The solute-binding proteins DppA1-5 of Pseudomonas aeruginosa have distinct substrate profiles.

Pseudomonas aeruginosa is an opportunistic pathogen with high antibiotic resistance that infects immunocompromised patients. Its survival in diverse environments relies on nutrient uptake systems such as the dipeptide permease (Dpp), an ATP-binding cassette transporter that imports di- and tripeptides. Unlike other bacteria, whose Dpp includes a single solute-binding protein that binds substrates in the periplasm, P. aeruginosa encodes five paralogs (DppA1-5), the functional significance of which remains unclear. Here, we systematically profile the ligand specificities of all five DppA proteins using Differential Scanning Fluorimetry with a library of 281 di- and tripeptides. We find that DppA1 and DppA3 preferentially bind dipeptides, whereas DppA2 and DppA4 favor tripeptides. DppA5 shows no detectable binding, suggesting a divergent function. Ligand binding is highly structure-sensitive and distinct across DppA paralogs. A comparative reanalysis of published nutrient utilization data for dppA1-5 suggests that previous studies underestimated this functional specialization. In contrast, our findings indicate that P. aeruginosa encodes multiple DppA paralogs to expand Dpp substrate scope and enhance nutrient acquisition. This work provides a foundation for further exploration of the Dpp in bacterial signaling and its exploitation for drug delivery via Trojan Horse antimicrobials.