Supplementary Information Structure of the VP16 ......Supplementary Information Structure of the VP16 Transactivator Target in ARC/Mediator Alexander G Milbradt1, Madhura Kulkarni2,3*,

Supplementary Information

Structure of the VP16 Transactivator Target in ARC/Mediator

Alexander G Milbradt1, Madhura Kulkarni2,3*, Tingfang Yi1*, Koh Takeuchi1,4, Zhen-Yu J Sun1,

Rafael E Luna1, Philipp Selenko1,4, Anders M Näär 2,3 and Gerhard Wagner1

1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School,

240 Longwood Avenue, Boston, MA 02115, 2Massachusetts General Hospital Cancer Center,

Building 149, 13th Street, Charlestown, MA 02129, 3Department of Cell Biology, Harvard Medical

School, 240 Longwood Avenue, Boston, MA 02115.

4Present addresses: Biomedical Information Research Center, National Institute of Advanced

Industrial Science and Technology, Aomi 2-3-26, Koto, Tokyo, 135-0064 Japan

[email protected], T.K.; Leibniz Institute of Molecular Pharmacology (FMP) Department

of NMR-assisted Structural Biology, Robert-Roessle-Strasse 10 13125 Berlin, Germany

[email protected], P.S.

* These authors contributed equally to this work.

Correspondence should be addressed to A.M.N. ([email protected]) or G.W. ([email protected]).

Nature Structural & Molecular Biology doi:10.1038/nsmb.1999

a

bCore domain TADVP16 1 490

MED25 NTD VBD391 553

1 747

411

420 430 440 450 460

HSV1 TAP TDVSLGDEL L GE V M DALDDFDL MLGD P PG T HD YS P H D D A AHA D GDS G F P SAP Chimpanzee_sv TAP TDVSLGDEL L GE V M DALDDFDL MLGD P PG T HD YS L R G E D TPV D VEF S M . PVP HSV2 TAP TDVSLGDEL L GE V M DALDDFDL MLGD P PG T HD YT I R D E D TPA E VES S M . PVS

470 480 490

HSV1 GALD DFEFEQMFTDALGID GG MA EYChimpanzee_sv GALD DFEFEQMFTDALGID GG VA DFHSV2 GALD DFEFEQMFTDALGID GG VD DF

MED25 NK LAWSGV EWQE P R LP YVN GE L T QWPGQQSVS L L KPK ASVDANTKLT S CQV H N K EPTOV1_NTD NK LAWSGV EWQE P R LP YVN GE L T QWP.EHRLS L L KRR YS.DSTAKLK T CQA Q N E DPTOV1_CTD NK LAWSGV EWQE P R LP YVN GE L T QWP..QIVN F M .PR ...EPNSRSK W SHV Q I R E

MED25 KL MQLIPQQLLTTL PLFRNS QFHFT D LK L RIM NGFAGC FQ I G RMV NK LES G Y G VH PTOV1_NTD KL MQLIPQQLLTTL PLFRNS QFHFT D LK L RIM NGFAGC FQ I G QLA NR CDS G C G ML PTOV1_CTD KL MQLIPQQLLTTL PLFRNS QFHFT D LK L RIM NGFAGC FR Y V RLV .K LET S C D VH

400 410 420 430 440

450 460 470 480 490 500

510 520 530 540 550

MED25 CE RVLMLLYSS KKIF GLIP DQ FV IR VI QPHTAP V K M Y SG NG Q TNHK VQQQKLEPTOV1_NTD CE RVLMLLYSS KKIF GLIP DQ FV IR VI QPHISP V K M Y SG SA Q TTRK A......PTOV1_CTD CE RVLMLLYSS KKIF GLIP DQ FV IR VI QSYKAS I E I H GN NG R ANQQ VLQRNLE

β1 β2

β3 α1 β4 α2

β6 α3

β5

β7

TADcTADn

TADc

Supplementary Figure 1. Sequence alignments of the MED25 VBD and VP16 TAD. (a) Sequence of the human MED25 VBD aligned with the homologous C- and N-terminal domain of human PTOV1. Secondary structure elements are shown above the sequences. (b) Sequence of the VP16 TAD from Herpes simplex virus I aligned with sequences of TADs from other viruses. Alignments were visualized with ESPript1. Conserved residues are highlighted in red.


