FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W3199685002> ?p ?o ?g. }

• W3199685002 abstract "Article13 September 2021Open Access Source DataTransparent process Porin threading drives receptor disengagement and establishes active colicin transport through Escherichia coli OmpF Marie-Louise R Francis Marie-Louise R Francis orcid.org/0000-0002-4257-0162 Department of Biochemistry, University of Oxford, Oxford, UK These authors contributed equally to this work Search for more papers by this author Melissa N Webby Melissa N Webby orcid.org/0000-0001-5721-0381 Department of Biochemistry, University of Oxford, Oxford, UK These authors contributed equally to this work Search for more papers by this author Nicholas G Housden Nicholas G Housden Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Renata Kaminska Renata Kaminska Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Emma Elliston Emma Elliston Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Boonyaporn Chinthammit Boonyaporn Chinthammit orcid.org/0000-0001-5433-7977 Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Natalya Lukoyanova Natalya Lukoyanova orcid.org/0000-0002-2051-0812 Department of Biological Sciences, ISMB, Birkbeck College, London, UK Search for more papers by this author Colin Kleanthous Corresponding Author Colin Kleanthous [email protected] orcid.org/0000-0002-3273-0302 Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Marie-Louise R Francis Marie-Louise R Francis orcid.org/0000-0002-4257-0162 Department of Biochemistry, University of Oxford, Oxford, UK These authors contributed equally to this work Search for more papers by this author Melissa N Webby Melissa N Webby orcid.org/0000-0001-5721-0381 Department of Biochemistry, University of Oxford, Oxford, UK These authors contributed equally to this work Search for more papers by this author Nicholas G Housden Nicholas G Housden Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Renata Kaminska Renata Kaminska Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Emma Elliston Emma Elliston Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Boonyaporn Chinthammit Boonyaporn Chinthammit orcid.org/0000-0001-5433-7977 Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Natalya Lukoyanova Natalya Lukoyanova orcid.org/0000-0002-2051-0812 Department of Biological Sciences, ISMB, Birkbeck College, London, UK Search for more papers by this author Colin Kleanthous Corresponding Author Colin Kleanthous [email protected] orcid.org/0000-0002-3273-0302 Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Author Information Marie-Louise R Francis1,3, Melissa N Webby1, Nicholas G Housden1, Renata Kaminska1, Emma Elliston1,4, Boonyaporn Chinthammit1,5, Natalya Lukoyanova2 and Colin Kleanthous *,1 1Department of Biochemistry, University of Oxford, Oxford, UK 2Department of Biological Sciences, ISMB, Birkbeck College, London, UK 3Present address: NovaBiotics, Aberdeen, UK 4Present address: Arnold & Porter Kaye Scholer LLP, London, UK 5Present address: University College London, London, UK *Corresponding author. Tel: +44 1865 613370; E-mail: [email protected] The EMBO Journal (2021)40:e108610https://doi.org/10.15252/embj.2021108610 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Bacteria deploy weapons to kill their neighbours during competition for resources and to aid survival within microbiomes. Colicins were the first such antibacterial system identified, yet how these bacteriocins cross the outer membrane (OM) of Escherichia coli is unknown. Here, by solving the structures of translocation intermediates via cryo-EM and by imaging toxin import, we uncover the mechanism by which the Tol-dependent nuclease colicin E9 (ColE9) crosses the bacterial OM. We show that threading of ColE9’s disordered N-terminal domain through two pores of the trimeric porin OmpF causes the colicin to disengage from its primary receptor, BtuB, and reorganises the translocon either side of the membrane. Subsequent import of ColE9 through the lumen of a single OmpF subunit is driven by the proton-motive force, which is delivered by the TolQ-TolR-TolA-TolB assembly. Our study answers longstanding questions, such as why OmpF is a better translocator than OmpC, and reconciles the mechanisms by which both Tol- and Ton-dependent bacteriocins cross the bacterial outer membrane. Synopsis Colicins were the first antibacterial agents found to be produced by bacteria, yet it has remained unclear how they cross the outer membrane of E. coli. Here, structural and imaging data reveal the mechanism of ColE9 toxin import via the OmpF translocon. Cryo-electron microscopy translocon structures of the bacteriocin colicin E9 reveals the early steps of translocation across the outer bacterial membrane. Translocon formation results in the passive displacement of the colicin from its receptor, BtuB. Subsequent import of colicin E9 through the lumen of a single OmpF subunit is driven by the proton-motive force. Electrostatic properties of the porin lumen explain why colicins are preferentially transported through OmpF rather than OmpC. Introduction The asymmetric outer membrane (OM) of Gram-negative bacteria, composed of lipopolysaccharides (LPS) in the outer leaflet and