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.

The Ins and Outs of Urea: Identification of Putative DUR3-like Urea Transporters in the Oligohaline Nerite Snail Theodoxus fluviatilis and Their Expression under Changing Salinities

“…We have not yet functionally characterized the putative DUR3-like urea transporters of T. fluviatilis, but the presence of most of the amino acid residues that were reported to be essential for the function of UreA, the respective DUR3 protein in A. nidulans [18], and that are present in DUR3-like of T. squamosa [20] (see Figure 1) strongly indicates functionality. In a recent publication, however, Sanguinetti et al [38] convincingly demonstrated that the amino acid residues W82, W84, N279, and T282 in UreA of A. nidulans are directly involved in urea binding and translocation. All four amino acid residues occur in Tf UT-B, but only W84 is present in Tf UT-A1-A3 and DUR3-like of T. squamosa (see Figure 1).…”

“…However, the residues W82, N279, T282, Y437, and S446 of UreA are missing in Tf UT-A1-A3 and the DUR3-like urea transporter of T. squamosa, but are present in Tf UT-B. For W82, N279, and T282, direct involvement in urea binding and translocation has recently been shown [38], whereas Y437 seems to be involved in substrate selectivity and S446 is necessary for the correct sorting of UreA to the membrane [39].…”