Snf5 and Swi3 subcomplex formation is required for

SWI/SNF complex function in yeast

Hao Zhoua, *, 1, Guidong Chena, 1, Chunming Dongb, 1, Xiaozhou Zhaoa, Zhongtian

Shena, Feilong Chena, Beibei liua, and Jiafu Longa, **

aState Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of

Protein Science, and College of Life Sciences, Nankai University, 94 Weijin Road,

Tianjin 300071, China

bState Key Laboratory of Food Nutrition and Safety, Tianjin University of Science &

Technology, Tianjin 300457, China

* Corresponding author.

** Corresponding author.

E-mail addresses: haozhou@nankai.edu.cn(H, Zhou), jflong@nankai.edu.cn(J, Long)

1These three authors contributed equally to this work.

Keywords: Chromatin remodeling complex, SWI/SNF complex, BAF complex,

Snf5-Swi3 subcomplex

Running title: Crystal structure of the Snf5-Swi3 subcomplex

Snf5_S.c E LVPIRL LRD N N E D ED T E D D W DKLI F D M A KLKVYKQAMNE S Q F QDR RFF TLL K KI D V D LR YRFED TREQHSnf5_C.e E LVPIRL LRD N N E D D NA DMEL DG K FTF EKMITP EIMCE LDL A F LEMSKRVMK EEG D .. I T K MIA PL V... SMARCB1_D.m E LVPIRL LRD N N E D DD NA Q DMEL EG K FTW E MITP F EVLCD LDL L F TDPTASLE A K C .. Q T K S Q A NP P... SMARCB1_D.r E LVPIRL LRD N N E D DD NA Q DMEI DG K FTW EKLMTP F EILCD LDL L F HDPAVIHE S Q V .. Q A M M A SP T... SMARCB1_M.m E LVPIRL LRD N N E D DD NA Q DMEI DG K FTW EKLMTP F EILCD LDL L F HDPAVIHE S P V .. Q A M M S NP T... SMARCB1_H.s E LVPIRL LRD N N E D DD NA Q DMEI DG K FTW EKLMTP F EILCD LDL L F HDPAVIHE S P V .. Q A M M S NP T...

Snf5_S.c I I Q D R KL I VG L DQ EWD S N PE F I Q I F I I I Q Q I F I A M DT C S QE QE QGNPY ELNQDRLGGD L R D V N NSD C E ES Snf5_C.e I I Q D R KL I VG L DQ EWD S N PE F PA A Q LE L Q AVL N N S V F M D S IQ TA N ASTEAPP DVN.....IC N Q PN E SRN SMARCB1_D.m I I Q D R KL I VG L DQ EWD S N PE F VPA QA Q IE F I Q VIV N N S V M E N A L A R A PNDPP LEE.....TC H T V KN E IK SMARCB1_D.r I I Q D R KL I VG L DQ EWD S N PE F VPA SA Q IE Y I Q VII N N S V F M E S A L A R S PTDS. LDE.....QM H I KE K LK SMARCB1_M.m I I Q D R KL I VG L DQ EWD S N PE F VPA SA Q IE Y I Q VII N N S V F M E S A L A R S PTDS. LED.....QS H I KE K LK SMARCB1_H.s I I Q D R KL I VG L DQ EWD S N PE F VPA SA Q IE Y I Q VII N N S V F M E S A L A R S PTDS. LED.....QS H I KE K LK

Snf5_S.c C EL L GEF IA SIR Q A F P T R P VT V KS S LD E F Q E A H E HMYH L LLGYN DG AIEDDDIRSRML TI DVY PAA SKI TSnf5_C.e C EL L GEF IA SIR Q A F P T R G G MS Y G LQW RTY SES L VD T EVE W K G NQ ..... ............ A CPF SS S GSMARCB1_D.m C EL L GEF IA SIR Q A F P T R G G VT Y G LSW RTY SE L ID N DAD W A A HC ..... A............ S VPF PS A ASMARCB1_D.r C EL L GEF IA SIR Q A F P T R G G VT Y G LSW RTY SEN L VE N DAD W S T HQ ..... ............ P IAI TG Q CSMARCB1_M.m C EL L GEF IA SIR Q A F P T R G G VT Y G LSW KTY SEN L VE N DAD W S T HQ ..... ............ P IAI TG Q CSMARCB1_H.s C EL L GEF IA SIR Q A F P T R G G VT Y G LSW KTY SEN L VE N DAD W S T HQ ..... ............ P IAI TG Q C

