HDX-MS performed on BtuB in E. coli outer membranes delineates the luminal domain’s allostery and unfolding upon B12 and TonB binding

Abstract

To import large metabolites across the outer membrane of gram-negative bacteria, TonB-dependent transporters (TBDTs) undergo significant conformational change. After substrate binding in BtuB, the Escherichia coli vitamin B12 TBDT, TonB binds and couples BtuB to the inner-membrane proton motive force that powers transport [N. Noinaj, M. Guillier, T. J. Barnard, S. K. Buchanan, Annu. Rev. Microbiol. 64, 43–60 (2010)]. However, the role of TonB in rearranging the plug domain of BtuB to form a putative pore remains enigmatic. Some studies focus on force-mediated unfolding [S. J. Hickman, R. E. M. Cooper, L. Bellucci, E. Paci, D. J. Brockwell, Nat. Commun. 8, 14804 (2017)], while others propose force-independent pore formation by TonB binding [T. D. Nilaweera, D. A. Nyenhuis, D. S. Cafiso, eLife 10, e68548 (2021)], leading to breakage of a salt bridge termed the “Ionic Lock.” Our hydrogen–deuterium exchange/mass spectrometry (HDX-MS) measurements in E. coli outer membranes find that the region surrounding the Ionic Lock, far from the B12 site, is fully destabilized upon substrate binding. A comparison of the exchange between the B12-bound and the B12+TonB–bound complexes indicates that B12 binding is sufficient to unfold the Ionic Lock region, with the subsequent binding of a TonB fragment having much weaker effects. TonB binding accelerates exchange in the third substrate-binding loop, but pore formation does not obviously occur in this or any region. This study provides a detailed structural and energetic description of the early stages of B12 passage that provides support both for and against current models of the transport process.