The KIX-domain of Gal11/MED15 was shown to interact with short transactivation domain of Pdr1 and Oaf1 (12 and 9 amino acids respectively) annotated to 9aaTAD family. Reported NMR data revealed a surprising sequence similarity of KIX domain and basic-leucine zipper (bZIP)

martin piskacek

English

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9aaTADs mimic DNA to interact with a pseudo-DNA Binding Domain KIX of Med15 (Molecular Chameleons)

Nature Precedings

9aaTAD mimics DNA to interact with a pseudo-DNA Binding Domain KIX 
of Med15
(Molecular Chameleon)
Martin Piskacek
MFPL, Department of Medical Biochemistry, Division of Molecular Genetics, Austria
The KIX-domain  of  Gal11/MED15  was  shown  to  interact  with  short 
transactivation domain of Pdr1 and Oaf1 (12 and 9 amino acids respectively) annotated to 
9aaTAD family.  Reported NMR data revealed a surprising sequence similarity of KIX 
domain and basic-leucine zipper (bZIP). 
Nine-amino-acid Trans-Activation Domain, 9aaTAD, defines a novel domain common 
to  a  large  super-family  of  eukaryotic  transcription  factors represented  in  yeast  by 
Gal4/Oaf1/Pip2/Leu3/Pdr1/Pdr3/Rtg3/Pho4/Gln3/Gcn4  and  in  mammals  by 
p53/E2A/NFAT/NFkB/HSF1/NF-IL6/KLF2/MLL-ALL1/EBNA2/VP16  . The  prediction  and 
online annotated results (including Pdr1 and Oaf1) are available on www.expasy.ch/tools.
Pdr1 and Oaf1 are 9aaTAD-transcription factors of the Gal4 family. Pdr1 and its close 
homolog Pdr3 mediate general drug resistance to many cytotoxic substances . Transactivation 
mediated by TFIID, SAGA and Mediator complexes was demonstrated previously for Pdr1 . 
Importantly, TAF9 interaction with Pdr1 was also reported , a typical feature for the number of 
the 9aaTAD transcription factors (is about 666, online annotation on www.expasy.ch/tools). 
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Oaf1 directly binds fatty acids, what is essential for effective Oaf1 transactivation and 
DNA binding .  However, close Oaf1 homolog Pip2 (with similar 9aaTAD, Fig.1) activates 
transcription  in  a  fatty-acid-independent  manner  .  Both  transcription  factors  include  very 
similar  and extremely effective transactivation domain 9aaTAD .  Pdr1 and Oaf1 9aaTADs 
activate transcription constitutively and do not respond to xenobiotic resp. fatty acids .
Gal11/Spt13/MED15  is  a  cofactor  of  general  mediator  complex  involved  in 
transcription  genome-wide  (originally  found  as  an  essential  factor  for  Gal4-dependent 
transactivation by T.Fukasawa and F.Winston labs)  .  Besides 9aaTAD transcription factors 
Pdr1/3 and Oaf1/Pip2, numerous transcription factors have physical and genetic interaction 
with  Gal11/MED15  (Gal4/Gcn4/VP16/Pho4/Msn2/Ino2/P201) . The  9aaTAD  and 
Gal11/MED15 provide transcription constitutive and are stimuli independent.
Notes to 9aaTAD regulation in the Gal4-family
Oaf1,  Pip2,  Pdr1,  Pdr3,  Leu3 and Gal4 are 9aaTAD-transcription factors with high 
sequence and structural  homology annotated to  the Gal4-family.  Each constitutively active 
9aaTAD  is  regulated  in  response  to  specific  stimuli  (fatty  acids,  xenobiotic  agents, -
isopropylmalate and galactose). The Middle-Homology-Region, MHR, has been implicated in 