phospholipids in the inner leaflet, shields the organism against environmental insults, the immune systems of plants and animals, bile salts in the human gut and several classes of antibiotics (Nikaido, 2003; Whitfield & Trent, 2014; Ranf, 2016; Vergalli et al, 2020). Bacteria have nevertheless evolved potent antibacterial systems to breach the OM that are integral to the internecine warfare between bacterial populations as they compete for space in which to grow and nutrients on which to feed (Granato et al, 2019). Attack strategies are typically of two types, those that depend on physical or close contact between bacterial cells and those mediated by diffusible molecules (Ruhe et al, 2020). The former includes contact-dependent inhibitors, where a toxin projected from an attacking cell binds a surface receptor of a recipient cell prior to import (Aoki et al, 2010), and type VI secretion, where toxins are delivered by a needle that punctures the OM (Basler et al, 2013). Diffusible toxins are generally referred to as bacteriocins and can be peptides or proteins. Here, we focus on a family of protein bacteriocins from the antimicrobial armoury of the Enterobacteriaceae and reveal their convoluted use of porins for OM transport. Protein bacteriocins from the Enterobacteriaceae and Pseudomonadaceae (hereafter referred to as bacteriocins) are plasmid or chromosomally encoded multidomain toxins that kill cells through the action of a C-terminal cytotoxic domain orchestrated by N-terminal regions of the toxin (Kleanthous, 2010). Cytotoxic activity is expressed either in the periplasm, through depolarisation of the cytoplasmic membrane or cleavage of the lipid II peptidoglycan precursor, or in the cytoplasm through cleavage of specific tRNAs, ribosomal RNA or chromosomal DNA. Passage across the OM is catalysed by one of two proton-motive force (PMF)-linked assemblies in the inner membrane. Ton-dependent (group B) bacteriocins contact TonB via TonB-dependent transporters (TBDTs) in the OM, whereas Tol-dependent (group A) bacteriocins contact one or more components of the Tol-Pal system (Cascales et al, 2007). Nearly all Tol-dependent bacteriocins identified to-date enter bacteria using a porin in combination with an outer membrane protein receptor, which can be a neighbouring TBDT or the porin itself (Kleanthous, 2010). Escherichia coli porins OmpF and OmpC are abundant trimeric β-barrel outer membrane proteins with narrow channels running through them that taper to an eyelet, 7–8 Å at the narrowest point (Nikaido, 2003). The dimensions and electrostatic nature of the eyelet allow the diffusion of nutrients and metabolites (< 600 Da) but excludes large antibiotics such as vancomycin and folded proteins. Recent work has shown that Ton-dependent bacteriocins specific for Pseudomonas aeruginosa are transported unfolded through TBDTs (White et al, 2017). Import is energised by the PMF via TonB and its associated ExbB-ExbD stator complex. By contrast, controversy surrounds how Tol-dependent bacteriocins exploit porins to cross the OM and whether this transport is energised. The present work focuses on the group A endonuclease (DNase) colicin, ColE9, a member of the E group of colicins (ColE2-E9), all of which use the same import machinery but target different nucleic acids in the cytoplasm (DNA, rRNA or tRNAs) (Kleanthous, 2010). We demonstrate using cryo-EM and live-cell imaging that ColE9 exploits the trimeric porin OmpF to cross the OM of E. coli by a combination of non-energised and energised transport. We show that following binding to its TBDT receptor, BtuB, ColE9 threads its disordered N-terminus through two subunits of OmpF both as a means of capturing a component of the PMF-coupled Tol-Pal system in the periplasm but also to disengage the toxin from its receptor. ColE9 then translocates unfolded through the narrow lumen of a single OmpF subunit by a process that is entirely driven by the PMF. We also demonstrate that large fluorophores normally excluded by the pores of OmpF can be forcibly brought into the cell by attaching them to the colicin. Results Cryo-EM structure of the ColE9 outer membrane translocon ColE9 is a plasmid-encoded heterodimer comprising a 60-kDa toxin bound to a small immunity protein, Im9. The two proteins are released together from E. coli cells in response to environmental stress (Cooper & James, 1984). Immunity proteins are high-affinity inhibitors that are co-expressed with the colicin and protect the producing cell from its cytotoxic action (Kleanthous et al, 1999), but are jettisoned during import of the bacteriocin (Vankemmelbeke et al, 2013). ColE9 has four functionally annotated domains (Fig 1A) (Klein et al, 2016). At its N-terminus is an 83 amino acid intrinsically unstructured translocation domain (IUTD) that houses three protein-protein interaction epitopes (Housden et al, 2010); a 16-residue TolB-binding epitope (TBE) associates with the periplasmic protein TolB which is flanked by two OmpF-binding sites (OBS1 and OBS2). The IUTD is followed by a folded α/β domain which is thought to be associated with inner membrane translocation and generally referred to as the translocation or T-domain (Sharp et al, 2017). Following