gnd-254904, fig. s1. zhi, et...
TRANSCRIPT
human --------------------MSKPAGSTS-----------RILDIPCKVCGDRSSGKHYGVYACDGCSGFFKRSIRRNRTYVCKSG---NQGGCPVDKTHmouse --------------------MSKPAGSTS-----------RILDIPCKVCGDRSSGKHYGVYACDGCSGFFKRSIRRNRTYVCKSG---NQGGCPVDKTHrat --------------------MSKPAGSTS-----------RILDIPCKVCGDRSSRKHYGVYACDGCSGFFKRSIRRNRTYVCKSG---NQGGCPVDKTHchicken --------------------MSKPAGSTS-----------RILDIPCKVCGDRSSGKHYGVYACDGCSGFFKRSIRRNRTYVCKSG---NQGGCPVDKTHcattle --------------------MSKPAGSTS-----------RILDIPCKVCGDRSSGKHYGVYACDGCSGFFKRSIRRNRTYVCKSG---NQGGCPVDKTHxenopus --------------------MSNPTGSTS-----------RILDIPCKVCGDRSSGKHYGVYACDGCSGFFKRSIRRNRSYVCKSG---NQGGCPVDKTHzebrafish --------------------MSKPAGSTSGCLDILNVKSSRILDIPCKVCGDRSSGKHYGVYACDGCSGFFKRSIRRNRTYVCKSG---TQGGCPVDKTHfruitfly --MQSSEGSPDMMDQKYNSVRLSPAASSR-----------ILYHVPCKVCRDHSSGKHYGIYACDGCAGFFKRSIRRSRQYVCKSQ---KQGLCVVDKTHred flour beetle MSEMQSVEGAMVHHLEPHRMQIKPQSPSS---------SSRILDIPCKVCGDFSSGKHYNIFACDGCAGFFKRSIRRNRQYVCKAK---DEGSCIIDKTH
human RNQCRACRLKKCLEVNMNKDAVQHERGPRTSTIR-KQVALYFRGHKEENGAAAHFPSAALPAPAFF------TAVTQ--LEPHGLELAAVSTTPERQTLVmouse RNQCRACRLKKCLEVNMNKDAVQHERGPRTSTIR-KQVALYFRGHKEDNGAAAHFPSTALPAPAFF------TAVTQ--LEPHGLELAAVSATPERQTLVrat RNQCRACRLKKCLEVNMNKGAVQHERGPRTSTIR-KQVALYFRGHKEDNGAAAHFPSAALPAPAFF------TAVTQ--LEPHGLELTAVSATPERQTLVchicken RNQCRACRLKKCLEVNMNKDAVQHERGPRTSTIR-KQVALYFRGHKEESSGAPHFPATALPAPAFF------TAVSQ--LEPHGLELAAVAGTPERQALVcattle RNQCRACRLKKCLEVNMNKDAVQHERGPRTSTIR-KQVALYFRGHKEENGAAAHFPSAALPAPAFF------TAVTQ--LDPHGLELAAVSATPERQTLVxenopus RNQCRACRLKKCLEVNMNKDAVQHERGPRTSTIR-KQVALYFRGHKEVNGSTQHFSSTALPTPTFF------TTVRQ--LEAHNLELAAISTVPERQTLVzebrafish RNQCRACRLKKCLEVNMNKDAVQHERGPRTSTIR-KQVALYFRGHKEVNGSSTHFPSTSIPGPPFF------TTVTQ--LEPHNLELNTVTSTPERQAIVfruitfly RNQCRACRLRKCFEVGMNKDAVQHERGPRNSTLR-RHMAMYKDAMMGAGEMPQIPAEILMNTAALTGFPGVPMPMPGLPQRAGHHPAHMAAFQPPPSAAAred flour beetle RNQCRACRLKKCQNVGMNKDAVQHERGPRNSTLRRQQMSSYYNESR---------VMMSPPGNVLN------LTMPK--YEPNPSIIDPGPALPPTGFLC
human SLAQPTPKYPHEVNGT---------------------PMYLYEVATESVCESAARLLFMSIKWAKSVPAFSTLSLQDQLMLLEDAWRELFVLGIAQWAIPmouse SLAQPTPKYPHEVNGT---------------------PMYLYEVATESVCESAARLLFMSIKWAKSVPAFSTLSLQDQLMLLEDAWRELFVLGIAQWAIPrat SLAQPTPKYPHEVNGT---------------------PMYLYEVATESVCESAARLLFMSIKWAKSVPAFSTLSLQDQLMLLEDAWRELFVLGIAQWAIPchicken GLAQPTPKYPHEVNGT---------------------PMYLYEVATESVCESAARLLFMSIKWAKSVPAFSTLSLQDQLMLLEDAWRELFVLGIAQWAIPcattle