this function . Pdr1 and Oaf1 regulate the 9aaTAD-mediated-transactivation by intramolecular 
masking in response to xenobiotic and fatty acids  in a similar way described for distal homolog 
Leu3.
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KIX-domain interaction with 9aaTAD
Yeast KIX domain of Gal11/MED15  reveals high sequence and structural similarities 
to mammalian MED15 and CBP .  hMED15 interactions with 9aaTAD-transcription factors 
VP16 and NFkB were  reported  .  Interactions  of  CBP/KIX-domain  with  further  annotated 
9aaTAD of either transcription factors p53 or MLL were reported from P.E.Wright lab . p53 
binding to CBP/KIX-domain was localized for residues:19-24 and 47-53 (*underlined in Fig.1). 
Both bind to CBP/KIX-domain at  two different binding sites with similar  Kd values.  This 
corresponds to a shared common motif annotated previously (residues:17-25 and 48-56) .
9aaTAD interactions with general coactivators (for review see Piskacek et al. 2009 ): 
• p53-9aaTAD1   interacts  with both  TAF9  (p53-residues:FSDLW,  NMR-data)   and  CBP/KIX-domain  (p53-
residues:FSDLWK, NMR-data)
• MLL/ALL1-transactivation domain comprising annotated 9aaTAD  interacts with both TAF9 (MLL-residues:2840-
58,  DDCGNILPSDIMDFVLK,  protein-protein-interaction  data) and CBP/KIX-domain  (MLL-residues:2841-61,  
DCGNILPSDIMDFVLKNTP, NMR-data) (*critical residues for MLL-transactivation  are underlined in Fig.1)
• VP16-transactivation domain  comprising annotated  9aaTAD (QMFTDALGI)   interacts with both TAF9  (VP16-
residues: MADFEFEQMFTDALGIDEYGG, NMR-data)  and Gal11/MED15  and hMED15 
• NFkB-transactivation domain  comprising annotated  9aaTAD (MDFSALLSQ)   interacts with both TAF9 (NFkB-
residues: EDFSSIADMDFSALLSQ, protein-protein interaction)  and CBP .
• E2A-transactivation domain (homolog to Rtg3) comprising annotated 9aaTAD (DLLDFSMMF)  interacts with both 
SAGA complex (critical residues LDFS) and CBP/KIX-domain (E2A-residues:PVGTDKELSDLLDFSMMFPLPVT, 
CD-spectroscopy) 
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The most studied 9aaTAD of 53 was reported to mimic DNA for single-stranded DNA 
binding protein RPA  and hallowed by the name "molecular chameleon". Interaction either RPA 
or KIX with 9aaTAD resembles bZIP interaction with DNA. bZIP domain provides specific 
DNA-binding of  transcription  factor  family  represented  by  CREB1,  ATF1/6,  FOS/JUN in 
mammals and by GCN4 in yeast. The bZIP DNA-binding domain of CREB1 resemble small 
region of KIX, where interactions with 9aaTADs take place (Fig.2). Therefore, it  could be 
hypothesize that bZIP and KIX are distal relative. In such a case, KIX and bZIP could originate 
from a single gene, which has undergo multiplication and each copy developed independently 
during evolution in a specific binding factor for 9aaTAD and DNA, respectively. 
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Fig.1
Annotated 9aaTAD KIX interaction (NMR)*
Oaf1 D LFDY DFLV DLFDYDFLV
Pip2 D FFDY DLLF Oaf1 homolog
Pdr1 E DLYS ILWS EDLYSILWSDWY
Pdr3 T DLYH TLWN Pdr1 homolog
p53 TAD1 E TFSD LWKL LSPEETF SDL  W K LPE 
p53 TAD2 D DIEQ WFTE QAMDDLMLSP D D I EQW  FTEDPGPD 
