The Correlation between Binding and Transport of a Siderophore Complex through Its TonB-Dependent Transporter

A promising approach to deliver antibiotics into Gram-negative bacteria is the “Trojan Horse” strategy, which exploits bacterial iron uptake systems to facilitate transport across the outer membrane. Only one “Trojan Horse” antibiotic has reached the market, underscoring the need to deepen our understanding of this mechanism. In this study, we investigate the molecular mechanisms underlying siderophore-mediated transport through PfeA, a TonB-dependent transporter from Pseudomonas aeruginosa, using a variety of modeling techniques, and analyze modalities of substrate translocation across the outer membrane. We reconstruct the passage of the Fe3+–enterobactin complex through PfeA, revealing distinct roles for the first and second binding sites. The first binding site initiates signal propagation toward the TonB box, while the deeper second binding site facilitates progressive ligand migration by weakening plug–barrel interactions. Thus, destabilization of plug–barrel hydrogen bonds─rather than movement of the plug domain─triggers substrate translocation. These findings provide mechanistic insight into the molecular basis of siderophore uptake and help clarify how specific binding and conformational events can facilitate substrate translocation through TonB-dependent transporters.