Competitive Binding of Magnesium to Calcium Binding Sites Reciprocally Regulates Transamidase and GTP Hydrolysis Activity of Transglutaminase 2
Abstract
:1. Introduction
2. Results
2.1. Identification of New Ca2+-Binding Sites in TG2
2.2. Ca2+ Binding to E437 and E539 Residues Is Required for the Transamidase Activity of TG2
2.3. Mg2+ Binding to E437 and E539 Inhibits the Transamidase Activity of TG2
2.4. Mg2+-Binding to E437 and E539 Promotes the GTP Binding and Hydrolysis Activity of TG2
2.5. Mg2+-Binding to E437 and E539 Is Critical for Preventing the Activation of Intracellular TG2
3. Discussion
4. Materials and Methods
4.1. Protein Expression and Purification
4.2. Crystallization and Data Collection
4.3. Structure Determination and Analysis
4.4. CD Analysis
4.5. Protein Data Bank Accession Code
4.6. ITC
4.7. In Vitro Transamidase Activity Assay
4.8. GTP Binding Assay
4.9. GTPase Activity Assay
4.10. Cell Culture
4.11. Intracellular Transamidase Activity Assay
4.12. Luciferase Reporter Assay
4.13. Western Blot Analysis
4.14. Statistical Analysis
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
TG2 | Transglutaminase 2 |
Ca2+ | Calcium ion |
Mg2+ | Magnesium ion |
ASU | Asymmetric unit |
CD | Circular dichroism |
WT | Wild type |
ITC | Isothermal titration calorimetry |
EC50 | Half-maximal effective concentration |
IC50 | Half-maximal inhibitory concentration |
NB | No binding |
HPSP | Human phosphoserine phosphatase |
IPTG | Isopropyl β-D-thiogalactopyranoside |
BP | Biotinylated pentylamine |
HDF | Human dermal fibroblasts |
CI | Confidence intervals |
n.s. | Not significant |
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Data Collection | Native |
---|---|
X-ray source | Synchrotron (PAL 5C) |
Detector | Eiger 9M |
Wavelength | 1.0000 |
Space group | C2221 |
Cell dimensions | |
a, b, c | 133.1 Å, 216.3 Å, 166.3 Å |
Resolution | 50–3.56 Å |
Wilson B-factor | 80.666 Å2 |
†No. of unique reflections overall | 29,265 |
† Rsym | 9.4% (34.5%) |
†I/I | 19.2 (3.9) |
†Completeness | 100% (99.9%) |
†Redundancy | 10.9 (11.0) |
Refinement | |
Resolution | 42.58–3.55 Å |
No. of reflections used (completeness) | 27,758 (99.59%) |
†Rwork | 22.5% (22.68%) |
†Rfree | 26.3% (26.85%) |
Average B-factors | |
Protein | 61.0 Å2 |
Other small molecules | 82 Å2 |
Root mean square deviations | |
Bond lengths | 0.013 Å |
Bond angles | 1.672° |
MolProbity analysis | |
Ramachandran outliers | 0.00% |
Ramachandran favored | 98.00% |
Ramachandran allowed | 2.00% |
Rotamer outliers | 6.00% |
Clashscore | 18.00 |
Divalent Ions | WT Kd (μM) | E437R Kd (μM) | E539R Kd (μM) | E437R/E539R Kd (μM) |
---|---|---|---|---|
Ca2+ | 0.027 ± 0.004 | 188 ± 24 | 0.047 ± 0.001 | NB |
Mg2+ | 0.215 ± 0.056 | 1.459 ± 0.523 | 0.282 ± 0.023 | NB |
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Jeong, E.M.; Lee, K.B.; Kim, G.E.; Kim, C.M.; Lee, J.-H.; Kim, H.-J.; Shin, J.-W.; Kwon, M.-a.; Park, H.H.; Kim, I.-G. Competitive Binding of Magnesium to Calcium Binding Sites Reciprocally Regulates Transamidase and GTP Hydrolysis Activity of Transglutaminase 2. Int. J. Mol. Sci. 2020, 21, 791. https://doi.org/10.3390/ijms21030791
Jeong EM, Lee KB, Kim GE, Kim CM, Lee J-H, Kim H-J, Shin J-W, Kwon M-a, Park HH, Kim I-G. Competitive Binding of Magnesium to Calcium Binding Sites Reciprocally Regulates Transamidase and GTP Hydrolysis Activity of Transglutaminase 2. International Journal of Molecular Sciences. 2020; 21(3):791. https://doi.org/10.3390/ijms21030791
Chicago/Turabian StyleJeong, Eui Man, Ki Baek Lee, Gi Eob Kim, Chang Min Kim, Jin-Haeng Lee, Hyo-Jun Kim, Ji-Woong Shin, Mee-ae Kwon, Hyun Ho Park, and In-Gyu Kim. 2020. "Competitive Binding of Magnesium to Calcium Binding Sites Reciprocally Regulates Transamidase and GTP Hydrolysis Activity of Transglutaminase 2" International Journal of Molecular Sciences 21, no. 3: 791. https://doi.org/10.3390/ijms21030791