A State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009

Investigation of the Intermolecular Recognition Mechanism between the E3 Ubiquitin Ligase and Substrate Based on Multiple Substrates Analysis: a case study of Keap1

Zheng-Yu Jiang, a,b Li-Li Xu, a,b Meng-Chen Lu, a,b Yang Pan,a,b Hao-Ze Huang,a,b Xiao-Jin Zhang, a,b,d Hao-Peng Sun, a,b,c,* Qi-dong You a,b,*

a State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China

b Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China

c Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China

d Department of Organic Chemistry, School of Science, China Pharmaceutical University, Nanjing 210009, China

Corresponding Author

* Corresponding author: Ph.D. Hao-Peng Sun

Address: China Pharmaceutical University, Nanjing 210009, China.

Tel/Fax: +86 025 83271216.

E-mail address:

*Corresponding author: Prof. Qi-Dong You

Address: China Pharmaceutical University, Nanjing 210009, China.

Tel/Fax: +86 025 83271351.

E-mail address:

Tables with binding free energies of Keap1-peptide complex structures (Table S1)

MM-GBSA

Energy Component / 1X2R / 2DYH / 2Z32 / 3ADE
VDWAALS / -47.8774±0.3743 / -48.3588±0.1137 / -46.9333±0.1574 / -42.5590±0.1391
EEL / -180.8141±2.3914 / -258.3758±0.8108 / -136.2385±1.0615 / -167.1382±1.2029
EGB / 167.0838±2.2147 / 287.2079±0.7336 / 138.9334±0.9386 / 173.3588±1.0355
ESURF / -8.8344±0.0312 / -8.0278±0.0081 / -8.1821±0.0126 / -7.1581±0.0078
DELTA G gas / -228.6915±2.3109 / -306.7346±0.8205 / -183.1718±1.0210 / -209.6971±1.1750
DELTA G solv / 158.2495±2.2196 / 279.1801±0.7308 / 130.7512±0.9421 / 166.2007±1.0331
DELTA TOTAL / -70.4421±0.459 / -27.5545±0.1959 / -52.4206±0.1871 / -43.4964±0.2003

MM-PBSA

Energy Component / 1X2R / 2DYH / 2Z32 / 3ADE
VDWAALS / -47.8774±0.3743 / -48.3588±0.1137 / -46.9333±0.1574 / -42.5590±0.1391
EEL / -180.8141±2.3914 / -258.3758±0.8108 / -136.2385±1.0615 / -167.1382±1.2029
EPB / 155.7737±2.1562 / 283.4598±0.7657 / 129.3605±0.8941 / 167.5701±1.1809
ENPOLAR / -41.7434±0.1653 / -36.7028±0.0589 / -39.8061±0.0623 / -34.1630±0.0477
EDISPER / 79.6061±0.235 / 70.6363±0.0730 / 77.2780±0.0932 / 65.0067±0.0739
DELTA G gas / -228.6915±2.3109 / -306.7346±0.8205 / -183.1718±1.0210 / -209.6971±1.1750
DELTA G solv / 193.6365±2.1215 / 317.3933±0.7776 / 166.8324±0.8908 / 198.4137±1.2055
DELTA TOTAL / -35.055±0.6027 / 10.6587±0.3453 / -16.3394±0.3003 / -11.2834±0.2431

Per-residue contribution for residues of substrate peptides bound to the Keap1 (Table S2)

