Allosteric signalling in the outer membrane translocation domain of PapC usher
- Birkbeck College, United Kingdom
- University of Houston, United States
- Stony Brook University, United States
- University of Würzburg, Germany
- Université Paris Descartes, France
Abstract
PapC ushers are outer-membrane proteins enabling assembly and secretion of P pili in uropathogenic E. coli. Their translocation domain is a large β-barrel occluded by a plug domain, which is displaced to allow the translocation of pilus subunits across the membrane. Previous studies suggested that this gating mechanism is controlled by a β-hairpin and an α-helix. To investigate the role of these elements in allosteric signal communication we developed a method combining evolutionary and molecular dynamics studies of the native translocation domain and mutants lacking the β-hairpin and/or α-helix. Analysis of a hybrid residue interaction network suggests distinct regions (residue 'communities') within the translocation domain (especially around β12-β14) linking these elements, thereby modulating PapC gating. Antibiotic sensitivity and electrophysiology experiments on a set of alanine-substitution mutants confirmed functional roles for four of these communities. This study illuminates the gating mechanism of PapC ushers and its importance in maintaining outer-membrane permeability.
Article and author information
Author details
Reviewing Editor
- Volker Dötsch, Goethe University, Germany
Version history
- Received: May 30, 2014
- Accepted: September 29, 2014
- Accepted Manuscript published: October 1, 2014 (version 1)
- Version of Record published: October 28, 2014 (version 2)
- Version of Record updated: September 27, 2016 (version 3)
Copyright
© 2014, Farabella et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Allosteric signalling in the outer membrane translocation domain of PapC usher
eLife 3:e03532.
https://doi.org/10.7554/eLife.03532
Further reading
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- Structural Biology and Molecular Biophysics
Bacteria utilize various strategies to prevent internal dehydration during hypertonic stress. A common approach to countering the effects of the stress is to import compatible solutes such as glycine betaine, leading to simultaneous passive water fluxes following the osmotic gradient. OpuA from Lactococcus lactis is a type I ABC-importer that uses two substrate-binding domains (SBDs) to capture extracellular glycine betaine and deliver the substrate to the transmembrane domains for subsequent transport. OpuA senses osmotic stress via changes in the internal ionic strength and is furthermore regulated by the 2nd messenger cyclic-di-AMP. We now show, by means of solution-based single-molecule FRET and analysis with multi-parameter photon-by-photon hidden Markov modeling, that the SBDs transiently interact in an ionic strength-dependent manner. The smFRET data are in accordance with the apparent cooperativity in transport and supported by new cryo-EM data of OpuA. We propose that the physical interactions between SBDs and cooperativity in substrate delivery are part of the transport mechanism.
