It is presumable thatthe mechanism of interaction between KIX and otherIDPs may display conserved features of a similar kind

THE ROLE OF KIX IN ALLOSTERY

KIX plays a crucial role from a regulatory perspectivealso since it has the capability of binding the same tran-scription factors in structurally distinct modes in additionto the capacity of binding two different transcriptionfactors at the same time By virtue of spatially separatedbinding sites KIX can directly mediate interactionsbetween bound transcription factors acting as a bridgebetween p53 and TAX or CREB and TAT The previouslydiscussed synergistic effect between the two KIX-bindingsites ie effect on binding of a transcription factor to onesite of CBP-KIX domain in the presence of another tran-scription factor already bound to the second site alsopromotes allosteric regulation Table 2 provides a list ofinteractions that have been observed and reported tobe involved in cooperative binding in KIX to dateInvestigations into thermodynamics of this cooperativityusing isothermal titration calorimetry have shown thatsecond binding event is accompanied by a 2-fold enhance-ment in binding affinity (7) This holds true in both direc-tions ie binding of MLL to KIX enhances the binding ofc-Myb at pKID site of KIX domain by 2-fold higher

Table 1 Details of known binding partners for the two different binding surfaces on the KIX domains of CBP

Sno Interacting protein Interacting domain Interaction site on CBPp300 KIX Phosphorylation dependence Reference

1 CREB KID c-Myb site + (38)2 c-Myb TAD c-Myb site (45)3 p53 TAD1 and TAD2 MLL c-Myb site + (61)4 MLL TAD MLL (59)5 FOXO3a CR2C and CR3 MLL c-Myb site + (62)6 TAX AD MLL site (60)7 c-Jun AD MLL site (54)8 BRCA1 BRCT domain c-Myb site (63)9 SREBP AD C-terminal of a3 (4)10 HIV-1 TAT AD MLL (17)



affinity and conversely binding of c-Myb to KIXenhances the MLL binding affinity to KIX whereuponthe ternary complex activates expression of various down-stream genes (75967) The same holds true for the ternarycomplex involving pKID instead of c-Myb although themechanism of stabilization is different suggesting a rolefor the KIX-binding sites as allosteric sites that undergoconformational changes leading to increased affinity withthe second ligand These conformational changes can beof two kinds First as observed for the ternary complex ofKIXc-MybMLL the favorable enthalpy associated withbinding of the second ligand to KIX domain decreaseswhen the complementary ligand occupies the firstbinding site implying an increase in the number or stabil-ity of electrostatic or polar interactions in the ternarycomplex Alternatively the observed cooperativity canarise on account of an increase in the favorable entropyupon ternary complex formation implying a contributionof hydrophobic interactions as observed for the ternarycomplex of KIXpKIDMLL (7) Other proteins likeHTLV-1 HTLV-1 basic leucine zipper factor (HBZ) andCREB-KID make ternary complexes when bound to KIXdomain and both phosphorylated CREB and HBZinteract with KIX domain of CBPp300 simultaneouslyat different binding sites and activate HTLV1 transcrip-tion (64) TAX also uses the same site as that of HBZ tobind KIX domain of CBP and to form ternary complexwith CREB (6068)

The allosteric binding sites further endow KIX withthe unique ability to communicate between these remoteand isolated regions on its surface providing a potentialmechanism to modulate transcriptional activity throughconformational transitions between bound and unboundstates Therefore KIX has the potential to act as apowerful regulator of gene transcription through its allo-steric binding sites and plays a key role in transcriptionalsynergy Biophysical characterization and quantification

of allosteric communication pathways is intimatelylinked to protein dynamics and subtle rotameric re-adjust-ments at the atomic level Although the high plasticity ofKIX essentially adds to its function enabling recognitionof a diverse array of activator sequences it also rendersthis domain intractable to crystallographic characteriza-tion and thus NMR methods have mostly been used tounderstand the intricate details of conformationaldynamics in KIX Bruschweiler et al (6769) used NMRrelaxation dispersion techniques and structural data todecipher the mechanism of allosteric transition in theMLLKIXpKIDc-Myb ternary complex They moni-tored the conformational rearrangements within theKIX domain between the unbound (KIX alone) binary(KIXMLL) and ternary (KIXMLLpKID) states andidentified a non covalent contiguous network of residuesconnecting the two remote sites through which KIX com-municates information about target presence at MLL siteto the allosteric c-MybpKID site Figure 4 depicts thishydrophobic network comprising of aliphatic side chainsin the ternary KIX complex (PDB ID 2LXT) and it canclearly be seen that it connects the two spatially separatebinding sites This network was found to be conservedacross yeast and mammalian KIX domains and wasproposed to form a defined pathway for transmission ofallosteric information by means of chemical shiftsresidual anisotropic interactions and rotameric changes(67) The 90 residue spanning KIX domain has a non-uniform distribution of long chained and cyclic aminoacids in its hydrophobic core the interior being formedmainly by non aromatic residues as compared to thesurface which in contrast is enriched for aromaticresidues Further the hydrophobic core displays greater