Nrf2 ETGE peptide

Residue / Location / Internal / van der Waals / Electrostatic / Polar Solvation / Non-Polar Solv. / TOTAL
Avg. / SD / SEM / Avg. / SD / SEM / Avg. / Std. Dev. / Std. Err. of Mean / Avg. / SD / SEM / Avg. / SD / SEM / Avg. / SD / SEM
LEU76 / L LEU 1 / 0 / 0 / 0 / -0.91083 / 0.199002 / 0.036333 / 0.961466667 / 0.38539 / 0.070362 / -0.7622 / 0.388770318 / 0.070979 / -0.13523 / 0.044295 / 0.008087 / -0.84679 / 0.272961 / 0.049836
ASP77 / L ASP 2 / 0 / 0 / 0 / -1.23913 / 0.107392 / 0.019607 / -8.3414 / 3.995827 / 0.729535 / 10.2388 / 4.040910058 / 0.737766 / -0.0539 / 0.024786 / 0.004525 / 0.604362 / 0.379827 / 0.069347
GLU78 / L GLU 3 / 0 / 0 / 0 / -2.78777 / 0.923472 / 0.168602 / 1.087066667 / 8.893903 / 1.623797 / 0.704833333 / 7.993800911 / 1.459462 / -0.65382 / 0.03822 / 0.006978 / -1.64968 / 0.899757 / 0.164272
GLU79 / L GLU 4 / 0 / 0 / 0 / -3.408 / 1.517513 / 0.277059 / -58.0114 / 3.823676 / 0.698104 / 50.81696667 / 2.709060975 / 0.494605 / -1.17319 / 0.031814 / 0.005808 / -11.7756 / 2.506149 / 0.457558
THR80 / L THR 5 / 0 / 0 / 0 / -4.49507 / 0.483002 / 0.088184 / -3.122933333 / 1.492312 / 0.272458 / 4.184066667 / 1.406834744 / 0.256852 / -0.64954 / 0.035504 / 0.006482 / -4.08348 / 0.48075 / 0.087772
GLY81 / L GLY 6 / 0 / 0 / 0 / -2.51133 / 0.356288 / 0.065049 / -1.046866667 / 0.680802 / 0.124297 / 2.141733333 / 0.91932446 / 0.167845 / -0.34141 / 0.02349 / 0.004289 / -1.75788 / 0.566885 / 0.103499
GLU82 / L GLU 7 / 0 / 0 / 0 / -3.2983 / 1.187471 / 0.216802 / -37.21736667 / 3.506191 / 0.64014 / 38.8923 / 2.88613548 / 0.526934 / -0.79712 / 0.033232 / 0.006067 / -2.42048 / 1.240502 / 0.226484
PHE83 / L PHE 8 / 0 / 0 / 0 / -2.56603 / 0.608366 / 0.111072 / -6.9414 / 1.455779 / 0.265788 / 6.828466667 / 1.042951061 / 0.190416 / -0.51755 / 0.112161 / 0.020478 / -3.19652 / 0.767401 / 0.140108
LEU84 / L LEU 9 / 0 / 0 / 0 / -2.02857 / 0.766856 / 0.140008 / 17.34833333 / 3.579984 / 0.653613 / -14.69563333 / 3.577694709 / 0.653195 / -0.47109 / 0.15434 / 0.028179 / 0.153045 / 1.371472 / 0.250395

Per-residue contribution for residues of substrate peptides bound to the Keap1

Nrf2 DLG peptide

Residue / Location / Internal / van der Waals / Electrostatic / Polar Solvation / Non-Polar Solv. / TOTAL
Avg. / SD / SEM / Avg. / SD / SEM / Avg. / SD / SEM / Avg. / SD / SEM / Avg. / SD / SEM / Avg. / SD / SEM
TRP24 / L TRP 1 1 / 0 / 0 / 0 / -5.1036 / 0.731821 / 0.023142 / -2.4884 / 3.144422 / 0.099435 / 4.8124 / 2.85157 / 0.090175 / -0.98815 / 0.114478 / 0.00362 / -3.76775 / 0.930072 / 0.029411
ARG25 / L ARG 2 2 / 0 / 0 / 0 / -1.1358 / 0.679053 / 0.021474 / -20.4982 / 5.875103 / 0.185787 / 23.315 / 6.350972 / 0.200835 / -0.1106 / 0.068622 / 0.00217 / 1.570401 / 1.321354 / 0.041785
GLN26 / L GLN 3 3 / 0 / 0 / 0 / -6.29828 / 0.817295 / 0.025845 / -1.45476 / 2.057269 / 0.065057 / 5.839617 / 1.81708 / 0.057461 / -1.04835 / 0.056475 / 0.001786 / -2.96177 / 1.434297 / 0.045356
ASP27 / L ASP 4 4 / 0 / 0 / 0 / -4.12253 / 0.407944 / 0.0129 / -18.8263 / 3.725821 / 0.117821 / 23.10801 / 3.70521 / 0.117169 / -0.60485 / 0.033396 / 0.001056 / -0.44569 / 1.011187 / 0.031977
ILE28 / L ILE 5 5 / 0 / 0 / 0 / -2.96234 / 0.44942 / 0.014212 / -0.27391 / 0.551191 / 0.01743 / 0.988118 / 0.62789 / 0.019856 / -0.50702 / 0.080147 / 0.002534 / -2.75515 / 0.510388 / 0.01614
ASP29 / L ASP 6 6 / 0 / 0 / 0 / -2.46949 / 1.032287 / 0.032644 / -56.2561 / 11.63114 / 0.367809 / 61.41131 / 10.7801 / 0.340897 / -0.8446 / 0.100019 / 0.003163 / 1.841126 / 1.760349 / 0.055667

