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

• Published in: Proceedings of the National Academy of Sciences - PNAS 2022-05, Vol.119 (20), p.1-8
• Main Authors: Zmyslowski, Adam M., Baxa, Michael C., Gagnon, Isabelle A., Sosnick, Tobin R.
• Format: Article
• Language: English
• Subjects: Allosteric Regulation; Bacteria; Bacterial Outer Membrane Proteins - chemistry; Bacterial Outer Membrane Proteins - metabolism; Binding; Biological Sciences; Biological Transport; Deuterium; Domains; E coli; Escherichia coli; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; Gram-negative bacteria; Hydrogen Deuterium Exchange-Mass Spectrometry; Hydrogen-deuterium exchange; Mass spectrometry; Mass spectroscopy; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Membrane Transport Proteins - chemistry; Membrane Transport Proteins - metabolism; Membranes; Metabolites; Outer membranes; Pore formation; Protein Binding; Protein Domains; Protein Folding; Protonmotive force; Substrates; Transport processes; Vitamin B 12 - metabolism; Vitamin B12
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.2119436119

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.