Substrate Recognition Properties from an Intermediate Structural State of the UreA Transporter

Abstract

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.

Keywords: AlphaFold2; Aspergillus nidulans; binding site; molecular docking; urea transport.

Figure 1

Comparison of the different UreA modeled conformations. (A) Schematic representation of the rocking-bundle mechanism where one domain (left) rocks against another less motile domain (right) to give access and release the substrate. Interface residues (green circles) from both domains form the substrate binding site, while substrate-triggered gating elements (yellow bars) can act as selectivity filters over or below the binding site. Specific and dynamic domains (brown triangle) operate as occlusion barriers to hinder substrate leakage. (B) Predicted outward-facing (orange) and inward-facing (blue) UreA structures with highlighted TMSs 2, 3, 7 and 11, respectively. (C) Comparison of outward- (orange) and inward-facing (blue) conformations of TMSs 2, 3, 7 and 11 that might compose the urea translocation pathway. Light gray structures are morphed (i.e., linearly interpolated) intermediates produced by UCSF Chimera’s morph conformations tool [37]. These frames indicate the possible movement in the TMSs when cycling between conformations. (D) One of the predicted UreA AF2 models (purple) with TMSs 2, 3, 7 and 11 (dark purple) highlighted. (E) Morphed intermediates (light gray structures) proposed that UreA’s AF2 (purple) and outward (orange) models have different TMSs 2 and 7 conformations. (F) Morphed intermediates (light gray) proposed a more modest mobility, but TMSs 2 and 7 were closer together in the AF2 model than in the inward model. Helices are shown as cylinders and less relevant protein loops were hidden to improve the visualization. An animation showing the continuous passage from one model to the other is provided in supplementary material to illustrate the differences between the different states.

Figure 2

W82 and W84 orientations in the UreA conformations. (A) Comparison between the orientations of W82 and W84 in each predicted conformation. In the intermediate UreA state, these two residues facing each other form a potential groove that may stabilize urea in the putative binding site. For a video of the orientation transitions between conformations, see the supplemental material information. (B) A representation of the possible urea gating mechanism showing the potential flexibility of TMSs 2, 3, 7, and 11, W82, and W84.

Figure 3

Docking of urea modulates the orientation of W82 and W84. (A) The radial plot of the angles between the planes defined by the indole rings of W82 and W84 and the schematic representation of angle (β) and distance (d) calculation. Red-bordered points show angles between 0° to 90° and distances up to 6 Å, thus, indicating that the sidechains could be potentially in an aromatic stacking orientation. Sampled angles are intercepted in all models between 45° and 67.5°, with the angles in the outward-facing model (B) reaching higher values than those sampled in the AlphaFold2 (C) and inward-facing (D) models.

Figure 4

Growth phenotypes of mutant UreA strains. Mutant strains were grown at 37 °C for 48 h on 0.625–5 mM urea as nitrogen source or on 0.625–5 mM 2-thiourea (2-TU) with 10 mM sodium nitrate (NaNO₃) as nitrogen source to test resistance to the analogue. Growth on acetamide as sole nitrogen source is not shown as no obvious phenotype of growth deficiency is evident when comparing wt and ureAΔ in all conditions tested. Growth on 5 mM ammonium (NH₄) and 10 mM NaNO₃ are used as controls. Wild type (wt) and ureAΔ strains are shown as positive and negative controls, respectively. Similar results were obtained at 25 °C (Supplementary Figure S8).

Figure 5

Docked urea, 2-TU, and ACM correlation and interaction profiles. (A) wt and nine mutations’ docking scores and experimental affinities correlated well for all ligands. Spearman correlation was 0.87, 0.95, and 0.90 for urea, 2-TU, and ACM, respectively. We highlighted all wt affinities, and urea results for W82F and N279A that have close affinity to wt. (B) Urea, 2-TU, and ACM had varied interaction profiles. Urea’s most prevalent interactions were amide-aromatic stacking and hydrogen bonding. Most residues in 2-TU and ACM have more hydrophobic interactions, while ACM’s W82 and W84 residues also achieved amide-aromatic stacking similar to urea.