Snf5_S.c P L AE E D DR TRR RR QIS L R K S N L A LD K D K QGR NRRG P18480Snf5_C.e P L AE E D DR TRR RR ETLTD I KKMR Q N M LV Y F GGGFN Q21831SMARCB1_D.m P L AE E D DR TRR RR ETLTD M KKIR Q N M LA T W F N TTG Q24090SMARCB1_D.r P L AE E D DR TRR RR ETLTD M KKIR Q N M LA T W L N APA Q5U39SMARCB1_M.m P L AE E D DR TRR RR ETLTD M KKIR Q N M LA T W SMARCB1_H.s P L AE E D DR TRR RR ETLTD M KKIR Q N M LA T W L N APA Q12824

450 460440 470 480 490 500

510 520 530 540 550 560 570

580 590 600 610 620 630 640

454

650 660 670 680

170 180 190 200 210 220 230

240 250 260 270 280 290

300 310 320 330 350340

360 370 380

656

Rpt1-β1 Rpt1-β2 Rpt1-α1 Rpt1-α2

Rpt1-α2 Rpt2-β3 Rpt2-β4 Rpt2-α4Rpt2-α3

Rpt2-α5 Rpt2-α6Rpt2-α7

Rpt2-α8

L N APA Q9Z0H3

Rpt2-β5α9 667

Supplementary Figure S1

Snf5(454-680)

Snf5(454-680)

Snf5(454-680)

Snf5(454-680)

* * *

**

Supplementary Figure S1. Structure-based sequence alignment of Snf5 from different species. Multiple sequence alignment of Snf5 or SMARCB1. The conserved residues are shaded red, and the secondary structure of Snf5 according to the crystal structure of Snf5(454-680)/Swi3(212-398) is shown at the top. Constructs in which highly conserved residues in Snf5 were mutated (Snf5(D475A, D483A, D498A), Snf5(T591A), Snf5(D475A, D483A, D498A, T591A)/Snf5(Rpt1*), Snf5(E582A)/Snf5(Rpt2*), or Snf5(D475A, D483A, D498A, E582A, T591A)/Snf5(Rpt1*, Rpt2*)) are indicated with black asterisks. Species abbreviations: H.s, Homo sapiens; M.m, Mus musculus; D.r, Danio rerio; D.m, Drosophila melanogaster; C.e, Caenorhabditis elegans; S.c, Saccharomyces cerevisiae. The GenBank numbers are shown at the end of the alignment.

Swi3_S.c S T S E G ES L A P IP A A I D V G E QE E M DN IF DTKS KQ GN SSV NT E D HK EQEDI EKTESV .KK DS ..E RN R I NDHSwi3_C.e S T S E KR RDDD DV D DD G A V VPK KD E KG LTDLD GA .R DG SM .G D . RS A VK E ..GKE A FSAP QK EGV......SMARCC2_D.r S T S E KRG REEEQ DL ELDEP VP V A LPK V KD D VKGG MTDLD QE E M TK Q E .. .K P A EE T T T.K S STP T D S ETVSMARCC2_M.m S T S E KRG REEEQ DL DMDEP VP V V LPK V KD E VKGG MTDLD QD E M TK H E .. .K P N EE T T NTK S SAP T D S ETTSMARCC2_H.s S T S E KRG REEEQ DL DMDEP VP V V LPK V KD E VKGG MTDLD QE E M TK H E .. .K P N EE T T NTK S SAP T D S ETT

Swi3_S.c E Q H I PSY WF H IE PEFF SKTP K T VI I L T R EVS SAN......PKK TITRVEP TFEIP A E SK NLEK S VQS N IP Swi3_C.e E Q H I PSY WF H IE PEFF SKTP T L NV E T VV A DYN I KRAM NGKNK DV............QS KEAPQ A G I Y G A Q SMARCC2_D.r E Q H I PSY WF H IE PEFF SKTP K L NV E T II A DYN V RRAL NGKNK EIG EEEEGSPSVKGEPVKGSD H D T H A S A SMARCC2_M.m E Q H I PSY WF H IE PEFF SKTP K Q L NV E T II A DYN V RRAL NGKNK EIG DEDENSTGNKGE TKNPD H D T H A S A SMARCC2_H.s E Q H I PSY WF H IE PEFF SKTP K Q L NV E T II A DYN V RRAL NGKNK EIG DEDENSTGNKGE TKNPD H D T H A S A