Per-residue contribution for residues of substrate peptides bound to the Keap1

Prothymosin α peptide

Residue / Location / Internal / van der Waals / Electrostatic / Polar Solvation / Non-Polar Solv. / TOTAL
Avg. / SD / SEM / Avg. / SD / SEM / Avg. / SD / SEM / Avg. / SD / SEM / Avg. / SD / SEM / Avg. / SD / SEM
GLN40 / L GLN 1 / 0 / 0 / 0 / -1.3232 / 0.201629 / 0.090171 / 4.921 / 2.0779 / 0.929265 / -2.5072 / 1.436518 / 0.642431 / -0.21193 / 0.051515 / 0.023038 / 0.878665 / 0.455903 / 0.203886
ASN41 / L ASN 2 / 0 / 0 / 0 / -1.5968 / 0.151612 / 0.067803 / 1.447 / 0.767075 / 0.343046 / -0.249 / 0.447253 / 0.200018 / -0.10159 / 0.018701 / 0.008363 / -0.50039 / 0.283195 / 0.126648
GLU42 / L GLU 3 / 0 / 0 / 0 / -2.9658 / 0.555648 / 0.248493 / 31.3742 / 1.987827 / 0.888983 / -28.0616 / 1.921515 / 0.859327 / -0.71614 / 0.06261 / 0.028 / -0.36934 / 0.629124 / 0.281353
GLU43 / L GLU 4 / 0 / 0 / 0 / -4.1756 / 0.761433 / 0.340523 / -33.3012 / 4.078282 / 1.823863 / 31.6818 / 2.867233 / 1.282265 / -1.08478 / 0.021176 / 0.00947 / -6.87978 / 1.019059 / 0.455737
ASN44 / L ASN 5 / 0 / 0 / 0 / -5.4314 / 0.560189 / 0.250524 / -7.3412 / 0.853396 / 0.38165 / 13.3492 / 0.98172 / 0.439039 / -0.87071 / 0.020224 / 0.009045 / -0.29411 / 0.343944 / 0.153816
GLY45 / L GLY 6 / 0 / 0 / 0 / -2.5524 / 0.278802 / 0.124684 / -0.6594 / 0.511677 / 0.228829 / 1.6154 / 0.451262 / 0.201811 / -0.3169 / 0.014719 / 0.006583 / -1.9133 / 0.323135 / 0.14451
GLU46 / L GLU 7 / 0 / 0 / 0 / -4.0384 / 1.015459 / 0.454127 / -35.3926 / 3.709086 / 1.658754 / 34.578 / 2.918554 / 1.305217 / -0.8307 / 0.023756 / 0.010624 / -5.6837 / 1.444849 / 0.646156
GLN47 / L GLN 8 / 0 / 0 / 0 / -2.9776 / 0.79624 / 0.356089 / -8.1714 / 3.769013 / 1.685554 / 8.7214 / 2.12488 / 0.950275 / -0.52892 / 0.169497 / 0.075801 / -2.95652 / 1.704841 / 0.762428
GLU48 / L GLU 9 / 0 / 0 / 0 / -1.2406 / 0.297299 / 0.132956 / 14.335 / 5.248745 / 2.34731 / -11.5746 / 5.174886 / 2.314279 / -0.22118 / 0.06072 / 0.027155 / 1.298617 / 0.954766 / 0.426984