SLAQPTPKYPHEVNGT---------------------PMYLYEVATESVCESAARLLFMSIKWAKSVPAFSTLSLQDQLMLLEDAWRELFVLGIAQWAIPxenopus GLAQPTPKYPHEVNGA---------------------PLYLYEFATESVCESAARLLFMSIKWAKSVPAFSTLSLQDQLMLLEDAWRELFVLGIAQWAIPzebrafish GLAQPTPKYPHEVSGT---------------------PMYLYEVATESVCESAARLLFMSIKWAKSVPAFSTLPLPDQLILLEDAWRELFVLGIAQWAIPfruitfly VLDLSVPRVPHHPVHQGHHGFFSPTAAYMNALATRALPPTPPLMAAEHIKETAAEHLFKNVNWIKSVRAFTELPMPDQLLLLEESWKEFFILAMAQYLMPred flour beetle NNYPPLPQVPPLPLPP---------------------IFPPTMINPSAICESAAQLIFMNVQWVRSIPAFTCLPLSDQLLLLEESWLDLFVLGAAQFLPL
human -VDANTLLAVSGMNGDNTDSQKLNKIISEIQALQEVVARFRQLRLDATEFACLKCIVTFKA---------------VPTHSGSELRS-------FRNAAAmouse -VDANTLLAVSGMNTDNTDSQKLNKIISEIQALQEVVARFRQLRLDATEFACLKCIVTFKA---------------VPTHSGSELRS-------FRNAAArat -VDANTLLAVSGMNSDNTDSQKLNKIISEIQALQEVVARFRQLRLDATEFACLKCTVTFKA---------------VPTHSGSELRS-------FRNAAAchicken -VDANTLLAVSGMNGDNTDSQKLNKIISEIQALQEVVARFRQLRLDATEFACLKCIVTFKA---------------VPTHSGSELRS-------FRNAAAcattle -VDANTLLAVSGMNGDNTDSQKLNKIISEIQALQEVVARFRQLRLDATEFACLKCIVTFKA---------------VPTHSGSELRS-------FRNAAAxenopus -VDASTLLAVSGMNNENTESPKLNKIISEIQALQDVVSRFRQLRLDATEFACLKCIVTFKAG--------------VSTHSGSELRN-------FRNAAAzebrafish -VDSSTLLAVSGMNTENTDSQRLNKIISEIQALQEVVTRFRQLRLDATEFACLKCIVTFKA---------------VPTHSGSELRS-------FRNASAfruitfly -MNFAQLLFVYE--SENANREIMGMVTREVHAFQEVLNQLCHLNIDSTEYECLRAISLFRKSPPSASSTEDLANSSILTGSGSPNSSASAESRGLLESGKred flour beetle -MDFSVLVEACGVLQQ--EPHRRDAFLKEVADFQETLKKISQFQLDAHEFACLRAIVLFKTSFE------------KPSSSSNQEKT-------TTESAK
human IAALQDEAQLTLNSYIHTRYPTQPCRFGKLLLLLP---ALRSISPSTIEEVFFKKTIGNVPITRLLSDMYK-----SSDImouse IAALQDEAQLTLNSYIHTRYPTQPCRFGKLLLLLP---ALRSISPSTIEEVFFKKTIGNVPITRLLSDMYK-----SSDIrat IAALQDEAQLTLNSYIHTRYPTQPCRFGKLLLLLP---ALRSISPSTIEEVFFKKTIGNVPITRLLSDMYK-----SSDIchicken IAALQDEAQLTLNSYIHTRYPTQPCRFGKLLLLLP---ALRSISPSTIEEVFFKKTIGNVPITRLLSDMYK-----SSDIcattle IAALQDEAQLTLNSYIHTRYPTQPCRFGKLLLLLP---ALRSISPSTIEEVFFKKTIGNVPITRLLSDMYK-----SSDIxenopus ISALQDEAQLTLNSYIHTRYPTQPCRFGKLLLLLP---ALRSINPSTIEEVFFKKTIGNVPITRVLSDMYK-----SSDIzebrafish IAALQDEAQLTLNSYIHTRYPTQPCRFGKLLLLLP---ALRSVGPSTIEEVFFKKTIGNVPITRLLSDMYK-----SSDIfruitfly VAAMHNDARSALHNYIQRTHPSQPMRFQTLLGVVQ---LMHKVSSFTIEELFFRKTIGDITIVRLISDMYS-----QRKIred flour beetle ISVIQDDAQMRLNKHVTTTYPKQPLRFGKILLLVSS--TFRTISGRTIEDLFFKKVIRDTPIVAIISNMYKNQILGNNNV
H3 H3’ H4 H5
H7 H8
H9 H10 H11 H12
100%99%98%97%99%
91%89%
40%45%
x1 x2 x3 x4 x5
x6 x7 x8 x9
DNA binding domain
DNA binding domain