Figure 4 The allosteric network of contiguous hydrophobic side chains(atom colored spheres) within KIX domain (PDB ID 2LXT) connect-ing the two remote binding sites Each binding site and the cationndashpibond are depicted in mesh representation The two ligands and theirrespective TADs are shown in similar colors at their cognate KIX-binding surfaces

Table 2 Examples of cooperative binding in KIX

Cooperative binding Cooperative effect

MLL-KIX-c-Myb Binding of MLL to KIX increases the affinityof KIX for c-Myb and thus facilitates normalblood cell development

HBZ-KIX-c-Myb HBZ interaction with KIX causes disruptionof cellular transcription by acting ascompetitive inhibitor to MLL It enhancesthe binding of c-Myb-AD to KIX and thusleads to continued proliferation of leukemiacells

HBZ-KIX-pKID HBZ interaction with KIX causes enhancedbinding of pKID to KIX and unregulatedcell proliferation causing T-cell leukemia

MLL-KIX-pKID MLLndashKIX interaction promotes favorableconformational rearrangements andenhanced binding of pKID to KIX thusincreased activation of CREB responsivegenes

TAX-KIX-pKID TAX interaction with KIX causes activationof HTLV-1 mediated genes thus causingT-cell leukemia



conservation of aliphatic residues than for aromaticresidues and it has been suggested that the observed evo-lutionary conservation of these aliphatic residues (namelyLeu-603 Leu-607 Ile-611 Leu-628 Leu-653 Ile-657 andIle-660) is due to the fact that they constitute the allostericregulatory network described above (69) Moleculardynamics (MD) studies further indicated that binding ofMLL to KIX produced a conformational redistributionwithin the protein favoring binding of the secondremote ligand (55) The a1ndasha2 linker region and G2helix of KIX have both been shown to play a key role inthe conformational control (5559) A detailed structuralcomparison of the binary and ternary KIX complexesshowed that rigidity of KIX protein backbone was notsignificantly altered between the two states but themethyl group nuclear overhauser effect patterns revealeda measurable rearrangement of the hydrophobic core andthe residues involved in this small but significant repack-ing of the core were identified to be the same as thosepreviously identified as part of the KIX allostericnetwork listed above (6769) Interestingly this allostericcommunication did not appear to be bi-directional in theinitial studies ie communication about presence of sub-strate at the c-MybpKID site to the MLL site could notbe detected on account of standard MD timescale limita-tions whereby only phenomena that take place on thescale of microseconds can be accessed while othersremain lsquoinvisiblersquo This limitation was recently overcomeby a detailed MD study of allosteric communication inKIX employing enhanced sampling methods thatallowed confirmation and visualization of the short-livedor invisible excited state of the ternary complex ofMLLKIXpKID (70) The authors confirmed the experi-mental results by Bruschweiler et al (6769) providing amechanistic model for the conformational transitionswithin the allosteric network showing that the structureof the invisible state is similar to that of the MLLKIXmoiety in the ternary complex Very recently the firstcrystal structure of KIX domain has been reported byusing a covalently linked small molecule ligand throughtethering methods (71) This structure was then used toperform MD simulations for identification of specific side-chain motions that remodel KIX binding sites formediating partner recognition through conformationallydynamic interfaces (71)

KIX INHIBITION AND TRANSCRIPTIONALSWITCHING

Unlike some coactivators that are specific to a singlefamily of transcription factors CBPp300 binds to aplethora of targets and initiates gene activation at variedpromoter sites integrating several signal transductionpathways For example CBPp300 also functions as acoactivator of NRs and thus despite being present insmall amount and limiting concentration CBP is at theepicenter of transduction events that lead to specific geneexpression in the cell Thus there is huge competition foracquiring it thereby entailing and even necessitating itsprecise regulation In this context again the KIX

domain plays a central role for discriminating betweenvarious cellular signals by virtue of its binding promiscuityand allostery The initial implications of two independentbinding surfaces on KIX included selective use of thesesites by transcription factors as both a mechanism to dis-criminate between inducible and constitutive activatorsand a mechanism for combinatorial recruitment of CBPby diverse TADs (54) In 2001 even before the secondKIX-binding site and its synergistic regulatory effectswere noticed the existence of a unique KIX based tran-scriptional lsquoswitchrsquo was uncovered that could toggle CBPfrom CREB-regulated to NR-regulated gene expressionwherein it was demonstrated that arginine specific methy-lation of CBPp300 inhibits cAMP-dependent gene regu-lation (72) The authors found that CBP could smoothlyswitch partners by recruiting coactivator associatedarginine methyltransferase 1 (CARM1) a type I proteinarginine methyltransferase (PRMT) based on evidencethat methylation could destabilize the structure of KIXdomain

Methylation is a common post-translational modifica-tion occurring mainly on arginine and lysine residues andPRMTs (of which 10 have been identified so far inmammals) methylate various proteins including histoneand non histones (73) PRMTs have conventionally beenclassified into two classes based on their nature of argininemethylation with type-I forming monomethylarginineand asymmetric dimethylarginine in contrast to type IIPRMTs that form monomethylarginine and symmetricdimethylarginine (74ndash76) CARM1 is a type I PRMTand it is known to methylate histone H3 CBPp300steroid receptor coactivator-3 and many other proteins(77ndash79)