Per-residue contribution for residues of substrate peptides bound to the Keap1

P62 peptide

Residue / Location / Internal / van der Waals / Electrostatic / Polar Solvation / Non-Polar Solv. / TOTAL
Avg. / SD / SEM / Avg. / SD / SEM / Avg. / SD / SEM / Avg. / SD / SEM / Avg. / SD / SEM / Avg. / SD / SEM
VAL348 / L VAL 1 / 0 / 0 / 0 / -0.82252 / 0.16923 / 0.023933 / 1.0268 / 0.522704 / 0.073922 / -0.69968 / 0.577065 / 0.081609 / -0.10723 / 0.026822 / 0.003793 / -0.60263 / 0.277562 / 0.039253
ASP349 / L ASP 2 / 0 / 0 / 0 / -0.9528 / 0.773173 / 0.02445 / -19.5796 / 7.839309 / 0.247901 / 18.15587 / 6.965346 / 0.220264 / -0.1966 / 0.049044 / 0.001551 / -2.57311 / 1.738659 / 0.054981
PRO350 / L PRO 3 / 0 / 0 / 0 / -2.88257 / 0.626127 / 0.0198 / -1.12331 / 1.302431 / 0.041186 / 1.430022 / 1.020655 / 0.032276 / -0.61508 / 0.065692 / 0.002077 / -3.19094 / 0.724615 / 0.022914
SER351 / L SER 4 / 0 / 0 / 0 / -4.16345 / 0.715058 / 0.022612 / -3.33246 / 1.880961 / 0.059481 / 5.969406 / 1.290164 / 0.040799 / -0.70956 / 0.03372 / 0.001066 / -2.23606 / 1.009954 / 0.031938
THR352 / L THR 5 / 0 / 0 / 0 / -4.3738 / 0.626663 / 0.019817 / -2.62788 / 1.723704 / 0.054508 / 3.749944 / 1.64459 / 0.052006 / -0.65916 / 0.039166 / 0.001239 / -3.91089 / 0.747853 / 0.023649
GLY353 / L GLY 6 / 0 / 0 / 0 / -2.49207 / 0.37249 / 0.011779 / -1.12807 / 0.794117 / 0.025112 / 2.297321 / 0.914517 / 0.02892 / -0.35612 / 0.027939 / 0.000884 / -1.67895 / 0.511723 / 0.016182
GLU354 / L GLU 7 / 0 / 0 / 0 / -3.27654 / 1.172693 / 0.037084 / -35.2376 / 5.093002 / 0.161055 / 37.34527 / 4.146613 / 0.131127 / -0.83268 / 0.035205 / 0.001113 / -2.00157 / 1.95766 / 0.061907
LEU355 / L LEU 8 / 0 / 0 / 0 / -2.33669 / 1.104836 / 0.034938 / -10.953 / 15.95657 / 0.504591 / 11.06473 / 14.36639 / 0.454305 / -0.59492 / 0.130827 / 0.004137 / -2.81986 / 1.851761 / 0.058558

per-residue contribution for residues of five sub-pockets in Keap1 (Table S3)

