Structural rewiring of the mannose-binding pocket reveals adaptive FimH mutations that enhance uroplakin-associated glycan recognition and uropathogenic adhesion.

Urinary tract infections are a major global health burden, with uropathogenic Escherichia coli (UPEC) as the leading causative agent. UPEC adhesion is mediated by the fimbrial lectin FimH, which recognizes high-mannose glycans on the urothelial uroplakin (UPK) complex. While FimH interactions with isolated glycans are structurally characterized, its recognition of UPK1A-tethered glycans within the intact human UPK architecture remains unclear. In addition, the mutational logic by which the Mannose-Binding Pocket (MBP) is rewired to enhance glycan affinity and adaptive fitness has not been systematically explored. Here, integrative structural modeling, interface-based protein redesign, and mutational energy profiling were used to define FimH-glycan-UPK interactions and identify affinity-modulating substitutions. Using the porcine UPK structure as a scaffold, human UPK assemblies complexed with oligomannose-3 (OM3) and oligomannose-6 (OM6) were modeled and analyzed. Comparative redesign of the FimH MBP across human and porcine UPK complexes generated 545 designs, which were prioritized using a robust median-based composite Z-score framework integrating affinity and stability effects. This identified adaptive high-affinity substitutions, notably N135S and Y137F in the human complex, and D54G and D47V in the porcine complex. Several affinity-reducing designs also showed strong concordance with experimentally-characterized FimH mutants and known glycan-binding trends, supporting the predictive robustness of the approach. Energy decomposition revealed increased hydrogen bonding and favorable van der Waals contacts in high-affinity designs, accompanied by enhanced conformational rigidity from normal mode analysis. These findings define the structural-energetic basis of FimH evolutionary fine-tuning toward UPK-associated glycans and provide a framework for rational anti-adhesive therapeutic design.