In case of CBPp300 the major site of methylation byCARM1 was shown to be Arg-600 in the KIX domain aresidue discussed earlier in this review as being criticalfor KIX stabilization Modification of the Arg-600 wasbelieved to destabilize the KIX domain by disrupting aburied cationndashpi interaction between positively chargedguanidinium group of this residue and the aromatic ringof Tyr-640 an interaction that is critical for specifyingthe unique cooperatively folded KIX structure as shownin Figure 2 (80) However it is noteworthy that in aparallel study and in multiple other studies (both in vitroand in vivo) carried out by independent groups followingthe initial observation of KIX methylation at Arg-600 aconsensus could not be reached for the exact region ofmethylation on CBP and it was argued that methylationby CARM1 actually occurs at an unstructured C-terminalextension of the CBP KIX domain (region 568ndash828)rather than its core region (638182) Predominant methy-lation at this unstructured segment of KIX washypothesized to affect the stability of CREBndashCBP inter-action leading to diminished recognition (63) Despite thecontroversy of exact methylation site it is quite clear thatprotein methylation at arginine residues within or nearCBP-KIX domain is an important mechanism for signaltransduction and CARM1 is critical for co-activatorsynergy in transcription Thus methylation of theCBP-KIX region (core or extended C terminal) byCARM1 can disrupt the CBPndashCREB interaction and



block transcription of CREB dependent genes which inturn may render the limited CBP pool free and availablefor NR dependent gene expression (728182) In thismanner KIX methylation effectively switches CBP tomediate NR-dependent gene transcription exclusivelyand this switch is especially significant in view of thelarge number of cellular events that CREB is associatedwith including glucose homeostasis immune responseand memory retention Interestingly the molecularswitch concept opened up a completely new paradigmfor interfering with gene regulation for medical purposesand led to new avenues of research concerning the revers-ibility of methylation based CREBndashCBP inactivationregulation of CARM1 itself as well as the connectionbetween methylation and ubiquitination of CBP ifany (83)

KIX DOMAIN AS A PREFERRED TARGET FORDRUG DEVELOPMENT

Small molecules that can interfere with the process oftranscription can be very useful as therapeutic or co-therapeutic drugs Moreover such molecules can also beused as an important research tool in the study of biolo-gical processes and mechanisms In the last decade severalsmall molecules have been found that can directly targetprotein-protein interactions involved in the process oftranscription (84) Being an important domain in the tran-scriptional regulators like p300CBP and MED15 KIXhas been a preferred choice for researchers to identify ordesign small molecules with agonistic or antagonisticeffect on it Initially protein grafting was used in combin-ation with molecular evolution by phage display toidentify phosphorylated miniature protein ligands thatcould bind to the CBP KIX with high nanomolaraffinity (85) Within a year in an NMR-based screeninga couple of compounds namely palmoic acid (KG-122)and naphthol AS-E-phosphate (KG-501) were foundthat could bind to CBP KIX domain (86) KG-122binds to the MLL site of CBP KIX but it alters the sidechain conformation of Lys-662 which forms an ion pairwith Ser-133 phosphate of CREB On the other handKG-501 docks in the c-Myb site of CBP KIX in thesame manner as the KID domain Thus upon bindingto the CBP KIX domain both these compounds disturbthe amino acids that are required for KIDndashKIX inter-action and so function as the inhibitors of CREBndashCBPinteraction attenuating the induction of cAMP responsivegenes (86) Later by using Renilla luciferase complemen-tation assay naphthol AS-E (unphosphorylated form ofKG-501) was identified as the first high-affinity smallmolecule ligand of CBP-KIX and a cell-permeablestrong inhibitor of KIXndashKID interaction (87) The struc-turendashactivity relationship studies of naphthol AS-E bymodifying the appendant phenyl ring revealed preferencefor a small electron-withdrawing group at the paraposition to inhibit KIXndashKID interaction (88) CREBnot only regulates hepatic gluconeogenesis during fastingand in diabetes (8990) but is also found to be over-expressed in different types of cancers (8891) Thus

small molecules such as naphthol AS-E that can act asinhibitors of KIXndashKID interaction are suitable structuraltemplates for the development of potential drugs to benefitcancer and diabetic patientsAs mentioned previously KIX domain of Gal11p

MED15 is targeted by AD of Pdr1p a zinc cluster tran-scription factor and its orthologs in yeast and fungi forup-regulation of multidrug resistance (2) Pathogenicfungi especially Candida species including C glabrataaccount for up to 9 of all blood stream infections withcrude mortality rate of 40 (92) So in an attempt topropose novel antifungal therapy as in the case ofCREBPndashCBP interaction using a high thorough-putfluorescence polarization assay few small molecular com-pounds like N16 K20 and I17 have been identified thatcan inhibit interaction between Pdr1p-AD and Gal11Ap-KIX of C glabrata (93) Though these studies are still ininfancy such molecules have the potential to be modifiedfor therapeutic or co-therapeutic purpose to check fungalinfections in immunocompromised patients or patientsundergoing chemotherapy

