Mutation-Induced Conformational Remodeling of Dihydropteroate Synthase Governs Sulfonamide Resistance in...

Streptococcus pneumoniae is a major bacterial pathogen, and dihydropteroate synthase (DHPS) is the primary target of sulfonamides (SAs). Environmental exposure to SAs promotes the emergence and spread of antibiotic resistance. However, the molecular basis of DHPS mutation-mediated resistance remains unclear. Derived from the folP sequences of trimethoprim-sulfamethoxazole-resistant clinical isolates, six clinically relevant DHPS mutants were constructed to investigate the molecular basis of resistance and to clarify how structure-activity relationships influence ligand recognition and binding modes. The results showed that the mutations altered the structural characteristics of the catalytic cavity and the substrate tunnel but exerted limited effects on the entry or binding of 6-hydroxymethyl-7,8-dihydropterin pyrophosphate (DHPPP). DHPPP binding induced conformational remodeling, including expansion of loop 2 or loop 5 and rotation of HIE153. Compared with the wild type, sulfamethoxazole (SMX) exhibited reduced binding affinities across all mutants, with loop 2 expansion and impaired loop closure identified as the primary structural determinants of resistance. Additionally, HIE153 indirectly contributed to SMX recognition by maintaining the local microenvironment, binding pocket conformation, and neighboring interaction networks. Structure-activity relationship analysis showed that fused-ring sulfonamides exhibited the strongest tolerance to resistance-associated mutations. These findings reveal how DHPS mutations preserve natural-substrate recognition while selectively weakening SA binding, providing molecular-level insight into SA resistance and its implications for environmental antimicrobial resistance.