1X2R / 2DYH / 2Z32 / 3ADE
P1
SER 508 / -2.84972 / -0.09423 / -0.98798 / -0.11311
PHE 478 / -0.30589 / -3.21186 / -0.1365 / -0.10366
ILE 461 / -0.09034 / -0.2599 / 0.030356 / 0.073375
ARG 483 / -8.99986 / 0.919502 / -6.57377 / 0.047374
ARG 415 / -5.1551 / -6.3665 / -4.22385 / -4.12611
GLY 462 / 0.071467 / 0.034497 / 0.079523 / 0.022608
-17.3294 / -8.97849 / -11.8122 / -4.19953
P2
SER 363 / -2.07851 / 0.115126 / -1.05463 / -1.8536
ARG 380 / -5.81376 / -4.40736 / -8.17194 / -5.55031
ASN 382 / -3.82245 / -1.78006 / -4.01081 / -4.78552
ASN 414 / 0.102264 / -0.86463 / 0.271332 / 0.212009
-11.6125 / -6.93693 / -12.966 / -11.9774
Backbone
GLY 509 / 0.019888 / -1.17496 / -0.27307 / -0.23844
ALA 556 / -0.87848 / -0.82889 / -1.00907 / -0.99669
SER 555 / -0.85807 / -0.20615 / -0.44346 / -0.83252
SER 602 / 0.055896 / 0.211186 / 0.139949 / 0.107301
GLY 603 / -0.09422 / 0.02516 / -0.12507 / -0.09037
GLY 571 / 0.14753 / 0.2006 / 0.241318 / 0.146058
-1.60746 / -1.77305 / -1.4694 / -1.90466
H1
TYR 525 / -2.54699 / -3.21047 / -3.59234 / -2.34828
GLN 530 / -1.75853 / -0.92434 / -1.90224 / -1.59134
ASP 573 / -0.01292 / -0.17668 / -0.16743 / -0.12016
GLY 574 / -0.19587 / -0.92398 / -0.38541 / -0.16989
TYR 572 / -4.50409 / -3.07227 / -4.97653 / -4.60514
-9.0184 / -8.30774 / -11.0239 / -8.83482
H2
THR 576 / 0.039308 / 0.001901 / 0.062017 / 0.018415
PHE 577 / -1.4718 / -0.52546 / -1.88125 / -1.45078
TYR 334 / -3.19928 / -0.57938 / -4.14677 / -2.76241
-4.63177 / -1.10294 / -5.966 / -4.19478

detailed information of the hydrogen bonds analysis (Table S4).

Nr2 ETGE peptide

Acceptor / Donor / %occupied / distance / angle
Glu79@O / Ser555@HG / 99.6 / 2.702 ( 0.11) / 22.65 (11.04)
Thr80@O / Ser602@HG / 98.8 / 2.773 ( 0.13) / 17.29 ( 9.35)
Glu78@O / Gln530@HE21 / 94.1 / 2.914 ( 0.12) / 24.30 (10.48)
Glu79@OE2 / Ser508@HG / 93.5 / 2.696 ( 0.13) / 17.51 (11.00)
Glu79@OE2 / Arg415@HH22 / 92.3 / 2.775 ( 0.10) / 25.08 ( 9.25)
Glu79@OE1 / Arg483@HH21 / 91.2 / 2.805 ( 0.12) / 30.30 ( 8.26)
Glu79@OE1 / Arg483@HE / 90.4 / 2.853 ( 0.12) / 28.91 ( 7.66)
Glu79@OE2 / Arg415@HH12 / 77.6 / 2.865 ( 0.14) / 34.30 ( 8.73)
Phe83@O / Asn382@HD21 / 76.6 / 2.936 ( 0.13) / 20.18 (10.65)
Glu82@OE1 / Arg380@HE / 67 / 2.858 ( 0.12) / 31.35 ( 8.75)
Glu82@OE2 / Ser363@HG / 64 / 2.672 ( 0.11) / 15.55 ( 8.99)
Glu82@OE1 / Arg380@HH21 / 63.9 / 2.796 ( 0.11) / 29.68 ( 9.07)
Glu82@OE1 / Ser363@HG / 35.8 / 2.671 ( 0.13) / 16.13 ( 8.47)
Glu82@OE2 / Arg380@HH21 / 35.6 / 2.795 ( 0.11) / 28.11 ( 9.72)
Glu82@OE2 / Arg380@HE / 32.8 / 2.848 ( 0.12) / 31.51 ( 8.67)
Glu82@OE1 / Asn382@HD22 / 28.5 / 2.878 ( 0.13) / 15.40 ( 8.51)
Glu79@OE1 / Ser508@HG / 27 / 2.916 ( 0.20) / 36.11 (16.15)
Glu78@OE1 / Arg483@HH22 / 23.8 / 2.848 ( 0.12) / 24.74 (12.49)
Glu78@OE2 / Arg483@HH22 / 21.5 / 2.830 ( 0.13) / 25.50 (13.35)