KIX IN PLANTS

As of date KIX domains have not been characterizedfrom plants although sequence data have shownevidence for existence of both CBP-like proteins andMED15 subunit of mediator complex in various speciesof plants (2194ndash96) However research in this area is fastgaining pace with availability of an increasing amount ofgenomic and transcriptomic data along with mutationalstudies from diverse plant species enabling reliable predict-ive analyses Already computational studies and proteinfamily databases like the InterPro have revealed a conser-vation of CBP KIX domain sequence as well as predictedsecondary structure in plants such as Oryza sativa Populustrichocarpa Medicago truncatula Glycine max andseveral others and these proteins are being identified asgene families in the plant kingdom (httpwwwebiacukinterpro) Initial reports suggested that KIX domain maynot be conserved in Arabidopsis thaliana but later studiesusing profile based search tools established the presence ofat least five KIX-like domains in proteins of this plantincluding three HATs namely HAT of CBP family 1(HAC1) HAC5 and HAC12 (949798) Examination ofthe domain architectures of these proteins revealed sub-stantial diversification from the animal sequences mostnotably the absence of bromodomains suggesting evolu-tionary plasticity and potential modification of functionalthough critical interacting residues were conserved(2194) Mutational studies on plant KIX containingproteins have revealed multiple developmental and pheno-typic defects and transcriptome analyses have suggestedimportant roles for these domains in development(leaf maturation and floral transition) and stress responses(abiotic) in both monocot and dicots (2099100)Further bioinformatics analyses have suggested amarked structural conservation of KIX domains inmultiple paralogs of rice and Arabidopsis CBPHACproteins and MED15 subunits when compared with



mouse CBP yeast Gal11 KIX and human ARC105 KIXdomains (2195) Very recently we performed an hiddenmarkov model based identification of KIX domains fromthese two plants that revealed 11 proteins containing KIXdomains each in rice and Arabidopsis expanding the pre-viously known repertoire Intriguingly in addition to CBPMED15 and RECQL5 KIX domain was found in F-box(FBX) proteins of rice Although functional analysis isyet to be done it seems that KIX domain in such FBXproteins can help them interact with the proteins targetedfor proteasomal degradation We also analyzed theseproteins in terms of gene structure domain architecturephylogenetics comparative transcriptomics and SNPbased association analysis (21) This study providedseveral new insights including existence of proteins withtandem KIX domains novel combinatorial arrangementsand correlation of KIX with seed size related agronomictraits in rice It remains to be seen whether the KIX genefamilies in plants have undergone sub-functionalizationand neo-functionalization and if so it needs to bedetermined how and where the requisite changes haveoccurred in terms of sequence and structure Insummary the plant kingdom offers a unique opportunityand enormous potential for research and exploration ofthe KIX domain

CONCLUSIONS

The KIX domain was discovered in p300CBP as a three-helix bundle important for proteinndashprotein interactionThis domain acts as docking site for ADs of several tran-scription factors including NRs NRs and p300CBPhomologs are not encoded by yeast and fungal genomesbut NR-like ligand-activated transcription factors whichtarget KIX domain of MED15 for their transcriptionalactivities were recently characterized in yeast MED15 iswell-conserved mediator subunit found in all the eukary-otes ranging from fungi to animals and plants Thus tran-scription factors targeting KIX domain seem to bevery ancient mechanism of transcriptional activationpredating divergence of fungi metazoan and plants Thishighlights very interesting evolutionary and functionalaspects of KIX domain which should be addressed bythe scientific community KIX domain is present acrossall the eukaryotic kingdoms ranging from yeast toanimals and plants but the proteins harboring thisdomain may not be conserved in all the eukaryotes Forinstance CBP is not found in fungi and FBX KIX isreported only in three rice FBX proteins but not in FBXproteins of Arabidopsis and metazoans We could not findany RecQL helicase with KIX domain in rice In additionthe proteins that target KIX domains are not conserved inall the eukaryotes NRs that target KIX domains of CBPand MED15 are found in metazoans but not in fungiand plants Zinc cluster transcription factors that targetMED15 KIX are specific to the kingdom of fungi Addingto this complexity is the specificity of KIXndashligand inter-action There are many transcription factors that interactwith MED15 KIX but not with the CBP KIX and viceversa Thus it is necessary to carry out a detailed in depth

structural analysis of KIX domains in all these diverseclass of proteins and their interaction with the cognateligands to help address the structural and functional con-vergence or divergence of this intriguing domain acrossdifferent kingdoms This understanding may also be veryuseful in designing different small molecules to target dif-ferent specific KIX domains for clinical therapeutic oragricultural applications In addition to CBP MED15and RecQL proteins recent discovery of KIX domain inthree rice FBX proteins raises the possibility of presenceof this domain in many other heretofore unknownproteins which may not have direct role in the assemblyof transcriptional machinery Future research programsinvolving structure-dependent computation algorithmmay help in characterization of all the members of KIXdomain containing protein family

ACKNOWLEDGEMENTS

Authors thank Director NIPGR for encouragement andsupport Computational resources provided under theBTISNET grant of DBT-DISC facility are gratefullyacknowledged

FUNDING

Innovative Young Biotechnologist Award (IYBA) grantfrom the Department of Biotechnology (DBT)Government of India awarded to [BTBI120452008 toJKT and BTBI120402005 to GY] Research grant[BTPR14519BRB108692010 from DBT to JKT]Recipient of the junior research fellowship (JRF) of theUniversity Grants Commission Government of India(to AV) Funding for open access charge NationalInstitute of Plant Genome Research