E 442

N467

A504

R 538

M470

H544

H435

V 508

K 398

S 416

G462

V 417

C 429

E 410

L513

K 411

T 424Q409

V 534

L448

R 488L461

S 396

T 476

Y 487

99

N477

N535

F533

E 553

Q548

V 431N438

W 402

K 545

V 395

204

S 482

A415

Q530

K 518

W NE 402

V 405F500

G524

202

Q455

T 441

S 468

F522

Q456

Q392

Y 432F473

I541

V 510R 469

V 47181

T 503

S 531

G436

S 394

I449

M450

Q430

V 540

R 425

T 421

W NE 444

R 509I537

E 507

K 447

T 542

F494

160

L457

K 440

58

C 497

N492

V 547

73

I453

L464Q446

L552Q451

K 484M490

I521

208

L525

Q546 107

Q443

L400

M523

T 459

S 517

V 433

G532

L514

V 498

K 520

A419W 408

A495

L406

199

163 L423 K 422

S 426

L439

N434

E 437

D418

N397

S 403

D479

L511

G491

G496

W NE 408

K 519

M512

G391

S 516

Q539

T 460

F465

L399

L486

Q393

K 551L480

E 407

K 478

F390

E 481

L427

G493

H499

L458

H474 Q472

I489

Y 528

G404

K 413

L483

N420

G485

N543

I526

D529

W 444Y 515

G536

A401

193

L452

R 466

F475

10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5

105

108

111

114

117

120

123

126

129

132

15N

(pp

m)

H (ppm)1

Supplementary Figure 2. 1H-15N-HSQC spectrum of the MED25 VBD with the assigned residue numbers.


Loop β2-β3435 - 442

Loop β5-β6500 - 508

0

20

40

60

80

140

160

180

200

391

394

397

400

403

407

410

413

416

419

422

425

428

431

434

437

440

443

447

450

453

456

459

462

466

469

472

475

478

481

484

487

490

493

496

499

502

507

510

513

516

519

522

525

528

531

534

537

540

543

546

549

552

a

b

Loop β2-β3435 - 442

Loop β2-β3435 - 442

Loop β5-β6500 - 508

Loop β5-β6500 - 508

T R

elax

tion

time

in m

s2

120

100

20

Supplementary Figure 3. 15N T2 relaxation time measurement of the MED25 VBD. (a) 15N T2 relaxation times of the free MED25 VBD are plotted against the residue number. Error bars represent s.d. The N- and C-terminus and the long loop between 1 and 2 display significantly longer T2 times, indicating local flexibility. Due to line broadening and peak overlap, T2 times of the residues forming loop 5/ 6 could only be determined for T503. (b) The 25 lowest energy structures of the MED25 VBD are shown overlaid on the secondary structure elements. Loop

2/ 3 and loop 5/ 6 close the barrel from the bottom. Nature Structural & Molecular Biology doi:10.1038/nsmb.1999

: : : : | : : : : : : : : | : MED25 VBD GEFGQQSVSNKLLAWSGVLEWQEKPKPASVDANTKLTRSLPCQVYVNHGENLKTEQWPQKLIMQLIPQQLLTTLGPLFRNSRMVQFHFTNKDLESLKGLYRIMGNGFAGCVHFPHTAPCEVRVLMLLYSSKKKIFMGLIPYDQSGFVNGIRQVITNHKQVQQQKLKU70/PDB:1JEQ -YISK---TRKRALSRLKLKLN--------------DIVISVGIYNVQKA-----FDDPGLMLGFKPLVLL--KKHHY-LRPSLFVYPEEGSSTLFSALLIKCLEEVAALCRYT-RNIP-PYFVALVPQEEPPGFQLVFL-----PFAD----------------SPOC/PDB:1OW1 -PVDM-LLKKYPIVWQGLLALK--------------NDTAAVQLHFV--GNNVHRSLPLPLRIQRMRLEAQLEGARRMTDYCLLLALPCSQTE-SLKAFITYLQAQAAGIINVPNP-----YVLQIFPPCISPHLMIVIASV-----------------------KU80/PDB:1JEQ FVQR----RHSIH-WPCRLTIG-------------SNLSIRIAAYK--------SEGK-CFSVGFCKSSQVQ--RRFFMGNQVLKVFAAAAAV-ALSSLIHALDDDMVAIVRY--KRAN-PQVGVAFPHIKYECLVYVQL-----PFME----------------

