Abstract
Membrane proteins play vital roles in inside-out and outside-in signal transduction by responding to inputs that include mechanical stimuli. Mechanical gating may be mediated by the membrane or by protein(s) but evidence for the latter is scarce. Here we use force spectroscopy, protein engineering and bacterial growth assays to investigate the effects of force on complexes formed between TonB and TonB-dependent transporters (TBDT) from Gram-negative bacteria. We confirm the feasibility of protein-only mediated mechanical gating by demonstrating that the interaction between TonB and BtuB (a TBDT) is sufficiently strong under force to create a channel through the TBDT. In addition, by comparing the dimensions of the force-induced channel in BtuB and a second TBDT (FhuA), we show that the mechanical properties of the interaction are perfectly tuned to their function by inducing formation of a channel whose dimensions are tailored to the ligand.
Highlights
Membrane proteins play vital roles in inside-out and outside-in signal transduction by responding to inputs that include mechanical stimuli
To investigate the mechanism of TonB-dependent transporters (TBDT) function and more generally, the role of protein–protein interactions in membrane protein gating, we first employ single-molecule force spectroscopy using an atomic force microscope (AFM) to show that the noncovalent interaction between the Ton box from BtuB (TBBtuB) and TonB is surprisingly durable under extension. Repeating this experiment using BtuB reconstituted in E. coli polar liposomes adsorbed onto a mica surface and TonB immobilized onto an AFM tip (Fig. 1c), we find that the TBBtuB–TonB complex is sufficiently durable under force to allow the unfolding of half of the plug domain before its dissociation
As a first step towards understanding the effects of mechanical extension on this network, we focussed on the non-covalent TonB–TBBtuB interaction using a minimal system comprising TonBDTMD
Summary
Membrane proteins play vital roles in inside-out and outside-in signal transduction by responding to inputs that include mechanical stimuli. While it is known that TonB-mediated transport requires TonB to be tethered to an energized IM19, the mechanism by which TonB remodels the plug domains of TBDTs remains unclear[20] Favoured models, such as the pulling hypothesis[21,22] or the rotational surveillance and energy transfer[23] model, suggest that TonB applies a mechanical remodelling force driven by its interaction with the ExbBD complex in the energized IM. This induces partial or full-plug domain unfolding via the non-covalent interaction of the Ton box with TonBCTD3,24–26. Mica surface intra-protein interactions are ideally oriented to engender these mechanical phenotypes[27,28] as shown using molecular dynamics (MD)[22]
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