Conflict of interest statement None declared

REFERENCES

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3 ThakurJK ArthanariH YangF ChauKH WagnerG andNaarAM (2009) Mediator subunit Gal11pMED15 is requiredfor fatty acid-dependent gene activation by yeast transcriptionfactor Oaf1p J Biol Chem 284 4422ndash4428

4 YangF VoughtBW SatterleeJS WalkerAK Jim SunZYWattsJL DeBeaumontR SaitoRM HybertsSG YangSet al (2006) An ARCMediator subunit required for SREBPcontrol of cholesterol and lipid homeostasis Nature 442700ndash704

5 NaarAM BeaurangPA RobinsonKM OlinerJDAvizonisD ScheekS ZwickerJ KadonagaJT and TjianR(1998) Chromatin TAFs and a novel multiprotein coactivatorare required for synergistic activation by Sp1 and SREBP-1ain vitro Genes Dev 12 3020ndash3031



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7 GotoNK ZorT Martinez-YamoutM DysonHJ andWrightPE (2002) Cooperativity in transcription factor bindingto the coactivator CREB-binding protein (CBP) the mixedlineage leukemia protein (MLL) activation domain binds to anallosteric site on the KIX domain J Biol Chem 27743168ndash43174

8 PaoGM JanknechtR RuffnerH HunterT and VermaIM(2000) CBPp300 interact with and function as transcriptionalcoactivators of BRCA1 Proc Natl Acad Sci USA 971020ndash1025

9 BannisterAJ OehlerT WilhelmD AngelP and KouzaridesT(1995) Stimulation of c-Jun activity by CBP c-Jun residues Ser6373 are required for CBP induced stimulation in vivo and CBPbinding in vitro Oncogene 11 2509ndash2514

10 Van OrdenK GieblerHA LemassonI GonzalesM andNyborgJK (1999) Binding of p53 to the KIX domain of CREBbinding protein A potential link to human T-cell leukemia virustype I-associated leukemogenesis J Biol Chem 274 26321ndash26328

11 ZhangJJ VinkemeierU GuW ChakravartiD HorvathCMand DarnellJE Jr (1996) Two contact regions between Stat1 andCBPp300 in interferon gamma signaling Proc Natl Acad SciUSA 93 15092ndash15096

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15 AkimaruH ChenY DaiP HouD-X NonakaMSmolikSM ArmstrongS GoodmanRH and IshiiS (1997)Drosophila CBP is a co-activator of cubitus interruptus inhedgehog signalling Nature 386 735ndash738

16 KwokRPS LauranceME LundbladJR GoldmanPSShihH-M ConnorLM MarriottSJ and GoodmanRH(1996) Control of cAMP-regulated enhancers by the viraltransactivator Tax through CREB and the co-activator CBPNature 380 642ndash646

17 VendelAC and LumbKJ (2004) NMR mapping of the HIV-1Tat interaction surface of the KIX domain of the humancoactivator CBP Biochemistry 43 904ndash908

18 LeeD LeeB KimJ KimDW and ChoeJ (2000) cAMPresponse element-binding protein-binding protein binds to humanpapillomavirus E2 protein and activates E2-dependenttranscription J Biol Chem 275 7045ndash7051

19 WoodMA AttnerMA OliveiraAM BrindlePK andAbelT (2006) A transcription factor-binding domain of thecoactivator CBP is essential for long-term memory and theexpression of specific target genes Learn Mem 13 609ndash617

20 CanetJV DobonA and TorneroP (2012) Non-recognition-of-BTH4 an Arabidopsis mediator subunit homolog is necessaryfor development and response to salicylic acid Plant Cell 244220ndash4235

21 ThakurJK AgarwalP ParidaS BajajD and PasrijaR (2013)Sequence and expression analyses of KIX domain proteinssuggest their importance in seed development and determinationof seed size in rice and genome stability in Arabidopsis MolGenet Genomics 288 329ndash346

22 KassubeSA JinekM FangJ TsutakawaS and NogalesE(2013) Structural mimicry in transcription regulation of humanRNA polymerase II by the DNA helicase RECQL5 Nat StructMol Biol 20 892ndash899

23 IslamMN FoxD GuoR EnomotoT and WangW (2010)RecQL5 promotes genome stabilization through two parallelmechanismsmdashinteracting with RNA Polymerase II and acting asa helicase Mol Cell Biol 30 2460ndash2472

24 KanagarajR HuehnD MacKellarA MenigattiM ZhengLUrbanV ShevelevI GreenleafAL and JanscakP (2010)RECQ5 helicase associates with the C-terminal repeat domain ofRNA polymerase II during productive elongation phase oftranscription Nucleic Acids Res 38 8131ndash8140

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29 NakajimaT UchidaC AndersonSF LeeCG HurwitzJParvinJD and MontminyM (1997) RNA helicase A mediatesassociation of CBP with RNA polymerase II Cell 90 1107ndash1112

