Substrate recognition properties from an intermediate structural state of the UreA transporter
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....
Saved in:
| Main Author: | |
|---|---|
| Other Authors: | , , , , , , |
| Format: | article |
| Language: | English |
| Published: |
2022
|
| Subjects: | |
| Online Access: | https://hdl.handle.net/20.500.12008/41370 |
| Tags: |
No Tags, Be the first to tag this record!
|
| _version_ | 1868890021440782336 |
|---|---|
| author | Sanguinetti Miralles, Manuel |
| author2 | Silva Santos, Lucianna Helene Dourron, Juliette Alamón Lima, Catalina Isadora Idiarte Montiel, Juan Amillis, Sotiris Pantano, Sergio Ramón, Ana |
| author2_role | author author author author author author author |
| author_browse | Alamón Lima, Catalina Isadora Amillis, Sotiris Dourron, Juliette Idiarte Montiel, Juan Pantano, Sergio Ramón, Ana Sanguinetti Miralles, Manuel Silva Santos, Lucianna Helene |
| author_facet | Sanguinetti Miralles, Manuel Silva Santos, Lucianna Helene Dourron, Juliette Alamón Lima, Catalina Isadora Idiarte Montiel, Juan Amillis, Sotiris Pantano, Sergio Ramón, Ana |
| author_role | author |
| collection | COLIBRI |
| dc.contributor.none.fl_str_mv | Sanguinetti Miralles Manuel, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Biología. Silva Santos Lucianna Helene, Instituto Pasteur (Montevideo). Dourron Juliette, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Biología. Alamón Lima Catalina Isadora, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Biología. Idiarte Montiel Juan, Instituto Pasteur (Montevideo). Amillis Sotiris Pantano Sergio, Instituto Pasteur (Montevideo). Ramón Ana, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Biología. |
| dc.creator.none.fl_str_mv | Sanguinetti Miralles, Manuel Silva Santos, Lucianna Helene Dourron, Juliette Alamón Lima, Catalina Isadora Idiarte Montiel, Juan Amillis, Sotiris Pantano, Sergio Ramón, Ana |
| dc.date.none.fl_str_mv | 2022 2023-11-21T12:01:21Z 2023-11-21T12:01:21Z |
| dc.format.none.fl_str_mv | 17 h. application/pdf |
| dc.identifier.none.fl_str_mv | Sanguinetti Miralles, M, Silva Santos, L, Dourron, J, [y otros autores]. "Substrate recognition properties from an intermediate structural state of the UreA transporter". International Journal of Molecular Science. [en línea] 2022, 23(24): 16039. 17 h. DOI: 10.3390/ijms232416039 1422-0067 https://hdl.handle.net/20.500.12008/41370 10.3390/ijms232416039 |
| dc.language.none.fl_str_mv | en_US eng |
| dc.publisher.none.fl_str_mv | MDPI |
| dc.relation.none.fl_str_mv | International Journal of Molecular Science, 2022, 23(24): 16039. |
| dc.rights.none.fl_str_mv | info:eu-repo/semantics/openAccess Licencia Creative Commons Atribución (CC - By 4.0) |
| dc.source.none.fl_str_mv | reponame:COLIBRI instname:Universidad de la República instacron:Universidad de la República |
| dc.subject.none.fl_str_mv | Aspergillus nidulans Urea transport AlphaFold2 Binding site Molecular docking |
| dc.title.none.fl_str_mv | Substrate recognition properties from an intermediate structural state of the UreA transporter |
| dc.type.none.fl_str_mv | Artículo info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion |
| description | 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. |
| eu_rights_str_mv | openAccess |
| format | article |
| id | anni_434550b599cdfee5d35b83e8c1941c4e |
| identifier_str_mv | Sanguinetti Miralles, M, Silva Santos, L, Dourron, J, [y otros autores]. "Substrate recognition properties from an intermediate structural state of the UreA transporter". International Journal of Molecular Science. [en línea] 2022, 23(24): 16039. 17 h. DOI: 10.3390/ijms232416039 1422-0067 10.3390/ijms232416039 |
