Going Outside the TonB Box: Identification of Novel FepA-TonB Interactions In Vivo

Abstract

In Gram-negative bacteria, the cytoplasmic membrane protein TonB transmits energy derived from proton motive force to energize transport of important nutrients through TonB-dependent transporters in the outer membrane. Each transporter consists of a beta barrel domain and a lumen-occluding cork domain containing an essential sequence called the TonB box. To date, the only identified site of transporter-TonB interaction is between the TonB box and residues ϳ158 to 162 of TonB. While the mechanism of ligand transport is a mystery, a current model based on site-directed spin labeling and molecular dynamics simulations is that, following ligand binding, the otherwise-sequestered TonB box extends into the periplasm for recognition by TonB, which mediates transport by pulling or twisting the cork. In this study, we tested that hypothesis with the outer membrane transporter FepA using in vivo photo-cross-linking to explore interactions of its TonB box and determine whether additional FepA-TonB interaction sites exist. We found numerous specific sites of FepA interaction with TonB on the periplasmic face of the FepA cork in addition to the TonB box. Two residues, T32 and A33, might constitute a ligandsensitive conformational switch. The facts that some interactions were enhanced in the absence of ligand and that other interactions did not require the TonB box argued against the current model and suggested that the transport process is more complex than originally conceived, with subtleties that might provide a mechanism for discrimination among ligand-loaded transporters. These results constitute the first study on the dynamics of TonB-gated transporter interaction with TonB in vivo. IMPORTANCE The TonB system of Gram-negative bacteria has a noncanonical active transport mechanism involving signal transduction and proteins integral to both membranes. To achieve transport, the cytoplasmic membrane protein TonB physically contacts outer membrane transporters such as FepA. Only one contact between TonB and outer membrane transporters has been identified to date: the TonB box at the transporter amino terminus. The TonB box has low information content, raising the question of how TonB can discriminate among multiple different TonBdependent transporters present in the bacterium if it is the only means of contact. Here we identified several additional sites through which FepA contacts TonB in vivo, including two neighboring residues that may explain how FepA signals to TonB that ligand has bound.KEYWORDS TonB, FepA, cork domain, p-benzoyl-L-phenylalanine, enterochelin, TonB box, energy transduction I ron is essential for growth of Gram-negative bacteria but can be difficult to acquire because of its insolubility under aerobic conditions, its scarcity in hosts, and the protective nature of the Gram-negative bacterial outer membrane (OM) (1). The majority of Gram-negative bacteria synthesize and secrete siderophores, which are high-

The Intrinsically Disordered Region of ExbD is Required for Signal Transduction

“…1B): ExbD (44-48ala), ExbD (49-53ala), ExbD (54-58ala), and ExbD (59-63ala). TonB system activity was determined by measuring the initial transport rates of two different iron-siderophores, 55 Fe-enterochelin and 55 Fe-ferrichrome (6, 40), to identify any TonB-dependent transporter-specific activities.…”

“…However, because iron-siderophore complexes are too large, too scare, and too important to passively diffuse through the outer-membrane porins, they require customized 22-stranded beta-barrel transporters called TonB-dependent transporters (TBDTs) to transition across the outer-membrane (1, 4, 5). After the siderophore captures an iron atom extracellularly, the iron-siderophore complex binds to a TDBT with sub-nanomolar affinity, causing the ligand-loaded TBDT to signal on its periplasmic face that ligand is bound (6–9). Energy is required to release the iron-siderophore from the TBDT (1–3).…”

“…This pattern is referred to as the TonB energization cycle (26). TonB binds to TBDTs both in vitro and in vivo whether or not it is “energized”, and therefore, it must be correctly configured during an energy transduction cycle to enable productive interaction with the TBDT (6, 27–29). Both ExbB and ExbD are required to correctly configure TonB to energize active transport across the outer membrane--ExbB indirectly, and ExbD directly (16, 30, 31).…”

Characterization ofEscherichia coliExbD protein modificationin vivo

“…The TBDTs consist of a 22-stranded β-barrel that is entirely occluded by an amino terminal globular domain, called the cork (12). At least one role of TonB is to energize movement of the cork to allow transport into the periplasm (13–17).…”

“…The integral cytoplasmic membrane proteins TonB, ExbB, and ExbD appear to form a complex in the cytoplasmic membrane with TonB extending across the periplasm to mechanically energize active transport through customized beta barrels in the outer membrane (11, 17, 27–29). TonB and ExbD are anchored in the cytoplasmic membrane by their N-termini, with the majority of each protein extending into the periplasmic space (Fig.…”