AMPA receptor ligand-binding domain: Site-directed mutagenesis
Solution structure of the Z-DNA binding domain of PKR-like...
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1 Solution structure of the Z-DNA binding domain of PKR-like protein kinase (PKZ) from Carassius auratus and quantitative analyses of the intermediate complex during B–Z transition Ae-Ree Lee 1,† , Chin-Ju Park 2,† , Hae-Kap Cheong 3 , Kyoung-Seok Ryu 3 , Jin-Wan Park 1,3 , Mun-Young Kwon 1 , Janghyun Lee 4 , Kyeong Kyu Kim 5 , Byong-Seok Choi 4 , and Joon-Hwa Lee 1, * 1 Department of Chemistry and RINS, Gyeongsang National University, Gyeongnam 52828, 2 Division of Liberal Arts and Sciences and Department of Chemistry, Gwangju Institute of Science and Technology, Gwangju 61005, 3 Division of Magnetic Resonance, KBSI, Chungbuk 28119, 4 Department of Chemistry, Korea Advanced Institute of Science and Technology, Daejeon 34141, 5 Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Gyeonggi 16419, Republic of Korea Supplementary Information Details of Derivation of Equations 2 Supplementary Figure S1 4 Supplementary Figure S2 5 Supplementary Figure S3 6 Supplementary Figure S4 7 Supplementary Figure S5 8 Supplementary Figure S6 9
Transcript of Solution structure of the Z-DNA binding domain of PKR-like...
1
Solution structure of the Z-DNA binding domain of PKR-like protein kinase (PKZ) from Carassius auratus and
quantitative analyses of the intermediate complex during B–Z transition
Ae-Ree Lee1,†, Chin-Ju Park2,†, Hae-Kap Cheong3, Kyoung-Seok Ryu3, Jin-Wan Park1,3, Mun-Young Kwon1, Janghyun Lee4, Kyeong Kyu Kim5, Byong-Seok Choi4, and Joon-Hwa Lee1,*
1Department of Chemistry and RINS, Gyeongsang National University, Gyeongnam 52828, 2Division of Liberal Arts and Sciences and Department of Chemistry, Gwangju Institute of Science and Technology, Gwangju 61005, 3Division of Magnetic Resonance, KBSI, Chungbuk 28119, 4Department of Chemistry, Korea Advanced Institute of Science and Technology, Daejeon 34141, 5Department of Molecular Cell Biology, Sungkyunkwan University
School of Medicine, Gyeonggi 16419, Republic of Korea
Supplementary Information
Details of Derivation of Equations 2 Supplementary Figure S1 4 Supplementary Figure S2 5 Supplementary Figure S3 6 Supplementary Figure S4 7 Supplementary Figure S5 8 Supplementary Figure S6 9
2
Details of Derivation of Equations. The possible pathways of B–Z transition of the 6-bp DNA duplex induced by Z-DNA binding proteins are given by:
2
2
ZPZPZ
BPBPB
PP
PP
(Scheme S1)
where P indicates the Z-DNA binding proteins and B and Z indicate the B-form and Z-form of 6-bp DNA duplexes, respectively. Based on the active B–Z transition mechanism reported previously,8 the B–Z transition pathway could be simply given by:
2ZPZPBPB PP (Scheme S2)
The total amount of added proteins ([P]tot) is the summation of the concentrations of the free protein (P) and protein complexes (BP, ZP, and ZP2):
][2][][][][ 2ZPZPBPPP tot (S1)
The dissociation constants for the BP and ZP2 complexes are given by:
][
]][[, BP
PBK BPd (S2)
][
]][[
22, ZP
PZPK ZPd (S3)