Swi3_S.c Y YRNFM YRLNP EY T RRN GD R H FL WGL NYQVD

M V S N S V A AL L I

R N F V TA S A F K TK SKL

Swi3_C.e Y YRNFM YRLNP EY T RRN GD R H FL WGL NYQVD

VA VDT VS AC LA VC IV L EQ L D

F A S S S A

SMARCC2_D.r Y YRNFM YRLNP EY T RRN GD R H FL WGL NYQVD

LA IDT Q LT AC LA VCAIM V EQ I E

S A S S

SMARCC2_M.m Y YRNFM YRLNP EY T RRN GD R H FL WGL NYQVD

LA IDT Q LT AC LA VCAIM V EQ I E

S A A S

SMARCC2_H.s Y YRNFM YRLNP EY T RRN GD R H FL WGL NYQVD

LA IDT Q LT AC LA VCAIM V EQ I E

S A A S

410 420400 430 440 450 460

340 350330 360 370 380 390

230 240220 250 260 270

280

470 480 490 500 510

212

398

αA αB

αC αD αE

300 310290 320 330 340

350 370 380360 390

P32591 G5EF87 E7FFZ4 Q6PDG5 Q8TAQ2

Supplementary Figure S2Swi3(212-398)

Swi3(212-398)

Swi3(212-398)

* **

Supplementary Figure S2. Structure-based sequence alignment of Swi3 from different species.Multiple sequence alignment of Swi3 or SMARCC2. The conserved residues are shaded red, and thesecondary structure of Swi3 according to the crystal structure of Snf5(454-680)/Swi3(212-398) is shown at the top. Residues R368, R380, and A376, which were mutated in the Swi3(R368A, R380A) or Swi3(A376E) constructs, are indicated with black asterisks. Species abbreviations: H.s, Homo sapiens; M.m, Mus musculus; D.r, Danio rerio; C.e, Caenorhabditis elegans; S.c, Saccharomyces cerevisiae. The GenBank numbers are shown at the end of the alignment.

Supplementary Figure S3

M(kDa)

14.4

18.4

25

35 (454-656) Snf5

(302-398)Swi3

(454-656) (302-398)Snf5 /Swi3

(454-680) Snf5(212-398)Swi3

M(kDa)

14.418.4

25

35

(454-680) (212-398)Snf5 /Swi3B

C

D

0.0 0.5 1.0-22.00-20.00-18.00-16.00-14.00-12.00-10.00

-8.00-6.00-4.00-2.000.00

-1.00

-0.80

-0.60

-0.40

-0.20

0.00

0 10 20 30 40

Time (min)

Molar Ratio

Rpt2Snf5

↓

Kd:1.18±0.18µM

µc

al/

se

cK

Ca

l/M

ole

of

inje

ca

nt

212 398

(212-398)Swi3

527 656

Supplementary Figure S3. Snf5(454–680) and Swi3(212–398) form a heteromeric complex.

(A) Gel filtration-based test of the interaction between the indicated coexpressed Snf5 and

Swi3 proteins. (B-C) SDS-PAGE of various recombinant, coexpressed Snf5 and Swi3

proteins after gel filtration. The fraction numbers shown here is corresponding to the the

numbers in the (A). (D) ITC-based measurements of the binding affinity between

Swi3(212–398) and Snf5(527–656) (Snf5Rpt2).

(454-680) (212-398)Snf5 /Swi3(454-656) (302-398)Snf5 /Swi3

A

5 10 15 20 25

0

200

400

600

800

1000

OD

28

0 (

mA

U)

Elution Volume (ml)

(454-680)Snf5(212-398)Swi3

fractions20 30 40 50

���������26�27�28�29�30�31�32�33�34�35fraction�number

��������29�30�31�32�33�34�35�36�37fraction�number

180°

A

B

CD

E

F

G

H

L

J

I

K

Snf5: , , , andA D G JSwi3: , , , , , , , andB C E F H I K LB

Supplementary Figure S4

Supplementary Figure S4. The asymmetric unit is composed of 4 copies of the

Snf5(454–680)/Swi3(212–398) complex. The asymmetric unit of the structure is composed of 4

copies of the Snf5(454–680)/Swi3(212–398) complex. The first copy is composed of chains A, B,

and C; the second copy is composed of chains D, E, and F; the third copy is composed of

chains G, H, and I; and the fourth copy is composed of chains J, K, and L.

