snf5 and swi3 subcomplex formation is required for swi/snf ...€¦ · snf5 and swi3 subcomplex...
TRANSCRIPT
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: [email protected](H, Zhou), [email protected](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