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Supporting Information for:

NMR Dynamics Study Reveals the Zα Domain of Human ADAR1

Associates with and Dissociates from Z-RNA More Slowly than Z-DNA

Ae-Ree Lee,† Jihyun Hwang,‡ Jeong Hwan Hur,§ Kyoung-Seok Ryu,# Kyeong Kyu Kim,§   

Byong-Seok Choi,‡ Nak-Kyoon Kim,*,¶ and Joon-Hwa Lee*,†

†Department of Chemistry and RINS, Gyeongsang National University, Gyeongnam 52828, South Korea

‡Department of Chemistry, Korea Advanced Institute of Science and Technology, Daejeon 34141, South Korea

§Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Gyeonggi 16419, South Korea

#Protein Structure Research Team, Korea Basic Science Institute, Chungbuk 28119, South Korea

¶Advanced Analysis Center, Korea Institute of Science and Technology, Seoul 02792, South Koreae

*To whom correspondence may be addressed.

E-mail: joonhwa@gnu.ac.kr (J.-H.L.); nkkim@kist.re.kr (N.-K.K.).

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Supporting Information Table S1. Two-state fast exchange model for the CPMG data of the hZαADAR1–d(CG)3 complex.a

Residue Δωmax (Hz)Individual fitting Global fitting

kex (s−1) ΔωFB (Hz) kex (s−1) ΔωFB (Hz) ΔΔω (Hz)b

Group I

K154 112 414±50 97±4

511±8

102±5 10

A158 125 301±51 115±6 129±4 4

S162 201 433±15 174±2 181±3 20

S178 286 541±13 299±3 295±2 9

L179 283 543±16 265±3 260±2 23

A180 176 419±32 153±4 161±3 15

Q186 163 434±23 159±3 166±3 3

A189 127 328±51 116±5 128±4 1

W195 148 457±26 127±3 130±4 18

K196 266 515±23 258±4 257±2 9

I197 151 449±39 145±4 150±4 1

Group II

K170 543 735±88 514±8

762±16

520±5 23

I172 467 710±24 402±5 412±6 55

R174 375 638±42 292±7 310±7 65

V175 622 836±43 518±11 500±5 122

T191 496 770±13 428±3 426±6 70

Group III

E171 1,123 1,815±175 955±53

1,530±104

871±34 248

N173 858 1,340±159 781±47 825±33 33

Y177 938 1,552±190 789±52 783±32 155

a During fitting, the pF values were restrained to 0.87. b ΔΔω = |ΔωFB – Δωmax|.

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Supporting Information Table S2. Pseudo-three-state exchange model for the CPMG data of the hZαADAR1–d(CG)3 complex.a

Residue

HSQC data CPMG data

Δδmax (ppm) ΔδBZ

(ppm)b kex (s−1) ΔωFB (Hz)d ΔδFB

(ppm) kZN (s−1) ΔωNZ (Hz)d

ΔδNZ (ppm)

K154 0.22 0.05

1,207±28

109±45 0.22

12.7±0.6

37±19 0.07

A158 0.25 0.13 123±40 0.25 54±14 0.11

S162 0.40 0.21 194±26 0.39 67±12 0.13

K170 1.08 0.17 502±13 1.00 306±12 0.61

E171 2.23 n.d.c 670±13 1.33 1,300±44 2.59

I172 0.93 0.10 327±17 0.65 308±12 0.61

N173 1.71 n.d. 606±14 1.21 706±32 1.41

R174 0.75 n.d. 336±16 0.67 83±14 0.17

V175 1.24 n.d. 420±17 0.84 501±24 1.00

Y177 1.87 n.d. 550±14 1.09 969±31 1.93

S178 0.57 0.09 338±16 0.67 100±11 0.20

L179 0.56 0.21 302±17 0.60 85±11 0.17

A180 0.35 0.03 172±29 0.34 59±13 0.12

Q186 0.32 0.08 177±28 0.35 61±13 0.12

A189 0.25 0.05 124±40 0.25 52±14 0.10

T191 0.99 0.14 354±16 0.70 313±12 0.62

W195 0.29 0.06 143±35 0.28 45±16 0.09

K196 0.53 0.05 290±18 0.58 89±11 0.18

I197 0.30 0.06 164±30 0.33 54±14 0.11

a During fitting, the pF values were restrained to 0.87. b Calculated by ΔδBZ = |ΔδZ – ΔδB| in Figure 4c. c Not determined. d Data at 800 MHz.

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Supporting Information Table S3. Pseudo-three-state exchange model for the CPMG data of the hZαADAR1–r(CG)3 complex.

