Crystal Structure-Based Exploration of Arginine-Containing Peptide Binding in the ADP-Ribosyltransferase Domain of the Type III Effector XopAI Protein
Abstract
:1. Introduction
2. Results and Discussion
2.1. Structure of XopAI
2.2. Structural Comparison of XopAI with mARTs
2.3. The Central Cleft of XopAI Has the Ability to Bind Peptides
2.4. MD Simulation Study Supports the Protein–Peptide Interaction of XopAI
2.5. MD Study for the PN Loop Dynamics of XopAI in Response to the Arg Peptide Binding
3. Materials and Methods
3.1. Plasmid Constructions
3.2. Protein Expression and Purification
3.3. Protein Crystallization
3.4. Data Collection and Structure Determination
3.5. Sedimentation-Velocity Analytical Ultracentrifugation
3.6. Fluorescence Spectroscopic Assay
3.7. Bioinformatic Analyses
3.8. MD Simulations
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
ARTT | ADP-ribosyl-turn-turn |
Br-MAD | bromide multiple-wavelength anomalous diffraction |
mART | mono-ADP-ribosyltransferase |
MD | molecular dynamics |
PN | phosphate-nicotinamide |
RMSD | root-mean-square deviation |
RMSF | root-mean-square fluctuation |
Xac | Xanthomonas axonopodis pv. citri |
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Dataset | Native | Br-MAD | ||
---|---|---|---|---|
λ1 (H rem) 1 | λ2 (infl) | λ3 (Peak) | ||
Wavelength (Å) | 0.9762 | 0.9056 | 0.9193 | 0.9190 |
Space group | P43212 | |||
Resolution (Å) | 2.0 | 2.3 | 2.4 | 2.5 |
Redundancy | 4.1 | 2.4 | 4.6 | 2.5 |
Completeness (%) | 93.7 (85.3) 2 | 98.5 (99.7) | 98.4 (99.7) | 98.2 (99.7) |
Average I/σ(I) | 11.1 (2.5) | 17.7 (3.5) | 19.5 (4.0) | 18.9 (3.8) |
Rmerge (%) | 8.2 (52.5) | 11.2 (49.2) | 10.2 (44.7) | 10.3 (43.6) |
Protein | XopAI | XopAI-ΔN70 | |
---|---|---|---|
Data collection | |||
Wavelength (Å) | 0.9762 | 0.9198 | 0.9762 |
Space group | P43212 | P41212 | P21 |
Cell parameters (Å) | a = b = 73.05 Å, c = 114.06 Å | a = b = 52.98 Å, c = 212.09 Å | a = 62.78 Å, b = 98.76 Å, c = 77.45 Å, β = 91.21° |
Resolution (Å) | 2.01 | 1.53 | 2.26 |
Mosaicity (°) | 1.07 | 0.29 | 1.23 |
Redundancy | 4.1 | 10.9 | 3.5 |
Completeness (%) | 93.7 (85.3) 1 | 100.0 (100.0) | 99.4 (99.3) |
Average I/σ(I) | 11.1 (2.5) | 9.6 (1.7) | 7.5 (1.8) |
Rmerge (%) | 8.2 (52.5) | 13.1 (106.4) | 14.2 (78.3) |
CC1/2 | 0.997 (0.712) | 0.997 (0.647) | 0.990 (0.609) |
Refinement | |||
Resolution limit (Å) | 26.56–2.01 | 36.89–1.53 | 31.53–2.26 |
Rwork (%) | 16.6 | 16.1 | 18.9 |
Rfree (%) | 19.5 | 17.1 | 23.2 |
Number of non-H atoms | |||
Protein | 1961 | 1973 | 7354 |
Water | 236 | 374 | 558 |
Ramachandran plot statistics | |||
Favored regions (%) | 99.15 | 98.73 | 98.43 |