The equilibrium constant between the BP and ZP complexes is given by:
][
][1, BP
ZPKBZ (S4)
The total concentration of DNA duplexes is given by:
][][][][][ 2ZPZPBPBN tot (S5)
Eq. S2 – S4 and S5 give rise to:
2,,
21,
,
1,
,
]][[]][[]][[][][
ZPdBPd
BZ
BPd
BZ
BPdtot KK
PBK
K
PBK
K
PBBN (S6)
The relative populations of each complex state compared to [N]tot are given by:
21,2,1,2,2,,
2,,
][][][][
][
PKPKKPKKK
KK
N
B
BZZPdBZZPdZPdBPd
ZPdBPd
tot (S7)
21,2,1,2,2,,
2,
][][][
][
][
][
PKPKKPKKK
PK
N
BP
BZZPdBZZPdZPdBPd
ZPd
tot (S8)
21,2,1,2,2,,
2,1,
][][][
][
][
][
PKPKKPKKK
PKK
N
ZP
BZZPdBZZPdZPdBPd
ZPdBZ
tot (S9)
3
21,2,1,2,2,,
21,2
][][][
][
][
][
PKPKKPKKK
PK
N
ZP
BZZPdBZZPdZPdBPd
BZ
tot (S10)
Eq. S7 – S10 and S1 give rise to:
21,2,1,2,2,,
21,2,1,2,
][][][
][2][][][][][
PKPKKPKKK
PKPKKPKNPP
BZZPdBZZPdZPdBPd
BZZPdBZZPdtottot
(S11)
Eq. S11 becomes Eq. S12:
0][})][]([)1]{([
})1()][][2({][][
2,,2,,2,1,
2,1,1,23
1,
totZPdBPdZPdBPdtottotZPdBZ
ZPdBZtottotBZBZ
PKKKKPNKKP
KKPNKPPK (S12)
Eq. S12 is simply expressed by:
0][][][ 23 cPbPaP (S13)
where 2,1,
11][][2 ZPd
BZtottot K
KPNa
,
1,
2,,2,
1,
)][]([1
1BZ
ZPdBPdtottotZPd
BZ K
KKPNK
Kb
,
totBZ
ZPdBPd PK
KKc ][
1,
2,,
Thus the closed-form solution of Eq. S13 has been reported as:31
3cos3
3
2
3][ 2
baa
P (S14)
where 32
3
32
2792arccos
ba
caba
10 9 8 7135
130
125
120
115
110
15N
(pp
m)
PKZfree caZα
PKZcaZα - dT(CG)3
10 mM NaClA
W60-Hε1
A23Q45
W60
F51
L62
R16
L44
L41D13
K34
V50
I30
G31
Q49 S47
G19G68
N43-Hδ2 T66
Q17T25
I26
N18-Hδ2
N52-Hδ2
Q49-Hε2
K10
N18
K74A3
K70
V73
T75Q71
S21
N54 L24
S35
K56
H40
R39
V59
E55
R9
T5S53
E29K28
Y42N38A27
E4
I22
N38-Hδ2
N52D61 N48
D33K20
M7E8
I69
R46K65
N67E64
L15
Q6I12
F14
I11
M63
T36 V37N43
L32
B
4
10 mM NaCl
∆δav
g (p
pm)
0.0
0.2
0.4
0.6
0.0
0.2
0.4100 mM NaCl
Residue0 10 20 30 40 50 60
0.0
0.2250 mM NaCl
α1 α2 β2α3β1 β3
1H (ppm)
Supplementary Fig. S1. (A) Superimposition of 1H/15N-HSQC spectra of free caZαPKZ (blue) and caZαPKZ–dT(CG)3 complex ([N[tot/[P]tot = 0.6, red) in NMR buffer (pH = 6.0) containing 10 mM NaCl at 35 oC. (B) The weighted average of 1H and 15N chemical shift changes (Δδavg) of caZαPKZ upon binding to dT(CG)3 at 10 (upper), 100 (middle), or 250 mM NaCl (lower). Residues whose cross-peaks disappear during titration are represented with green square symbols. The color used to illustrate the Δδavg is: red or blue, > 0.18 ppm; orange or cyan, 0.12 – 0.18 ppm; and yellow or pale green, 0.08 – 0.12 ppm.
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■■■■
■■
■■
■■■■■■■
[N]tot / [P] tot
0
1.0
2.0
10 mM NaClA
135
130
125
120
115
110
15N
(pp
m)
10 9 8 7 6
Q49
H40
I30
A23
L24
K56
V59
D33
T25
A27
N18
G19
K20
E29S35
Q45
S53
E55
W60
D13
I22
N54
B 100 mM NaCl
10 9 8 7 6
135
130
125
120
115
110
15N
(pp
m)
1H (ppm)
Q49
H40
I30
A23
L24
K56
V59
D33
T25
A27
N18
G19
K20
E29
S35
Q45
S53
E55
W60
D13
I22
N54
Supplementary Fig. S2 . The change in 1H/15N-HSQC spectra of caZαPKZ by addition of dT(CG)3 in NMR
buffer (pH = 6.0) containing (A) 10 or (B) 100 mM NaCl at 35 oC. The cross-peak color changes gradually from blue (free) to red (bound) according to the [N]tot/[P]tot ratio.
