gnd-254904, fig. s1. zhi, et...

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.


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


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


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.


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.


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


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


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


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



5% input IP: anti-Flag

WT ∆A

tro WT ∆A

tro

Atrophin-1+SHP



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.

gnd-254904, fig. s1. zhi, et...

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