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35 YangX-J OgryzkoVV NishikawaJ-i HowardBH andNakataniY (1996) A p300CBP-associated factor thatcompetes with the adenoviral oncoprotein E1A Nature 382319ndash324

36 BannisterAJ and KouzaridesT (1996) The CBP co-activator isa histone acetyltransferase Nature 384 641ndash643

37 OgryzkoVV SchiltzRL RussanovaV HowardBH andNakataniY (1996) The transcriptional coactivators p300 andCBP are histone acetyltransferases Cell 87 953ndash959

38 RadhakrishnanI Perez-AlvaradoGC ParkerD DysonHJMontminyMR and WrightPE (1997) Solution structure of theKIX domain of CBP bound to the transactivation domain ofCREB a model for activatorcoactivator interactions Cell 91741ndash752

39 RadhakrishnanI Perez-AlvaradoGC ParkerD DysonHJMontminyMR and WrightPE (1999) Structural analyses ofCREB-CBP transcriptional activator-coactivator complexes byNMR spectroscopy implications for mapping the boundaries ofstructural domains J Mol Biol 287 859ndash865

40 ParkerD JhalaUS RadhakrishnanI YaffeMB ReyesCShulmanAI CantleyLC WrightPE and MontminyM (1998)Analysis of an activatorcoactivator complex reveals an essentialrole for secondary structure in transcriptional activation MolCell 2 353ndash359

41 GangulyD and ChenJ (2009) Atomistic details of thedisordered states of KID and pKID Implications in coupledbinding and folding J Am Chem Soc 131 5214ndash5223

42 GarzaAS AhmadN and KumarR (2009) Role of intrinsicallydisordered protein regionsdomains in transcriptional regulationLife Sci 84 189ndash193



43 GreigKT CarottaS and NuttSL (2008) Critical roles for c-Myb in hematopoietic progenitor cells Seminars in immunology20 247ndash256

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45 ZorT De GuzmanRN DysonHJ and WrightPE (2004)Solution structure of the KIX domain of CBP bound to thetransactivation domain of c-Myb J Mol Biol 337 521ndash534

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48 AygunO XuX LiuY TakahashiH KongSEConawayRC ConawayJW and SvejstrupJQ (2009) Directinhibition of RNA polymerase II transcription by RECQL5J Biol Chem 284 23197ndash23203

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50 ScogginKE UlloaA and NyborgJK (2001) The oncoproteinTax binds the SRC-1-interacting domain of CBPp300 to mediatetranscriptional activation Mol Cell Biol 21 5520ndash5530

51 ColginMA and NyborgJK (1998) The human T-cell leukemiavirus type 1 oncoprotein Tax inhibits the transcriptional activityof c-Myb through competition for the CREB binding proteinJ Virol 72 9396ndash9399

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53 ZorT MayrBM DysonHJ MontminyMR and WrightPE(2002) Roles of phosphorylation and helix propensity in thebinding of the KIX domain of CREB-binding protein byconstitutive (c-Myb) and inducible (CREB) activators J BiolChem 277 42241ndash42248

54 CampbellKM and LumbKJ (2002) Structurally distinct modesof recognition of the KIX domain of CBP by Jun and CREBBiochemistry 41 13956ndash13964

55 KorkmazEN NussinovR and HalilogluT (2012)Conformational control of the binding of the transactivationdomain of the MLL protein and c-Myb to the KIX domain ofCREB PLoS Comput Biol 8 e1002420

56 GrafT (1992) Myb a transcriptional activator linkingproliferation and differentiation in hematopoietic cells CurrOpin Genet Dev 2 249ndash255

57 HessJL BittnerCB ZeisigDT BachC FuchsUBorkhardtA FramptonJ and SlanyRK (2006) c-Myb is anessential downstream target for homeobox-mediatedtransformation of hematopoietic cells Blood 108 297ndash304

58 JinS ZhaoH YiY NakataY KalotaA and GewirtzAM(2010) c-Myb binds MLL through menin in human leukemia cellsand is an important driver of MLL-associated leukemogenesisJ Clin Invest 120 593ndash606

59 De GuzmanRN GotoNK DysonHJ and WrightPE (2006)Structural basis for cooperative transcription factor binding to theCBP coactivator J Mol Biol 355 1005ndash1013

60 RamirezJA and NyborgJK (2007) Molecular characterizationof HTLV-1 Tax interaction with the KIX domain of CBPp300J Mol Biol 372 958ndash969

61 LeeCW AraiM Martinez-YamoutMA DysonHJ andWrightPE (2009) Mapping the interactions of the p53transactivation domain with the KIX domain of CBPBiochemistry 48 2115ndash2124

62 WangF MarshallCB YamamotoK LiGY Gasmi-SeabrookGM OkadaH MakTW and IkuraM (2012)Structures of KIX domain of CBP in complex with two FOXO3atransactivation domains reveal promiscuity and plasticity in

coactivator recruitment Proc Natl Acad Sci USA 1096078ndash6083

63 LeeYH BedfordMT and StallcupMR (2011) Regulatedrecruitment of tumor suppressor BRCA1 to the p21 gene bycoactivator methylation Genes Dev 25 176ndash188

64 CookPR PolakowskiN and LemassonI (2011) HTLV-1 HBZprotein deregulates interactions between cellular factors and theKIX domain of p300CBP J Mol Biol 409 384ndash398