Supplementary Figure 1. The full length sequence alignment of TLX from human (NP_003260), mouse (NP_689415), rat (NP_001106668), chicken (NP_990501), cattle (NP_001179582), zebrafish (NP_001003608), xenopus (NP_001079280), fruitfly (NP_524596), and red flour beetle (NP_001034502). The percentages left to species stand for sequence identity to human TLX. TLX has the DNA binding domain, followed by the ligand binding domain that may not contain traditional helices H1 and H2. Helices H3-H12 are labeled by H and numbers. In front of helix H3, a series of human TLX N-terminal trunctions were made (designated by arrowheads and named the letter x followed by numbers). MBP-TLXx9 yielded initial crystals that were optimized by homologous surface muta-tions. Several prolines are present in front of human or red flour beetle TLX helix H3 and encircled by red boxes. They do not favor the formation of helices.The amino acids involved in TLX/Atrophin binding are conserved and encircled by black boxes. The residues potentially involved in TLX dimerization are encircled by yellow box.
GND-254904, Fig. S1. Zhi, et al.
DTPALRTLSEYARPHV
DTPALRTLSEYARPHV
DTPALRTLSEYARPHA
DTPALRTLSEYARPHV
mouse
rat
cattle
human
mouse
rat
cattle
human
red flour beetle
fruitfly
DTPALRTLSEYARPHV
DTPALRTLSEYARPHV
DTPALRTLSEYARPHA
DTPALRTLSEYARPHV
DTPALRQLSEYARPHA
DTPALRQLSEYARPHV
YLGP MSPG
MSPGYLGP
YLGP MSPG
YLGP MSPG
YIGP MSPT
YIGP MSPT
Atrophin-1
Atro box
YIGP MSPT
YIGP MSPT
PPYA
PGFN GFSP
AFSP
=peptide Atr1
=peptide Atr2
=peptide dAtro
Atrophin-2
Atrophin
Supplementary Figure 2. The Atro box sequence alignment of Atrophin-1 from human (NP_001007027), mouse (NP_031907), rat (NP_058924), cattle (NP_001192677), Atrophin-2 from human (NP_001036146), mouse (NP_001078961), rat (NP_446337), cattle (XP_005217062), Atrophin from fruitfly (NP_659574) and red flour beetle (XP_008194914). The Atro box sequences are highlighted in grey. The peptide sequences used in crystallization and biochemical assays are underlined.
GND-254904, Fig. S2. Zhi, et al.
Supplementary Figure 3. Homologous competitive binding of peptides dAtro, Atr1 and Atr2 to TLXLBD in the AlphaScreen assay. Biotinylated dAtro (A), Atr1 (B) or Atr2 peptide (C) (200 nM) from Fig. 1a was incubated with the TLXLBD (residues 182–385) HisMBP fusion protein and increasing concentrations of nonbiotinylated dAtro (IC =32.27 µM), Atr1(IC =29.87 µM) or Atr2 (IC =74.34 µM) peptide . Error bars = SD (n = 3).