| instacron_str | Universidad de la República |
| institution | Universidad de la República |
| instname_str | Universidad de la República |
| language | eng |
| language_invalid_str_mv | en_US |
| network_acronym_str | anni |
| network_name_str | oai-lr-anni |
| oai_identifier_str | oai:colibri.udelar.edu.uy:20.500.12008/41370 |
| publishDate | 2022 |
| publishDateSort | 2022 |
| publisher.none.fl_str_mv | MDPI |
| reponame_str | COLIBRI |
| repository.mail.fl_str_mv | |
| repository.name.fl_str_mv | |
| repository_id_str | |
| rights_invalid_str_mv | Licencia Creative Commons Atribución (CC - By 4.0) |
| spelling | Substrate recognition properties from an intermediate structural state of the UreA transporterSanguinetti Miralles, ManuelSilva Santos, Lucianna HeleneDourron, JulietteAlamón Lima, Catalina IsadoraIdiarte Montiel, JuanAmillis, SotirisPantano, SergioRamón, AnaAspergillus nidulansUrea transportAlphaFold2Binding siteMolecular dockingThrough 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.ANII: FCE_3_2018_1_148002MDPISanguinetti Miralles Manuel, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Biología.Silva Santos Lucianna Helene, Instituto Pasteur (Montevideo).Dourron Juliette, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Biología.Alamón Lima Catalina Isadora, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Biología.Idiarte Montiel Juan, Instituto Pasteur (Montevideo).Amillis SotirisPantano Sergio, Instituto Pasteur (Montevideo).Ramón Ana, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Biología.2023-11-21T12:01:21Z2023-11-21T12:01:21Z2022Artículoinfo:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersion17 h.application/pdfSanguinetti Miralles, M, Silva Santos, L, Dourron, J, [y otros autores]. "Substrate recognition properties from an intermediate structural state of the UreA transporter". International Journal of Molecular Science. [en línea] 2022, 23(24): 16039. 17 h. DOI: 10.3390/ijms2324160391422-0067https://hdl.handle.net/20.500.12008/4137010.3390/ijms232416039reponame:COLIBRIinstname:Universidad de la Repúblicainstacron:Universidad de la Repúblicaen_USengInternational Journal of Molecular Science, 2022, 23(24): 16039.Las obras depositadas en el Repositorio se rigen por la Ordenanza de los Derechos de la Propiedad Intelectual de la Universidad de la República.(Res. Nº 91 de C.D.C. de 8/III/1994 – D.O. 7/IV/1994) y por la Ordenanza del Repositorio Abierto de la Universidad de la República (Res. Nº 16 de C.D.C. de 07/10/2014)info:eu-repo/semantics/openAccessLicencia Creative Commons Atribución (CC - By 4.0)oai:colibri.udelar.edu.uy:20.500.12008/413702026-04-14T10:10:21Z |
| spellingShingle | Substrate recognition properties from an intermediate structural state of the UreA transporter Sanguinetti Miralles, Manuel Aspergillus nidulans Urea transport AlphaFold2 Binding site Molecular docking |
| status_str | publishedVersion |
| title | Substrate recognition properties from an intermediate structural state of the UreA transporter |
| title_full | Substrate recognition properties from an intermediate structural state of the UreA transporter |
| title_fullStr | Substrate recognition properties from an intermediate structural state of the UreA transporter |
| title_full_unstemmed | Substrate recognition properties from an intermediate structural state of the UreA transporter |
| title_short | Substrate recognition properties from an intermediate structural state of the UreA transporter |
| title_sort | Substrate recognition properties from an intermediate structural state of the UreA transporter |
| topic | Aspergillus nidulans Urea transport AlphaFold2 Binding site Molecular docking |
| url | https://hdl.handle.net/20.500.12008/41370 |