65 FerreonJC LeeCW AraiM Martinez-YamoutMADysonHJ and WrightPE (2009) Cooperative regulation of p53by modulation of ternary complex formation with CBPp300 andHDM2 Proc Natl Acad Sci USA 106 6591ndash6596

66 GianniS MorroneA GiriR and BrunoriM (2012) A folding-after-binding mechanism describes the recognition between thetransactivation domain of c-Myb and the KIX domain of theCREB-binding protein Biochem Biophys Res Commun 428205ndash209

67 BruschweilerS SchandaP KloiberK BrutscherBKontaxisG KonratR and TollingerM (2009) Directobservation of the dynamic process underlying allosteric signaltransmission J Am Chem Soc 131 3063ndash3068

68 VendelAC McBryantSJ and LumbKJ (2003) KIX-mediatedassembly of the CBP-CREB-HTLV-1 tax coactivator-activatorcomplex Biochemistry 42 12481ndash12487

69 BruschweilerS KonratR and TollingerM (2013) Allostericcommunication in the KIX domain proceeds through dynamicrepacking of the hydrophobic core ACS Chem Biol 81600ndash1610

70 PalazzesiF BarducciA TollingerM and ParrinelloM (2013)The allosteric communication pathways in KIX domain of CBPProc Natl Acad Sci USA 110 14237ndash14242

71 WangN MajmudarCY PomerantzWC GagnonJKSadowskyJD MeagherJL JohnsonTK StuckeyJABrooksCL 3rd WellsJA et al (2013) Ordering a dynamicprotein via a small-molecule stabilizer J Am Chem Soc 1353363ndash3366

72 XuW ChenH DuK AsaharaH TiniM EmersonBMMontminyM and EvansRM (2001) A transcriptional switchmediated by cofactor methylation Science 294 2507ndash2511

73 LeeDY TeyssierC StrahlBD and StallcupMR (2005) Roleof protein methylation in regulation of transcription EndocrRev 26 147ndash170

74 McBrideAE and SilverPA (2001) State of the arg proteinmethylation at arginine comes of age Cell 106 5ndash8

75 KatzJE DlakicM and ClarkeS (2003) Automatedidentification of putative methyltransferases from genomic openreading frames Mol Cell Proteomics 2 525ndash540

76 BedfordMT (2007) Arginine methylation at a glance J CellSci 120 4243ndash4246

77 StallcupMR (2001) Role of protein methylation in chromatinremodeling and transcriptional regulation Oncogene 203014ndash3020

78 BedfordMT and RichardS (2005) Arginine methylationan emerging regulator of protein function Mol Cell 18263ndash272

79 LeeYH and StallcupMR (2009) Minireview protein argininemethylation of nonhistone proteins in transcriptional regulationMol Endocrinol 23 425ndash433

80 WeiY HorngJ-C VendelAC RaleighDP and LumbKJ(2003) Contribution to stability and folding of a buried polarresidue at the CARM1 methylation site of the KIX domain ofCBP Biochemistry 42 7044ndash7049

81 Chevillard-BrietM TroucheD and VandelL (2002) Control ofCBP co-activating activity by arginine methylation EMBO J 215457ndash5466

82 LeeYH CoonrodSA KrausWL JelinekMA andStallcupMR (2005) Regulation of coactivator complexassembly and function by protein arginine methylationand demethylimination Proc Natl Acad Sci USA 1023611ndash3616

83 NishiokaK and ReinbergD (2001) Transcription Switchingpartners in a regulatory tango Science 294 2497ndash2498

84 ArndtHA (2006) Small molecule modulators of transcriptionAngew Chem Int Ed 45 4552ndash4560



85 RutledgeSE VolkmanHM and SchepartzA (2003) Molecularrecognition of protein surfaces high affinity ligands for the CBPKIX domain J Am Chem Soc 125 14336ndash14347

86 BestJL AmezcuaCA MayrB FlechnerL MurawskyCMEmersonB ZorT GardnerKH and MontminyM (2004)Identification of small-molecule antagonists that inhibit anactivator coactivator interaction Proc Natl Acad Sci USA101 17622ndash17627

87 LiBX and XiaoX (2009) Discovery of a small moleculeinhibitor of the KIX-KID interaction ChemBioChem 102721ndash2724

88 LiBX YamanakaK and XiaoX (2012) Structure-activityrelationship studies of naphthol AS-E and its derivatives asanticancer agents by inhibiting CREB-mediated genetranscription Bioorg Med Chem 20 6811ndash6820

89 HerzigS LongF JhalaUS HedrickS QuinnR BauerARudolphD SchutzG YoonC and PuigserverP (2001) CREBregulates hepatic gluconeogenesis through the coactivator PGC-1Nature 413 179ndash183

90 YoonJC PuigserverP ChenG DonovanJ WuZ RheeJAdelmantG StaffordJ KahnCR and GrannerDK (2001)Control of hepatic gluconeogenesis through the transcriptionalcoactivator PGC-1 Nature 413 131ndash138

91 XiaoX LiBX MittonB IkedaA and SakamotoKM (2010)Targeting CREB for cancer therapy friend or foe Curr CancerDrug Targets 10 384ndash391

