| Autor: |
Sanguinetti M; Sección Bioquímica, Departamento de Biología Celular y Molecular, Facultad de Ciencias, Universidad de la República, Iguá 4225, Montevideo 11400, Uruguay., Silva Santos LH; Biomolecular Simulations Group, Institut Pasteur de Montevideo, Mataojo 2020, Montevideo 11400, Uruguay., Dourron J; Sección Bioquímica, Departamento de Biología Celular y Molecular, Facultad de Ciencias, Universidad de la República, Iguá 4225, Montevideo 11400, Uruguay., Alamón C; Sección Bioquímica, Departamento de Biología Celular y Molecular, Facultad de Ciencias, Universidad de la República, Iguá 4225, Montevideo 11400, Uruguay.; Neurodegeneration Laboratory, Institut Pasteur de Montevideo, Mataojo 2020, Montevideo 11400, Uruguay., Idiarte J; Sección Bioquímica, Departamento de Biología Celular y Molecular, Facultad de Ciencias, Universidad de la República, Iguá 4225, Montevideo 11400, Uruguay.; Columbia University Irving Medical Center, Columbia University, New York, NY 10032, USA., Amillis S; Department of Biology, National and Kapodistrian University of Athens, Panepistimioupolis, 15784 Athens, Greece., Pantano S; Biomolecular Simulations Group, Institut Pasteur de Montevideo, Mataojo 2020, Montevideo 11400, Uruguay., Ramón A; Sección Bioquímica, Departamento de Biología Celular y Molecular, Facultad de Ciencias, Universidad de la República, Iguá 4225, Montevideo 11400, Uruguay. |
| Abstrakt: |
Through a combination of comparative modeling, site-directed and classical random mutagenesis approaches, we previously identified critical residues for binding, recognition, and translocation of urea, and its inhibition by 2-thiourea and acetamide in the Aspergillus nidulans urea transporter, UreA. To deepen the structural characterization of UreA, we employed the artificial intelligence (AI) based AlphaFold2 (AF2) program. In this analysis, the resulting AF2 models lacked inward- and outward-facing cavities, suggesting a structural intermediate state of UreA. Moreover, the orientation of the W82, W84, N279, and T282 side chains showed a large variability, which in the case of W82 and W84, may operate as a gating mechanism in the ligand pathway. To test this hypothesis non-conservative and conservative substitutions of these amino acids were introduced, and binding and transport assessed for urea and its toxic analogue 2-thiourea, as well as binding of the structural analogue acetamide. As a result, residues W82, W84, N279, and T282 were implicated in substrate identification, selection, and translocation. Using molecular docking with Autodock Vina with flexible side chains, we corroborated the AF2 theoretical intermediate model, showing a remarkable correlation between docking scores and experimental affinities determined in wild-type and UreA mutants. The combination of AI-based modeling with classical docking, validated by comprehensive mutational analysis at the binding region, would suggest an unforeseen option to determine structural level details on a challenging family of proteins. |