Residue HSQC data CPMG data

Δδmax (ppm) kex (s−1) ΔωFB (Hz)a ΔδFB (ppm) kZN (s−1) ΔωNZ (Hz)a ΔδNZ (ppm)

K154 0.23

3.03±0.07

97±6 0.19

2.15±0.08

< 10 < 0.02

A158 0.20 110±6 0.22 < 10 < 0.02

S162 0.45 198±11 0.39 < 10 < 0.02

E171 2.74 645±16 1.28 1,240±36 2.47

I172 1.18 327±17 0.65 272±13 0.54

N173 1.59 435±18 0.87 819±37 1.63

R174 0.93 472±15 0.94 270±13 0.54

V175 1.52 335±11 0.67 688±30 1.37

Y177 1.84 488±17 0.97 816±36 1.62

S178 0.48 217±12 0.43 112±10 0.23

L179 0.50 262±9 0.52 145±10 0.29

A180 0.45 208±12 0.41 < 10 < 0.02

Q186 0.27 157±35 0.31 50±46 0.10

A189 0.72 255±9 0.51 143±10 0.28

T191 2.85 831±27 1.65 1,175±38 2.34

W195 0.54 238±10 0.47 < 10 < 0.02

K196 0.50 249±10 0.50 114±9 0.23

a Data at 800 MHz.

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Supporting Information Figure S7. 1D imino proton spectra of the mixture of d(CG)3 and r(CG)3 upon titration with hZαADAR1 in NMR buffer (pH = 6.0) at 35 °C. The resonances from B-form DNA are labeled as dG2b and dG4b, those from A-form RNA are labeled as rG2a and rG4a, and those from Z-form DNA and RNA are labeled as dG2z, dG4z, rG2z and rG4z, respectively.

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Supporting Information Figure S8. Bio-Layer Interferometry sensograms of (a) DNA [d(CG)6] (b) RNA [r(CG)6] binding of hZαADAR1 at various concentrations at 35 °C.

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Supporting Information Figure S9. Superimposition of 1H/15N-HSQC spectra of free hZαADAR1 (blue), hZαADAR1−d(CG)3 complex (green) and hZαADAR1−d(CG)3 complex (red) at 35 °C. The 1H/15N-HSQC spectra, in which the threshold of spectra is decreased, are shown in the inset boxes.

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Supporting Information Figure S10. The change in 1H/15N-HSQC spectra of hZαADAR1 upon addition of d(CG)3 in NMR buffer (pH = 6.0) at 35 °C. The cross-peak color changes gradually from blue (free) to red (bound) according to the [N]t/[P]t ratio.

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Supporting Information Figure S11. The change in 1H/15N-HSQC spectra of hZαADAR1 upon addition of r(CG)3 in NMR buffer (pH = 6.0) at 35 °C. The cross-peak color changes gradually from blue (free) to red (bound) according to the [N]t/[P]t ratio.

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Supporting Information Figure S12. The 15N CPMG relaxation dispersion data for (a) residues K154, A158, S162, S178, L179, A180, Q186, A189, W195, K196 and I197 (group I), (b) residues K170, I172, R174, V175 and T191 (group II) and (c) residues E171, N173 and Y177 (group III) in the hZαADAR1–d(CG)3 complex. Blue and red solid lines indicate the best individual and global fits to eq 7, respectively.

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Supporting Information Figure S13. Global fitting analysis of the 15N CPMG relaxation dispersion data for (a) 3 residues in group III and (b) 8 residues in groups II and III in the hZαADAR1–d(CG)3 complex using a pseudo-three-state conformational exchange model including two independent two-state exchange processes. Solid lines indicate the best global fits to eq 1.

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Supporting Information Figure S14. Global fitting analysis of the 15N CPMG relaxation dispersion data for all 19 residues in the hZαADAR1–d(CG)3 complex using a pseudo-three-state conformational exchange model including two independent two-state exchange processes. Solid lines indicate the best global fits to eq 1.

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Supporting Information Figure S15. Global fitting analysis of the 15N CPMG relaxation dispersion data for all 17 residues in the hZαADAR1–r(CG)3 complex using a pseudo-three-state conformational exchange model including two independent two-state exchange processes. Solid lines indicate the best global fits to eq 2.

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Supporting Information Figure S16. Comparison of the structures of hZαADAR1 in the d(CG)3-binding forms (left, cyan, PDB ID: 1QBJ) and r(CG)3-binding (right, magenta, PDB ID: 2GXB). The atom colors used to illustrate the Δδavg are: blue, >0.08 ppm; and cyan, 0.04–0.08 ppm (the same color coding is used in panel A). Intermolecular H-bonds are indicated by green solid lines. The water molecules in key positions within the protein–DNA or protein–RNA interface are indicated by yellow spheres.