92 PfallerMA and DiekemaDJ (2007) Epidemiology of invasivecandidiasis a persistent public health problem Clin MicrobiolRev 20 133ndash163

93 NaarAM WagnerG NishikawaJ and ArthanariH (2010)High throughput screens for small-molecule inhibitors of thenuclear receptor-like pathway regulating multidrug resistance infungi United States Patent Number US8557746B2

94 PandeyR MallerA NapoliCA SelingerDA PikaardCSRichardsEJ BenderJ MountDW and JorgensenRA (2002)Analysis of histone acetyltransferase and histone deacetylasefamilies of Arabidopsis thaliana suggests functional diversificationof chromatin modification among multicellular eukaryotesNucleic Acids Res 30 5036ndash5055

95 MathurS VyasS KapoorS and TyagiAK (2011) TheMediator complex in plants structure phylogeny andexpression profiling of representative genes in a dicot(Arabidopsis) and a monocot (rice) during reproduction andabiotic stress Plant Physiol 157 1609ndash1627

96 PasrijaR and ThakurJK (2013) Tissue specific expression profileof Mediator genes in Arabidopsis Plant Signal Behav 8 5 e23983

97 BordoliL NetschM LethiU LutzW and EcknerR (2001)Plant orthologs of p300CBP conservation of a core domain inmetazoan p300CBP acetyltransferase-related proteins NucleicAcids Res 29 589ndash597

98 EcknerR (1996) p300 and CBP as transcriptional regulatorsand targets of oncogenic events Biol Chem 377 685ndash688

99 HanSK SongJD NohYS and NohB (2007) Role of plantCBPp300-like genes in the regulation of flowering time PlantJ 49 103ndash114

100 PasrijaR and ThakurJK (2012) Analysis of differentialexpression of Mediator subunit genes in Arabidopsis PlantSignal Behav 7 1676ndash1686



mammals (125) KIX has been shown to interact with arange of transcription factors other than CREB such asc-Myb (6) mixed lineage leukemia protein (MLL) (7)breast cancer 1 (BRCA1) (8) c-Jun (9) p53 (10) signaltransducers and activators of transcription (STAT1) (11)and sterol responsive element-binding protein (SREBP)(12) of humans (Figure 1AndashC) to Gal4p (13) Gcn4 (14)Pdr13 (2) and Oaf1 (3) of yeast (Figure 1C) cubitusinterruptus (15) of Drosophila and human t-lymphotropicvirus type 1 (HTLV-1) Tax (16) human immunodeficiencyvirus type 1 (HIV-1) Tat (17) and E2 (18) of viruses(Figure 1B) Direct involvement of KIX domain hasbeen demonstrated in long term storage of hippocam-pus-dependent memory in mammalian brains (19) lipidhomeostasis in mammals (4) multi-drug resistance (2)and fatty acid metabolism (3) in yeast and salicylic acidmediated defense response in Arabidopsis (20) Importanceof KIX domain in determination of seed size of rice hasbeen postulated by presence of single nucleotide poly-morphisms (SNPs) differentiating the genotypes of longgrain from that of short grain (21) KIX domain hasrecently been reported from human recQ protein-like 5(RECQL5) helicase as an RNA Pol II-binding domain(Figure 1D) which is in contrast to the regular role ofKIX domains of CBP ARC105 and Gal11p that bind

to ADs of transcription activators (2ndash422) In this casethe KIX domain of RECQL5 interacts with Rpb1 jawdomain of RNA Pol II to repress transcription (22)Thus the proteins that possess KIX domain namelyCBP mediator subunit MED15 and most recentlyhelicase RecQL5 are mainly transcriptional regulatorsand the major role of KIX is to act as a docking site fortranscription factors and for stabilization of proteinndashprotein interactions (1252324) Solution structures ofseveral KIX domains in mouse humans and yeast areavailable in the protein data bank (PDB) both inligand-bound complexes and in the unbound formsAccording to the Pfam database KIX domain alsoshows conservation in the plant kingdom and othergroups such as arthropods and nematodes although thestructure of KIX in these groups is not yet characterizedDespite elucidation of its crucial role in transcription

several gaps remain in our knowledge of the KIX domainspecifically in terms of its ability to recognize and assist infolding of intrinsically disordered proteins its role in sec-ondary structure determination its varying bindingmodes mechanism of allostery high structural plasticityand even some of its cellular functions Furthermore verylittle is known about KIX domain proteins in plantsalthough a recent association analysis of SNP loci in

Figure 1 Cartoon depicting the varied interactions and overall roles played by KIX in (A) the general transcriptional apparatus (B) regulation viaCBP and its coactivators (C) the mediator complex and (D) the RecQL5 helicase










































































KIX IN PLANTS





CONCLUSIONS



ACKNOWLEDGEMENTS


FUNDING



REFERENCES





















































































































































































KIX IN PLANTS





CONCLUSIONS



ACKNOWLEDGEMENTS


FUNDING



REFERENCES















































































































CONCLUSIONS



ACKNOWLEDGEMENTS


FUNDING



REFERENCES














































































































survey and summary molecular recognition by the kix domain

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