TonB Binding Produces Allosteric Changes in the Outer Loops and Substrate Binding Site of the TBDT BTUB

Abstract

Outer-membrane TonB-dependent transporters function in the uptake of essential nutrients, and are important for the success of many bacterial pathogens. During transport, these proteins undergo a cycle of binding and unbinding to the inner membrane protein TonB, through an interaction that is mediated by the Ton box, an energy-coupling segment at the periplasmic interface. Previous work has shown that in BtuB, the vitamin B12 transporter in Escherichia coli, substrate binding unfolds the Ton box, an event which regulates the interaction between BtuB and TonB.In previous work (Joseph et al. (2015) Angew Chem Int Ed Engl. 54:6196) we demonstrated that electron-electron resonance (DEER) data could be obtained on BtuB in native outer membrane preparations and whole cells, and that distances were comparable in native and reconstituted systems. In the present study DEER was used in outer membranes to examine structural heterogeneity in the loops and the effect of ligands on loop configuration. The data show that the loops sample a large conformational space, but that the co-ligand calcium selects for one conformational substrate in the 2nd external loop. The ordering of this loop is correlated with a dramatic increase in the affinity of BtuB for B12. Interestingly, the binding of a C-terminal fragment of TonB to the periplasmic interface of BtuB produces significant changes in the exterior loops to generate a more open substrate binding pocket. Moreover, spectra from the spin-labeled cobalamin analog indicate that TonB weakens the affinity of the substrate and can displace vitamin B12 from BtuB. These data indicate that signal transduction moves in both directions in BtuB and that reduced B12 binding by TonB is a step in the transport process.This work was supported by NIGMS, GM035215.