Pept
ide
Bin
ding
(bin
ding
sig
nals
x 1
000)
0
2
4
6
10
8 IC =29.87 µM50
0
1
2
3
4IC =74.34 µM50
Pept
ide
Bin
ding
(bin
ding
sig
nals
x 1
000)
A
B
50
C
0
5
10
15
20
Pept
ide
Bin
ding
(bin
ding
sig
nals
x 1
000)
10 10 10 10 10 10 10-8 -7 -6 -5 -4 -3 -2
peptide (concentrations, M)
IC =32.27 µM50
dAtro
Atr1
Atr2
50
50
GND-254904, Fig. S3. Zhi, et al.
S
TLX --------MS-------KPAGSTSR----------------ILDIPCKVCGDRSSGKHYGVYACDGCSGFFKRSIRRNRTYVCKSGNQGGCPVDKTHRNQCRACRLKKCLEVNMNKDAVQ
PNR METRPTALMSSTVAAAAPAAGAASRKESPGRWGLGEDPTGVSPSLQCRVCGDSSSGKHYGIYACNGCSGFFKRSVRRRLIYRCQVG-AGMCPVDKAHRNQCQACRLKKCLQAGMNQDAVQ
HERGPR-TSTIRKQVALYFRGHKEENGAAAHFPSAALP-APAFFTAVTQLEPH---GLELAAVSTTPERQTLVSLAQPTPKYPHEVNGTPMYLYEVATESVCESAARLLFMSIKWAKSVPNERQPRSTAQVHLDSMESNTESRPESLVAPPAPAGRSPRGPTPMSAARALGHHFMASLITAETCAKLEPEDADENIDVTSNDPEFPSSPYSSSSPCGLDSIHETSARLLFMAVKWAKNLP
AFSTL LQDQLMLLEDAWRELFVLGIAQWAIPVDANTLLAVSGMNGDNTDSQKLNKIISEIQALQEVVARFRQLRLDATEFACLKCIVTFKAVPTHSGSELRSFRNAAAIAALQDEAQLTVFSSLPFRDQVILLEEAWSELFLLGAIQWSLPLDSCPLLAPPEASAAGGAQGRLTLASMETRVLQETISRFRALAVDPTEFACMKALVLFKP-------ETRGLKDPEHVEALQDQSQVM
LNSYIHTRYPTQPCRFGKLLLLLPALRSISPSTIEEVFFKKTIGNVPITRLLSDMYKSSDILSQHSKAHHPSQPVRFGKLLLLLPSLRFITAERIELLFFRKTIGNTPMEKLLCDMFKN---
TLXPNR
TLXPNR
TLXPNR
100%44%
H3
H3’ H4 H5 H7 H8 H9
H9 H10 H11 H12A B C
D E F
A
C
0
2
4
6
8
RLU
vector WT BDVp16-hTLXLBD
0
0.1
0.2
0.3
0.4
RLU
vector WT BDGal4-hTLXLBD
5.3x4.9x
repression fold
B D
50µm
Supplementary Figure 4. Obtainment of diffractable human TLX (hTLX) LBD crystals using the homologous surface mutation strategy. (A) Based on Amino acid sequence alignment of human TLX (NP_003260) and PNR (NP_055064, PDB ID code 4LOG, sequence identity to TLX=44%, six sites (marked A–G) predicted to be on the TLX surface were mutated to the corresponding amino acids in PNR (circled in black box) individually or in combination. Helices H3-12 are labeled by H and numbers. (B) MBP-hTLXLBD-BD (the combinatorial mutations of sites B and D in TLX (residues 182-385) in complex with the peptide dAtro yielded half-moon-like crystals (pointed by arrowheads) that diffracted up to 3.55 Å). The crystals were grown at 20 °C in sitting drops containing 1.0 μL of the protein solution (10 mg/mL) and 1.0 μL of the well solution containing 0.2 M sodium/potassium phosphate, 20% (wt/vol) PEG3350. (C) The combinatorial mutations of sites B and D did not affect the interaction between hTLX and Atrophin-1 or -2 in mammalian two hybrid assays. Mouse Atrophin-1 (residues 823-950) and mouse Atrophin-2 (residues1197-1334) were fused to Gal4. (D) The combinatorial mutations of sites B and D did not affect repressive activity of hTLX in cell reporter assays using the Gal4 fused receptor LBD. All error bars = SD (n = 3).