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10 mM NaCl
Residue0 10 20 30 40 50 60
N (p
pm)
-2
0
2
B-DNA boundZ-DNA bound
∆δ
0 10 20 30 40 50 60-2
0
2
Residue
N (p
pm)
∆δ
100 mM NaCl
B-DNA boundZ-DNA bound
0 10 20 30 40 50 60-0.4
0.0
0.4
Residue
H (p
pm)
∆δ
0 10 20 30 40 50 60-0.4
0.0
0.4
Residue
H (p
pm)
∆δ
A
B
α1 α2β2
α3β1 β3
α1 α2β2
α3β1 β3
α1 α2β2
α3β1 β3
α1 α2β2
α3β1 β3
0 10 20 30 40 50 60
-1
0
1
freeB-DNA boundZ-DNA bound
Cα1 α2
β2α3
β1 β3α1 α2
β2α3
β1 β3
B-DNA boundZ-DNA bound
B-DNA boundZ-DNA bound
0 10 20 30 40 50 60
-0.2
0.0
0.2
freeB-DNA boundZ-DNA bound
Residue
N (p
pm)
∆δ
Residue
H (p
pm)
∆δ
Supplementary Fig. S3 . (A,B) The 15N (left) and 1H (right) chemical shift differences between the free and
bound form for B-DNA (red) and Z-DNA binding (blue) of caZαPKZ to dT(CG)3 at (A) 10 and (B) 100 mM NaCl at 35 oC. (C) The 15N (left) and 1H (right) chemical shift differences between [NaCl] of 10 and 100 mM for free caZαPKZ (yellow circle) and caZαPKZ bound to B-DNA (magenta circle) and Z-DNA (cyan circle) at 35 oC.
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10 9 8 7 6135
130
125
120
115
110250 mM NaCl100 mM NaCl10 mM NaCl
L24
W60-Hε1
A23
K28N38
A27
L41
R9
N43-Hδ2
R39
pH = 6.0
15N
(pp
m)
V50G68 G19
G31S47
Q49
T66
T25Q17
I26
W60
K74
K70
A3
Q45
L44
R16
K34
L62
F51
N54
S35
S21
D13S2 E4
I22
N18
N52
S53 T5
T72N48
D61
Y42H40
E29
V59
K10E8K20
D33K56 M7
Q6
V73
R46T75
T36 I11
E64
Q71
F14
I12 I69 L32
V37 N43M63K65
E55
1H (ppm)
Supplementary Fig. S4. Superimposition of 1H/15N-HSQC spectra of free caZαPKZ in NMR buffer (pH 6.0) containing 10 (red), 100 (blue), or 250 mM NaCl (green) at 25 oC.
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A
0.0 0.5 1.0 1.5 2.0 2.5
Con
c. (m
M)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
[P]tot
[N]tot
[P][B][BP][ZP][ZP2]
[N]tot / [P] tot
0.0 0.5 1.0 1.5 2.0 2.5
Con
c. (m
M)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
B
10 mM NaCl
100 mM NaCl
[N]tot / [P] tot
Supplementary Fig. S5. (A,B) The concentrations of the total caZαPKZ ([P]tot), total dT(CG)3 ([N]tot), free
caZαPKZ ([P]), free B-DNA dT(CG)3 ([B]), and each states of the caZαPKZ–dT(CG)3 complex ([BP], [ZP], and [ZP2]) as a function of the [N]tot/[P]tot ratio at (A) 10 or (B) 100 mM NaCl.
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∆δH
(ppm
)
∆δN
(ppm
)
D13N18G19K20I22A23L24T25A27E29I30D33S35H40Q45Q49S53N54E55K56V59 W60
dT(CG)3 pH = 8.0
10 mM NaCl
A
100 mM NaCl
d(CG)3 pH = 8.0B
[N]tot / [P]tot
0.0 0.5 1.0 1.5 2.0-0.2
-0.1
0.0
0.1
0.2
∆δH
(ppm
)
10 mM NaCl
0.0 0.5 1.0 1.5 2.0
-1.0
-0.5
0.0
0.5
1.0
∆δN
(ppm
)
-0.2
-0.1
0.0
0.1
0.2
-1.0
-0.5
0.0
0.5
1.0
0 1 2 3-0.2
-0.1
0.0
0.1
0.2
0 1 2 3
-1.0
-0.5
0.0
0.5
1.0
[N]tot / [P]tot
∆δH
(ppm
)
∆δN
(ppm
)
[N]tot / [P]tot [N]tot / [P]tot
Supplementary Fig. S6. (A) Global fitting of the 1H/15N-HSQC titration curves for caZαPKZ with dT(CG)3 as a
function of [N]tot/[P]tot ratio at pH 8.0. Data for the global fitting derived from 1H (left) and 15N (right) chemical shift changes of HSQC cross peaks of caZαPKZ at 10 (upper) or 100 mM NaCl (lower). (B) Global fitting of the 1H/15N-HSQC titration curves for caZαPKZ with d(CG)3 as a function of [N]tot/[P]tot ratio at pH 8.0. Data for the global fitting derived from 1H (left) and 15N (right) chemical shift changes of HSQC cross peaks of caZαPKZ at 10 mM NaCl.