Nrf2 DLG peptide

Acceptor / Donor / %occupied / distance / angle
Gln26@O / Ser555@HG / 94.00 / 2.681 ( 0.11) / 22.34 (11.45)
Arg25@O / Gln530@HE21 / 90.50 / 2.924 ( 0.12) / 30.39 (10.66)
Asp29@OD1 / Arg380@HH11 / 52.30 / 2.875 ( 0.13) / 23.46 (12.15)
Asp29@OD1 / Asn382@HD22 / 29.00 / 2.906 ( 0.14) / 31.92 (12.63)
Asp29@OD2 / Arg380@HH11 / 21.80 / 2.889 ( 0.15) / 28.95 (14.12)
Gly574@O / Arg25@HH11 / 55.9 / 2.892 ( 0.13) / 28.28 (13.65)

Prothymosin α peptide

Acceptor / Donor / %occupied / distance / angle
Glu43@O / Ser555@HG / 99.8 / 2.670 ( 0.10) / 18.54 ( 9.95)
Asn44@O / Ser602@HG / 99.6 / 2.737 ( 0.12) / 16.60 ( 8.82)
Glu42@O / Gln530@HE21 / 92.4 / 2.903 ( 0.13) / 30.69 (11.29)
Glu46@OE1 / Arg380@HH21 / 74.4 / 2.825 ( 0.12) / 22.06 (11.16)
Glu46@OE2 / Ser363@HG / 67.9 / 2.652 ( 0.11) / 15.88 ( 8.11)
Glu43@OE2 / Arg483@HH12 / 54.4 / 2.770 ( 0.09) / 21.59 ( 9.66)
Glu48@OE1 / Arg380@HH22 / 53.7 / 2.835 ( 0.14) / 23.94 (11.70)
Glu43@OE1 / Arg415@HH22 / 53.5 / 2.785 ( 0.11) / 26.40 ( 9.74)
Glu48@OE1 / Arg380@HH12 / 49.8 / 2.879 ( 0.14) / 28.28 (12.61)
Glu46@OE1 / Asn382@HD22 / 47.5 / 2.881 ( 0.13) / 17.26 ( 9.61)
Glu42@OE2 / Tyr525@HH / 46.1 / 2.718 ( 0.14) / 23.23 (10.58)
Glu43@OE1 / Arg415@HH12 / 45 / 2.857 ( 0.13) / 33.61 ( 8.77)
Glu43@OE1 / Arg483@HH12 / 44.2 / 2.781 ( 0.11) / 21.28 (10.17)
Glu48@OE2 / Arg380@HH22 / 38.1 / 2.833 ( 0.13) / 22.22 (11.43)
Glu43@OE2 / Arg415@HH22 / 38 / 2.776 ( 0.10) / 24.15 ( 9.82)
Glu48@OE2 / Arg380@HH12 / 36.5 / 2.886 ( 0.15) / 26.48 (12.40)
Glu43@OE2 / Arg483@HH22 / 33.5 / 2.892 ( 0.14) / 33.52 ( 8.04)
Glu43@OE2 / Arg415@HH12 / 31.5 / 2.866 ( 0.14) / 33.87 ( 9.43)
Glu42@OE1 / Tyr525@HH / 29.4 / 2.722 ( 0.14) / 20.66 (10.61)
Glu46@OE2 / Arg380@HH21 / 27 / 2.840 ( 0.12) / 21.55 (11.70)
Glu43@OE1 / Arg483@HH22 / 25.1 / 2.895 ( 0.14) / 32.90 ( 9.08)
Glu46@OE2 / Asn382@HD22 / 22.6 / 2.881 ( 0.13) / 18.74 (10.20)

P62 peptide