: : : : | : : : : 100 : : : : | : MED25 VBD LLLLLLLLLLEEEEEEEEEEEELLLLLLLLLLLLLLEEEEEEEEEEELLLLLLHHHLLLEEEEEEEEHHHHHHHHHHHLLEEEEEEEELLLLHHHHHHHHHHHHHHEEEEEELLLLLLLLLLEEEEEELLLLLLEEEEEELLHHHHHHHHHHHHHHHHLLLLLLLKU70/PDB: 1JEQ -HHHH---LLLLLLEEEEEELL--------------LLEEEEEEELLLLL-----LLLLEEEEEEEEHHHL--LLLLL-LLLLEEEEELLLHHHHHHHHHHHHHHLEEEEEEEE-LLLL-LEEEEEEEELLLLEEEEEEL-----LLHH----------------SPOC/PDB: 1OW1 -LLLL-HHHHLLEEEEEEEEEL--------------LEEEEEEEEEE--ELHHHHHLLLLEEEEEEELLHHHHHHHHLLLEEEEEEEELHHHH-HHHHLHHHHHHLEEEEEEELLL-----EEEEEELLLLLLLEEEEEEEL-----------------------KU80/PDB: 1JEQ HHHH----LLLLL-EEEEEEEL-------------LLEEEEEEEEE--------LLLL-EEEEEEEEHHHLL--HHHLEEEEEEEEEELHHHH-HHHHHHHHHHHLEEEEEEE--LLLL-LEEEEEEEEELLEEEEEEEL-----LLHH----------------

α2α1 α3β1 β2 β3 β4 β5 β6 β7

10 20 9060504030 1301201101008070 140 150 160

Supplementary Figure 4. Structural homology search by DALI2. Alignment of amino acid sequences (upper panel) and structural elements (lower panel) with the three structural homologous proteins of MED25 VBD: KU70/PDB: 1JEQ3, SPOC/PDB: 1OW14, KU80/PDB:1JEQ3. Extended regions are highlighted in red and helical portions are shown in blue (lower panel).


a

2.00.0 3.01.0 4.0

Molar ratio

kcal

mol

e in

ject

ant

0.0

-0.5

K =1.6 μMd

-10

-8

-6-4

-2

0μ

cal s

ec

Time (min)0 20 40 60 80 100

K ~50 nMd

Molar ratio

kcal

mol

e in

ject

ant

-10-8-6-4-20

-12

μca

l sec

0.0

-0.2

-0.4

0 20 40 60 80 100Time (min)

2.00.0 3.01.0

b

-1-1

-1-1

Supplementary Figure 5. Isothermal calorimetry titration. (a) Isothermal calorimetry titration of MED25 VBD with VP16 TADn revealed a Kd of 1.6 μM. (b) Isothermal calorimetry titration of MED25 VBD with VP16 full-length TAD revealed a Kd of approximately 50 nM. The C-terminal portion of the VP16 TAD contributes to the binding to MED25 VBD and likely accounts for the difference in Kd between TADn and TAD.