Gal4
Gal4-Atrophin-1
Gal4-Atrophin-2
GND-254904, Fig. S4. Zhi, et al.
MBP(A)
MBP(B)
MBP(D)
MBP(C)
MBP(D)
MBP(A)
MBP(B)
MBP(C)
180 0
hTLX(B)
hTLX(D)
hTLX(A)
hTLX(B)
hTLX(D)
hTLX(C)
hTLX(A)hTLX(C)
dAtro(C)
dAtro(D)
dAtro(C)
dAtro(D)
Supplementary Figure 5.There are four MBP (light blue) fused hTLX LBDs in one noncrystallographic symmetric unit. Two LBDs (TLX(C) in pink and TLX(D) in purple) are in complex with peptide dAtro (red) and arranged in a non-symmetric manner. The other two LBDs (TLX(A) in green and TLX(B) in light green) are not in complex with peptide dAtro and arranged in a symmetric manner.
GND-254904, Fig. S5. Zhi, et al.
900hTLX/RXR
SRC1-2
dAtro
H12H12
H1
RA
RA
SRC1-2
dAtro
H12
H12
H10
H11
H10
H11
kink
Supplementary Figure 6. Superposition of hTLX(pink) onto ligand-bound RXR (gold, PDB code=1FM6). Compared to RXR helices H10 and H11, there is a kink between TLX helices H10 and H11, which causes the space hindrance to ligand binding. RA , cis-retinoid acid in yellow, is ligand for RXR. hTLX does not have helix H1. Peptide dAtro clashes with RXR helix H12/H3.The RXR-interacting peptide SRC1-2 (orange) overlaps with hTLX helix H12.
GND-254904, Fig. S6. Zhi, et al.
0
5
10
15
20
25
30
35Pe
ptid
e B
indi
ng(b
indi
ng s
igna
ls x
100
0)
Vector hTLX rfbTLX
A B
50µm
Supplementary Figure 7. Obtainment of diffractable insect TLX crystals. (A) red flour beetle TLX (rfbTLX) binds more strongerly than hTLX to peptide dAtro in an AlphaScreen assay. The TLX LBDs fused to HisMBP were used in experi-ments. Error bars = SD (n=3). (B) Representative picture of MBP-rfbTLX/dAtro crystals that diffracted up to 2.6 Å.The crystals were grown at 20 °C in sitting drops containing 1.0 μL of the protein solution (14 mg/mL) and 1.0 μL of the well solution containing 0.04 M citric acid, 0.06 M BIS-TRIS propane, pH 6.4, 20% (wt/vol) PEG3350.
HisMBP
GND-254904, Fig. S7. Zhi, et al.
MBP(a)
rfbTLX(a)rfbTLX(b)
MBP(b)
MBP(a)
rfbTLX(a) MBP(b)
dAtro(a) dAtro(b)
dAtro(a)
dAtro(b)
900
A
B
Supplementary Figure 8. Structural analysis of the red flour beetle TLX (rfbTLX) LBD. (A) There are two MBP (light blue) fused hTLX LBDs (cyan) in one noncrystallographic symmetric unit. Both are in complex with peptide dAtro (red) and form a symmetrical dimer via the helix H10-H10 interaction. (B) Representative Fo-Fc electron density omit map contoured at 1.0 σ for peptide dAtro(b) (red). Of note, the dAtrophin-contact residues on the rfbTLX(b) H12 are different from those on the rfbTLX(a) H12 and labeled.
rfbTLX(b)
dAtro(b)
Y396A390
H12
H3
GND-254904, Fig. S8. Zhi, et al.
