Structural Insights into the Active Site Formation of DUSP22 in N-loop-containing Protein Tyrosine Phosphatases
Abstract
:1. Introduction
2. Results
2.1. Characteristics of the DPN–triloop Interaction
2.2. The Role of DPN–triloop Interaction in Phosphatase Activity
2.3. D57, S93, and N128 Mutants Perturbed the Active Site Conformation in the Solution Structure
2.4. The Structural Role of the DPN–triloop Interaction in Crystal Structures
3. Discussion
4. Materials and Methods
4.1. Protein Expression and Purification
4.2. Kinetic Assay
4.3. NMR Spectroscopy
4.4. Protein Crystallization
4.5. X-Ray Data Collection and Structure Determination
4.6. Data Availability
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
Cys-based PTPs | Cysteine-based protein tyrosine phosphatases |
DUSPs | Dual-specificity phosphatases |
NMR | Nuclear magnetic resonance |
P-loop | Phosphate binding loop |
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KM (mM) | kcat (s−1) | kcat/KM (s−1·M−1) | |
---|---|---|---|
WT | 0.64 ± 0.03 | 0.65 ± 0.01 | 1.0 × 103 |
D57A | 5.6 ± 0.7 | 0.025 ± 0.001 | 4.5 |
D57N | 0.83 ± 0.07 | 0.021 ± 0.0005 | 25 |
S93A | 12 ± 2 | 0.045 ± 0.003 | 3.8 |
S93N | 8.8 ± 0.5 | 0.057 ± 0.001 | 6.5 |
N128A | 26 ± 3 | 0.26 ± 0.01 | 10 |
N128D | 21 ± 2 | 0.041 ± 0.002 | 2.0 |
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Lai, C.-H.; Chang, C.-C.; Chuang, H.-C.; Tan, T.-H.; Lyu, P.-C. Structural Insights into the Active Site Formation of DUSP22 in N-loop-containing Protein Tyrosine Phosphatases. Int. J. Mol. Sci. 2020, 21, 7515. https://doi.org/10.3390/ijms21207515
Lai C-H, Chang C-C, Chuang H-C, Tan T-H, Lyu P-C. Structural Insights into the Active Site Formation of DUSP22 in N-loop-containing Protein Tyrosine Phosphatases. International Journal of Molecular Sciences. 2020; 21(20):7515. https://doi.org/10.3390/ijms21207515
Chicago/Turabian StyleLai, Chih-Hsuan, Co-Chih Chang, Huai-Chia Chuang, Tse-Hua Tan, and Ping-Chiang Lyu. 2020. "Structural Insights into the Active Site Formation of DUSP22 in N-loop-containing Protein Tyrosine Phosphatases" International Journal of Molecular Sciences 21, no. 20: 7515. https://doi.org/10.3390/ijms21207515