MLL S DIMD FVLK DCGNILPSDIMDFVLKNTP
E2A D LLDF SMMF PVGTDKELSDLLDFSMMFPLPVT
Rtg3 E TLDF SLVT E2A homolog
CREB R KILN DLSS RREILSRRPSYRKILNDLSSDAP
CREB B 6 E AILA ELKK CREB-mutant binding to KIX 
Gli3 D DVVQ YLNS TAD homology to CREB/KIX
Gal4 D DVYN YLFD Pdr1 and Oaf1 homolog
KIX interactions with annotated 9aaTADs. Critical residues for interaction with KIX domain (NMR data) are 
underline. Alignment of p53 and MLL/ALL were derived previously from NMR data by P.E. Wright lab  and 
correlate with our prediction (annotated 9aaTAD result online 2007, supplementary data).
Fig.2
yMED15-KIX Pdr1-binding IRI HA KNF EAA LFAKSSS KKEYM DSM NE KVAV
yMED15-KIX Oaf1-binding IRI HA KNF EAA LFAKSSS KKEYM DSM NE KVAV 
hMed15-KIX SKS  S KDM ESH VFLKAKT RDEYL SLV A  RLII
CREB1-bZIP DNA-binding VRL  M KNR EAA RECRRK  KKEYV KCL EN RVAV
ATF1-bZIP DNA-binding IRL  M KNR EAA RECRRK  KKEYV KCL EN RVAV
ATF6-bZIP DNA-binding QRM  I KNR ESA CQSRKK  KKEYM LGL EA RLKA
FosB-bZIP DNA-binding RRE    RNK LAA AKCRNR  RRELT DRL QA ETDQ
AP1 -bZIP DNA-binding RKR  M RNR IAA SKCRKR  KLERI ARL EE KVKT
GCN4-bZIP DNA-binding LKR  A RNT EAA RRSRAR  KLQRM KQL ED KVEE
bZIP-pattern 
 Rx .. KNx xAA xxxRxK        
Alignment of KIX and DNA binding domains. Interactions of Oaf1and Pdr1 with amino acids of KIX domain 
of Gal11 are in red and blue respectively. DNA binding domains of bZIP transcription factors are in green.
Pattern for bZIP/KIX family:
[LIFVMASTYRKHQN][KR][LIFVMASTYRKHQNDE]x(0,2)[KR][NQDE][LIFVMASTYRKHQN][EDLIVMST]x(5)[KR][KRASTC](2,3
)[KR][LIFVMASTYRKHQNDE][EDNQA][YFLIKRDEST][LIFVMASTY]x(4,5)[RK]
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Supplementary data
Annotated 9aaTAD
 Prediction and Experimental Data
 2007
Deposit Pattern RC-out # Predicted 9aa TAD:  Reference
Yeast:
Gal4 P04386 Y - D DVYN YLFD  
Lac9 P08657 Y - D DVYN YIFD  
Oaf1 P39720 Y - D LFDY DFLV
Pdr1 P12383 Y - E DLYS ILWS
Leu3 P08638 Y - D ILMN EFAF
Tea1 P47988 Y - D DLIR ELFG and ETVVIEIID
Rtg3 P38165 Y 4 E TLDF SLVT
Gcn4 P03069 Y - D AVVE SFFS and SNLDFDFAL
Pho4 P07270 M 1,4,5 D DLDR AFEL   
Gln3 P18494 M - G EIAQ LWDF
Plats:
HSF P41152 Y 4 D DIWE ELLSE and overlapping repeat ELLSEDLIS
Mammalian:
p53, TADI+II P04637 M 1,4,5 E TFSD LWKL, QAMDDLMLS,DDIEQWFTE   
E2A P15923 M - D LLDF SMMF and QAVSVILNL
HSF1 Q00613 V 5 S ALLD LFSP and 5x false-positive hits
KLF2 O43474 M - D LLDL DFIL
NF-IL6 P49715 M - D EFLA DLFQ
NF-κB Q04206 M,V - M DFSA LLSQ and S IADM  D F SA   overlapping repeat
NFAT1 Q13469 M - D ELDF SILF
MLL/ALL1 Q03164 M - S DIMD FVLK and QVLTALLNS
ESX P78545 M - S WIIE LLEK and TQVLDWISY
Viral:
EBNA2 P12978 V, M 4 E SWDY IFET 
VP16 P04486 V - Q MFTD ALGI and 6x false-positive hits
#RC-out  represents  Refinement  Criteria (RC),  which should be omitted for  prediction.  RC1, RC4 and RC5 
caused collision for some of well experimentally characterized 9aa TADs and must be applied with concern.  
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Refferences
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http://precedings.nature.com/documents/3939/version/1

9aaTADs mimic DNA to interact with a pseudo-DNA Binding Domain KIX of Med15 (Molecular Chameleons)

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