Protonmotive force, ExbB and ligand‐bound FepA drive conformational changes in TonB

Abstract

SummaryTonB couples the cytoplasmic membrane protonmotive force (pmf) to active transport across the outer membrane, potentially through a series of conformational changes. Previous studies of a TonB transmembrane domain mutant (TonB-⌬V17) and its phenotypical suppressor (ExbB-A39E) suggested that TonB is conformationally sensitive. Here, two new mutations of the conserved TonB transmembrane domain SHLS motif were isolated, TonB-S16L and -H20Y, as were two new suppressors, ExbB-V35E and -V36D. Each suppressor ExbB restored at least partial function to the TonB mutants, although TonB-⌬V17, for which both the conserved motif and the register of the predicted transmembrane domain ␣-helix are affected, was the most refractory. As demonstrated previously, TonB can undergo at least one conformational change, provided both ExbB and a functional TonB transmembrane domain are present. Here, we show that this conformational change reflects the ability of TonB to respond to the cytoplasmic membrane proton gradient, and occurs in proportion to the level of TonB activity attained by mutant-suppressor pairs. The phenotype of TonB-⌬V17 was more complex than the -S16L and -H20Y mutations, in that, beyond the inability to be energized efficiently, it was also conditionally unstable. This second defect was evident only after suppression by the ExbB mutants, which allow transmembrane domain mutants to be energized, and presented as the rapid turnover of TonB-⌬V17. Importantly, this degradation was dependent upon the presence of a TonB-dependent ligand, suggesting that TonB conformation also changes following the energy transduction event. Together, these observations support a dynamic model of energy transduction in which TonB cycles through a set of conformations that differ in potential energy, with a transition to a higher energy state driven by pmf and a transition to a lower energy state accompanying release of stored potential energy to an outer membrane receptor.

Agrobacterium VirB10, an ATP energy sensor required for type IV secretion

“…Upon sensing of proton motive force (pmf) through partner protein interactions at the IM, TonB undergoes a structural transition as monitored by a change in protease susceptibility (19). Similarly, we found that VirB10 adopts alternative protease-susceptible and -resistant conformations as a function of cellular energetic status.…”

“…Similarly, TonB possesses motifs enabling contacts with its partner proteins at both membranes. For TonB, these transenvelope protein contacts are thought to form simultaneously or through a shuttle mechanism to mediate coupling of IM transmembrane potential to the gating of OM transporters (17)(18)(19)). Thus, we tested whether VirB10 might also contribute to T4SS machine assembly or function through an energy-sensing mechanism.…”

“…It is interesting to note that VirD4 and VirB11 most probably repetitively bind and hydrolyze ATP during the course of machine assembly or substrate transfer, raising the possibility that this transenvelope bridge forms only transiently. In this context, it is intriguing that ABC transporters, and possibly also the TonB-dependent systems, form transient transenvelope structures specifically in response to energy and ligand binding (19,41). Studies examining the possible contribution of substrate binding to biogenesis of the VirB͞D4 T4SS are thus warranted.…”

Studies on colicin B translocation: FepA is gated by TonB

“…The DfepA strain KP1490 was constructed by a FLP recombinaseassisted homologous recombination (Datsenko and Wanner, 2000). Briefly the chromosomal fepA in KP1270 [W3110 aroB ( Larsen et al, 1999)] was replaced by kan r gene, resulting in strain KP1488. The kan r gene was subsequently excised using FLP recombinase, resulting in strain KP1490.…”

Movements of the TolR C-terminal Domain Depend on TolQR Ionizable Key Residues and Regulate Activity of the Tol Complex

“…ExbBD proteins are auxiliary partners of TonB protein, which is responsible for the energy-dependent uptake of iron-charged siderophores (15). As for TolA, activity of TonB is dependent upon PMF, ExbB, ExbD, and a conserved motif within the TonB inner membrane anchor, regulating conformational modifications (16,17). MotA and MotB are the two components of the flagellar stator, responsible for coupling PMF with flagella rotation (18).…”