Substrate Recognition Properties from an Intermediate Structural State of the UreA Transporter
Abstract
:1. Introduction
2. Results
2.1. Characterization of a Theoretical Intermediate State of UreA and Its Binding Site
2.2. Site-Directed Mutagenesis of Identified Residues in the Putative Binding Site
2.3. Insights into Ligand Binding by the Intermediate State of UreA
3. Discussion
4. Materials and Methods
4.1. Structure Prediction
4.2. Molecular Docking
4.3. Structural Analysis
4.4. A. nidulans Strains, Media and Transformation Procedures
4.5. Construction of ureA Mutants by Site-Directed Mutagenesis
4.6. [14C]-Urea Uptake Measurements
4.7. Protein Extraction and Western Blot Assays
4.8. Epifluorescence Microscopy
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Allele | Location in Protein | Vo (%) | Urea Km (µM) | Thiourea Ki (µM) | Acetamide Ki (µM) |
---|---|---|---|---|---|
wt | 100 | 26 ± 2 | 528 ± 42 | 237 ± 14 | |
W82F * | TMS2 | 50 ± 8 | 23 ± 5 | 447 ± 24 | 198 ± 12 |
W82Y | TMS 2 | 25 ± 2 | 42 ± 5 | >1000 | 101 ± 16 |
W84A | TMS 2 | 20 ± 2 | 193 ± 12 | >2000 | 489 ± 39 |
W84F | TMS 2 | 15 ± 2 | 96 ±13 | >2000 | 524 ± 42 |
W84Y | TMS 2 | 46 ± 4 | 159 ± 11 | >2000 | >1000 |
N279A | TMS 7 | 100 ± 5 | 30 ± 3 | >1000 | 51 ± 5 |
N279Q | TMS 7 | 70 ± 3 | 91 ± 6 | 813 ± 59 | 87 ± 9 |
T282A | TMS 7 | 14 ± 2 | 80 ± 27 | >1000 | 361 ± 85 |
T282S | TMS 7 | 65 ± 3 | 130 ± 22 | >1000 | 290 ± 12 |
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Sanguinetti, M.; Silva Santos, L.H.; Dourron, J.; Alamón, C.; Idiarte, J.; Amillis, S.; Pantano, S.; Ramón, A. Substrate Recognition Properties from an Intermediate Structural State of the UreA Transporter. Int. J. Mol. Sci. 2022, 23, 16039. https://doi.org/10.3390/ijms232416039
Sanguinetti M, Silva Santos LH, Dourron J, Alamón C, Idiarte J, Amillis S, Pantano S, Ramón A. Substrate Recognition Properties from an Intermediate Structural State of the UreA Transporter. International Journal of Molecular Sciences. 2022; 23(24):16039. https://doi.org/10.3390/ijms232416039
Chicago/Turabian StyleSanguinetti, Manuel, Lucianna Helene Silva Santos, Juliette Dourron, Catalina Alamón, Juan Idiarte, Sotiris Amillis, Sergio Pantano, and Ana Ramón. 2022. "Substrate Recognition Properties from an Intermediate Structural State of the UreA Transporter" International Journal of Molecular Sciences 23, no. 24: 16039. https://doi.org/10.3390/ijms232416039