Supplementary Figure S5. Snf5Rpt1-Rpt2/Swi3SWIRM heterotrimer and SFH1Rpt1-

Rpt2/RSC8SWIRM heterotrimer have a similar assembly mode.

(A) Stereo view of the superimposed structures of Rpt1 (cyan) and Rpt2 (orange) from

Snf5. (B) Structure-based sequence alignment of Rpt1 and Rpt2 from Swi3. The

Supplementary Figure S5

secondary structures of Rpt1 and Rpt2 according to the crystal structure of Snf5(454-

680)/Swi3(212-398) are shown at the top and bottom, respectively, and the conserved

residues are shaded red. (C) Comparison of the structures of Snf5Rpt1-Rpt2/Swi3SWIRM

heterotrimer (color coded) and SFH1-RSC8 subcomplex (color coded) in RSC

substrate-recruitment module (PDB ID 6K15). For simplify, SFH1 and RSC are shown

in cartoon mode and the rest subunits of the substrate-recruitment module are shown in

surface mode. (D) Stereo view of the superimposed structures of the Snf5Rpt1-

Rpt2/Swi3SWIRM heterotrimer (same color coded in (C)) and SFH1Rpt1-Rpt2

(gray)/RSC8SWIRM (light blue and marine) heterotrimer.

Table S1. Data collection and refinement statistics.

Crystal name K2PtCl6 SAD data Native data

Data collection Space group C121 C121

Wavelength (Å) 0.9789 0.9789

Unit cell

a, b, c (Å) 202.60, 146.04, 152.96 205.52, 146.46, 154.33

α, β, γ () 90.00, 130.69, 90.00 90.00, 131.20, 90.00

Resolution range (Å) 50.0-2.90(2.95-2.90) a 50.0-2.65(2.70-2.65) a

No. of unique reflections 74,849 99,168

Rsym (%)b 12.4(62.8) a 14.0(66.3) a

I/σ 13.3(1.8) a 8.8(1.2) a

Completeness (%) 100.0(100.0) a 99.3(98.2) a

Redundancy 5.5(5.7) a 4.3(4.2) a

Refinement Resolution range (Å) 48.6-2.65

Rcrystal (%)c 22.82

Rfree (%)d 27.62

r.m.s.d

Bond length (Å) 0.009

Bond angle () 0.901

Number of

Protein atoms 13,381

Ligends 8

Water moleculars 244

B factor (Å2)

Protein

44.73

Ramachandran plot (%)

Favored 92.3

Allowed 7.4

Disallowed 0.3

a the highest resolution shell.

b IIIRj jsym

c Rcrystal= hkl obshkl calcobs FFF

d Rfree, calculated the same as Rcrystal, but from a test set containing 5% of data excluded from the refinement calculation

Table S2. Yeast strains used in this study.

Strain name Genotype Reference

BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 [1]

YXB01 BY4741 snf5::URA3 This study

YXB02 BY4741 snf5::URA3 pP1K- SNF5::LEU2 This study

YXB03 BY4741 snf5::URA3 pP1K-SNF5(E582A)::LEU2 This study

YXB04 BY4741 snf5::URA3

pP1K-SNF5(D475A,D483A,D498A,T591A)::LEU2

This study

YXB05 BY4741 snf5::URA3 pP1K-

SNF5(D475A,D483A,D498A,E582A,T591A)::LEU2

This study

YXB06 BY4741 URA3 LEU2 This study

YXB07 BY4741 snf5::URA3 LEU2 This study

YXC01 BY4741 swi3::URA3 This study

YXC02 BY4741 swi3::URA3 pP1K- SWI3::LEU2 This study

YXC03 BY4741 swi3::URA3 pP1K- SWI3(A376E)::LEU2 This study

YXC04 BY4741 swi3::URA3 pP1K- SWI3(R368A,R380A)::LEU2 This study

YXC05 BY4741 swi3::URA3 LEU2 This study

P promoter

1. Sen P, Ghosh S, Pugh BF and Bartholomew B (2011) A new, highly conserved domain in

Swi2/Snf2 is required for SWI/SNF remodeling. Nucleic Acids Res 39:9155-66. doi:

10.1093/nar/gkr622