9.5 9.0 8.5 8.0 7.5 7.0 6.5

130

125

120

115

110

105

9.5 9.0 8.5 8.0 7.5 7.0 6.5

130

125

120

115

110

105

++

WT VBD Q451E VBDN TAD15

N TAD15

N TAD15

15N

(pp

m)

15N

(pp

m)

15N

(pp

m)

H (ppm)1 H (ppm)1

9.5 9.0 8.5 8.0 7.5 7.0 6.5

130

125

120

115

110

105

H (ppm)1

a b c

Supplementary Figure 6. The MED25 VBD Q451E mutation on 3 adjacent to the hydrophobic pocket also disrupts binding of full-length VP16 TAD to MED25 VBD. 1H-15N-HSQC spectra of free VP16 TAD (a), at 1:1 ratio of wild-type MED25 VBD (b) and with 1:1.5 excess of Q451E MED25 VBD (c) show that VP16 TAD only loosely binds, as seen by minor chemical shift changes, to mutant MED25 VBD without adopting a folded conformation. Far-shifted and broadened signals caused by the addition of wild-type MED25 VBD (b), are missing when the mutant MED25 VBD is added to 15N-labeled VP16 TAD (c).


9.5 9.0 8.5 8.0 7.5 7.0 6.5130

125

120

115

110

Phe442

Leu444

Gly448

Met447Asp441

Leu446

105

15N

(pp

m)

H (ppm)1

Asp445

Asp439

Supplementary Figure 7. 1H-15N-HSQC spectra of the VP16 TAD (black signals) and the VP16 TADn (red signals) both in the presence of 1.3 equivalents MED25 VBD. Far-shifted resonances overlap for both peptides and are indicated by circles and assignments.


10 9 8 7

130

125

120

115

110

Gly524

Leu525

Gln456

Ile521

Val471

TADc

TADc

TADn

1 H (ppm)

15N

(pp

m)

Gln456

Gln456Ile521

Ile521

V471

Leu525

Gly524

a

b

β4

β7α1

β6

β4 β7

α3

β6

α3α2

α1

α2

β3

β5

β5β1

105

Supplementary Figure 8. Mapping the TADc binding site on MED25 VBD. (a) Overlay of 1H-15N-TROSY-HSQC5 spectra of the MED25 VBD bound to the VP16 TADn (red) and the MED25 VBD bound to the VP16 TAD (black). (b) A cartoon representation of MED25 VBD shows the clustering of the residues experiencing distinct chemical shift changes upon interaction with VP16 TADc on 4 and 7. These residues are highlighted in blue. The TADn and TADc binding sites are located on different sides of the MED25 VBD barrel.


WT K447E Q451E H499E K545E

anti-Flag

Supplementary Figure 9. Wild-type and mutant MED25 VBD displayed comparable level of expression in transfected HEK293T cells when immunoblotted with anti-Flag antibody.


Supplementary Table 1. Ramachandran Plot Summary of the MED25 VBD structure from PROCHECK6.

Ramachandran Plot Summary from PROCHECK

Most favoured regions 89.1%

Additionally allowed regions 10.4%

Generously allowed regions 0.5%

Disallowed regions 0.0%

Supplementary References 1. Gouet, P., Courcelle, E., Stuart, D.I. & Metoz, F. ESPript: analysis of multiple sequence

alignments in PostScript. Bioinformatics 15, 305-308 (1999). 2. Holm, L. & Sander, C. Protein structure comparison by alignment of distance matrices. J

Mol Biol 233, 123-138 (1993). 3. Walker, J.R., Corpina, R.A. & Goldberg, J. Structure of the Ku heterodimer bound to

DNA and its implications for double-strand break repair. Nature 412, 607-614 (2001). 4. Ariyoshi, M. & Schwabe, J.W. A conserved structural motif reveals the essential

transcriptional repression function of Spen proteins and their role in developmental signaling. Genes Dev 17, 1909-1920 (2003).

5. Pervushin, K., Riek, R., Wider, G. & Wüthrich, K. Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci U S A 94, 12366-12371 (1997).

6. Laskowski, R.A., Rullmannn, J.A., MacArthur, M.W., Kaptein, R. & Thornton, J.M. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR 8, 477-486 (1996).


Documents

Supplementary Information Structure of the VP16 ......Supplementary Information Structure of the VP16 Transactivator Target in ARC/Mediator Alexander G Milbradt1, Madhura Kulkarni2,3*,