A B
TLX(a)/PNR
L359(a)
L360(a)
L360(b)
L359(b)H10
H10
TLX(a) TLX(b) TLX(b)/PNR
H10
H10
C
TLX(b)/PNR TLX(a)/PNR
L359/L372
L360/L373 L360/L373
L359/L372
H10 H10
DH10
PTQPCRFGKL LL LLPALRhTLX
rfbTLX PKQPLRFGKI LL LVSSTFR
hPNR PSQPVRFGFL LL LLPSLRF
334 351
349 367
146 164
E F
Supplementary Figure 9. Dimerization analysis of TLX. (A) The dimerization interface in rfbTLX is mediated by resiudes from helices H10 of both monomers. (B) Superposition of rfbTLX (cyan) and PNR (yellow). Dimerization in both receptors are mediated by the helix H10-H10 interaction. (C) Close-up presentation of the dimerization inter-face in superposed rfbTLX and PNR. The residues from helices H10 that form the dimer are conserved in rfbTLX and PNR. The first residue stands for rfbTLX and the second represents PNR. (D) The sequence alignment of helix H10 from hTLX, rfbTLX and hPNR. The residues involved in receptor dimerization are conserved and encircled by red box. Numbers refer to the amino acid position in receptors. (E) hTLX forms a dimer in a representative size exclusion chromatography profile. (F) Mutation of residues involved in hTLX dimerization impaired hTLX repressive activity.
0
2
4
6
8
10
Rep
ress
ion
fold
Vector
WT
L344S
/
L345S
Gal4-hTLXLBD
mAU
400
500
600
700
V0 ml0 40 80 120 160 200 240280 320360
monomer
dimer
GND-254904, Fig. S9. Zhi, et al.
EID1
AtrophinTLX
W199
V203
Q232
A286
C287
K289
F294
L317
A
C
Atrophin
EID1
SHP
H12
D
5% input IP: anti-Flag
Flag-Atrophin-2 (WB: anti-Flag)
HA-SHP (WB: anti-HA)
E5% input IP: anti-Flag
vecto
r
Atrophin-2+
SHPve
ctor
Atrophin-2+
SHP
WT ∆A
tro WT ∆A
tro
Atrophin-2+SHP
Flag-Atrophin-2 (WB: anti-Flag)
HA-SHP (WB: anti-HA)
5% input IP: anti-Flag
WT ∆A
tro WT ∆A
tro
Atrophin-1+SHP
Flag-Atrophin-1 (WB: anti-Flag)
HA-SHP (WB: anti-HA)
F5% input IP: anti-Flag
WT ∆H
12 WT ∆H
12
SHP+Atrophin-2
Flag-SHP(WB: anti-Flag)
HA-Atrophin-2 (WB: anti-HA)
Supplementary Figure 10. Two unconventional modes of repression could be present in the same orphan nuclear receptor. (A) Superposition of hTLX (pink) and SHP (hidden)/EID1 (purple) reveals a potential H1 pocket in TLX. Residues that form this potential H1 pocket are highlighted in yellow and labeled. (B) Mutations in the correspond-ing TLX H1 pocket affected TLX repressive activity. Repression fold was calculated by comparing RLU with TLX to RLU without TLX. The results were then analyzed using Student’s independent-sample t test. The statistical signifi-cance level was set to be P < 0.05 (one asterisk). The numbers above asterisks indicate P values. Error bars = SD (n = 3). Western blot was performed to check expression level of SHP mutants in cells. The same amount of cell lysates was loaded. (C) Superposition of SHP (green) and TLX (hidden)/Atrophin (red) reveals a potential auto-repressed pocket in SHP. (D) SHP (NP_ 035980) interacted with Atrophin-2 (NP_001036146, residues 1087-1566) in a co-immunoprecipitation assay. (E) Deletion of the Atro box (residues 1249-1264) in Atrophin-2 compromised the Atrophin-2-SHP interaction (left panel), while deletion of the Atro box (resiudes 879-894) in Atrophin-1 (NP_001007027, residues 720-1191) barely affected the Atrophin-1-SHP interaction (right panel). (F) Deletion of helix H12 in SHP decreased the Atrophin-2-SHP interaction.
0
1
2
3
4
Rep
ress
ion
fold
5
6B
vecto
rWT
W199R
V203R
Q232E
A286D
/C287D
K289A
F294S
L317R
Gal4-hTLXLBD
Flag-hTLXFL (WB: anti-Flag)
0.014*
0.015*
0.048*
GND-254904, Fig. 10. Zhi, et al.