Allowed regions (%) | 0.85 | 1.27 | 1.46 |
Disallowed regions (%) | 0 | 0 | 0.11 |
Average B factor (Å2) | 25.9 | 22.7 | 35.1 |
R.m.s. deviation from ideality | |||
Bond length (Å) | 0.004 | 0.010 | 0.004 |
Bond angle (˚) | 0.655 | 1.024 | 0.561 |
Protein Data Bank (PDB) code | 6KLY | 6K93 | 6K94 |
Domain Name | Binding Energy (kcal/mol) | Number of Bound Residues | Interface Area (Å2) | PDB ID |
---|---|---|---|---|
XopAI (P41212) | −8.3 | 12 | 673 | 6K93 (this study) |
XopAI (P43212) | −7.6 | 7 | 470 | 6KLY (this study) |
PDZ | −3.7 ± 2.3 | 4~9 (6) 1 | 432 ± 92 | 1BE9, 1L6O, 1MFG, 1N7F, 1OBX, 1OBZ, 1Q3P, 2I0I, 2QT5, 3DIW, 3LNY |
IRS | −4.9 ± 1.3 | 9 | 617 ± 20 | 1UEF, 3ML4 |
VHS | −5.5 ± 3.2 | 5~7 (6) | 420 ± 45 | 1JUQ, 1UJK |
14-3-3 | −5.7 ± 1.7 | 5~10 (7) | 554 ± 122 | 1QJB, 2BTP, 2C74, 3MHR, 3UBW |
WH1 | −6.6 ± 2.6 | 5~6 (6) | 315 ± 35 | 1DDV, 1EVH, 1QC6 |
Skp1 | −6.9 ± 2.3 | 6~12 (10) | 548 ± 151 | 2AST, 2OVQ, 2P1Q |
SH2 | −7.0 | 4 | 363 | 1FYR |
TPR | −7.0 ± 1.1 | 5~8 (7) | 502 ± 29 | 1ELR, 1ELW |
PID | −7.7 ± 2.7 | 9~10 (10) | 717 ± 28 | 1AQC, 1M7E, 1NTV |
BIR | −8.3 ± 1.7 | 4~7 (6) | 420 ± 54 | 1JD5, 1SE0, 3D9T |
WD40 | −8.4 ± 2.3 | 8~12 (9) | 567 ± 44 | 2CE8, 4ERY, 5IGO, 5IGQ |
SH3 | −9.4 ± 0.6 | 9~10 (10) | 480 ± 24 | 1CKA, 1N5Z, 1W70 |
SPRY | −10.0 ± 0.5 | 7 | 352 ± 2 | 2JK9, 3EMW |
Complex | ΔEvdW 1 | ΔEelec | ΔGpolar | ΔGnonpolar | ΔGbinding |
---|---|---|---|---|---|
P41212 | −248.2 ± 25.2 | −582.9 ± 42.2 | 496.4 ± 42.8 | −33.8 ± 2.1 | −368.5 ± 40.3 |
P43212 | −173.3 ± 22.1 | −539.2 ± 40.4 | 505.1 ± 38.9 | −27.7 ± 2.0 | −235.1 ± 30.9 |
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Share and Cite
Liu, J.-H.; Yang, J.-Y.; Hsu, D.-W.; Lai, Y.-H.; Li, Y.-P.; Tsai, Y.-R.; Hou, M.-H. Crystal Structure-Based Exploration of Arginine-Containing Peptide Binding in the ADP-Ribosyltransferase Domain of the Type III Effector XopAI Protein. Int. J. Mol. Sci. 2019, 20, 5085. https://doi.org/10.3390/ijms20205085
Liu J-H, Yang J-Y, Hsu D-W, Lai Y-H, Li Y-P, Tsai Y-R, Hou M-H. Crystal Structure-Based Exploration of Arginine-Containing Peptide Binding in the ADP-Ribosyltransferase Domain of the Type III Effector XopAI Protein. International Journal of Molecular Sciences. 2019; 20(20):5085. https://doi.org/10.3390/ijms20205085
Chicago/Turabian StyleLiu, Jyung-Hurng, Jun-Yi Yang, Duen-Wei Hsu, Yi-Hua Lai, Yun-Pei Li, Yi-Rung Tsai, and Ming-Hon Hou. 2019. "Crystal Structure-Based Exploration of Arginine-Containing Peptide Binding in the ADP-Ribosyltransferase Domain of the Type III Effector XopAI Protein" International Journal of Molecular Sciences 20, no. 20: 5085. https://doi.org/10.3390/ijms20205085