Computational design and experimental validation of peptide inhibitors to disrupt urease enzyme maturation in pathogenic bacteria <i>Proteus mirabilis</i>

Urease, an essential virulence factor in pathogens such as Proteus mirabilis, requires nickel incorporation into its active site for maturation. This process is mediated by accessory proteins, with a key role of UreE–UreG interaction representing an early step in nickel delivery. Disruption of this interaction impairs enzyme maturation, reduces urease secretion, and limits microbial survival under acidic conditions. In this study, peptide inhibitors were designed and evaluated to target the protein-protein interaction within the UreE–UreG complex. Protein structures were predicted using AlphaFold2, and hotspot analysis identified critical interface residues. Peptides generated via the PeptiDerive server were refined and prioritized using HPEPDOCK, HADDOCK Z-scores, Prodigy binding-affinity estimates, and molecular dynamics simulation data. The top five peptides were synthesized for in vitro evaluation. UreE–UreG binding was assessed using Surface Plasmon Resonance (SPR), and Differential Scanning Fluorimetry (DSF) was used to evaluate stabilization of the protein-peptide complexes. Molecular dynamics simulations and MM-GBSA analysis demonstrated stable peptide binding, with favorable free energies for G11-UreE (−38.737 kcal/mol) and E09-UreG (−27.491 kcal/mol). Per-residue energy decomposition revealed key hotspot residues driving stable peptide binding in G11-UreE and E09-UreG complexes, primarily via electrostatic and van der Waals interactions. Consistently, DSF revealed positive ΔTm shifts for E09-UreG (+2.0 °C) and UreE-G11 (+2.5 °C), confirming complex formation. These results suggest that engineered peptides may interfere with urease maturation and provide insights into the mechanisms underlying protein-protein interaction modulation. The findings support peptide-based strategies can be promising approaches to target bacterial urease virulence.