the T-domain is a long coiled-coil receptor-binding (R-) domain, the apex of which binds the vitamin B12 transporter BtuB on the cell surface (Fig 1B). Finally, at the C-terminus of the colicin is a cytotoxic DNase to which the immunity protein Im9 is bound. Following import and ejection of Im9, the internalised DNase elicits cell death through random cleavages of the bacterial genome. The ColE9 DNase is a member of the H-N-H/ββα-Me class of metal-dependent endonucleases (Pommer et al, 2001), which includes apoptotic nucleases and the core nuclease of CRISPR/Cas9. Other cytotoxic domains are also delivered by the same toxin chassis, including the RNase of ColE3 that inhibits protein synthesis by site-specific cleavage of the ribosomal A-site (Ng et al, 2010). The structures of ColE3 (Soelaiman et al, 2001) and ColE9 (Klein et al, 2016) have been reported previously as have the binary complexes of ColE3 R-domain-BtuB (Kurisu et al, 2003), ColE9 OBS1-OmpF (Housden et al, 2010) and ColE9 TBE-TolB (Loftus et al, 2006). Figure 1. Cryo-EM structures of the ColE9 outer membrane translocon Schematic of the ColE9 sequence showing its constituent domains: an intrinsically unstructured translocation domain (IUTD) at the N-terminus is followed by three structured domains involved in translocation (T), receptor (R) binding and cytotoxicity (C). The IUTD houses three linear protein-protein interaction epitopes, two OmpF-binding sites (OBS1, OBS2) flank a TolB-binding epitope (TBE). Residue numbers denote position in ColE9 sequence. Cartoon of the ColE9 OM translocon. ColE9 (orange) exploits the vitamin B12 transporter BtuB (grey) as its extracellular receptor and the porin OmpF (green) for threading its N-terminal IUTD (solid black line) through to the periplasm where it captures TolB (blue). Star represents the primary site on the ColE9 DNase domain (K469C) where fluorophores were covalently attached throughout this study. Cryo-EM map of the fully assembled ColE9 translocon, with local resolution range 4.5–16 Å. Component proteins are coloured as in panel B. The structure shows extracellular ColE9 creating a protein bridge between the two OMPs, BtuB and OmpF. The β-barrel of BtuB is tilted 35° relative to that of OmpF. TolB is located on the periplasmic side of OmpF, with no associations to BtuB. ColE9 and TolB regions of the map have weaker density than those of the β-barrels. Model of the intact ColE9 translocon generated after docking and rigid-body refinement of individual structures of ColE9 residues 85–580 (PDB ID 5EW5), OmpF (3K19), BtuB (PDB ID 2YSU) and TolB (PBD ID 4JML). Cryo-EM map of the partial ColE9 translocon with an average resolution of 3.7 Å, which has density consistent with OmpF, TolB, and ColE9 residues 3–314. Map is coloured based on component parts shown in panel B. TolB is much better resolved here than in the full translocon map in panel C, although ColE9 density is weaker. ColE9 and TolB density align on the extracellular and periplasmic side of OmpF, respectively. The refined structure of the partial translocon, generated by docking and refinement of ColE9 residues 85–580 (PDB ID 5EW5), OmpF (PDB ID 3K19) and TolB-ColE9 TBE (PDB ID 4JML). ColE9 residues 3–67 were built de novo. Download figure Download PowerPoint Tol-dependent colicins assemble a translocon at the cell surface that involves its periplasmic target along with the OM receptor and translocator which together establish the import pathway of the toxin. No structures have yet been reported for any colicin translocon and so there is little understanding of how these assemblies underpin OM transport. We have reported previously the isolation and purification of the entire OM translocon for ColE9 in which the toxin, bound to TolB, BtuB and OmpF, is solubilised in 1% β-octylglucoside (β-OG) (Housden et al, 2013). In addition, the translocon contains an engineered disulphide bond at the periphery of the ColE9 TBE-TolB interface, designed to stabilise the assembled complex without impacting the protein-protein interface (Housden et al, 2013). We followed the same procedure, which combines in vivo and in vitro approaches, but, for ease of assembly (see Materials and Methods), using ColE9 truncated at the end of the R-domain and hence missing both the DNase and Im9. The assembled translocon was transferred into amphipols and vitrified on graphene oxide coated grids for visualisation of individual particles by cryo-EM (see Materials and Methods for details). Single particle analysis revealed the presence of two populations of ColE9 translocon complexes, one where the translocon was intact and a second class where BtuB was absent, which we refer to as the partial translocon (Appendix Figs S1 and S2). The final post-processed map of the full translocon (Fig 1C and D ) is at an overall resolution of 4.7 Å, according to the gold-standard FSC method, with local resolutions ranging from 4.7 to 16 Å. The absence of BtuB in the partial translocon, most likely due to receptor dissociation during its deposition on grids, resulted in an improvement in map resolution (Fig 1E and F), which varies from 3.7 to 6 Å (FSC). Masked subtraction of BtuB from the full translocon map and refinement of these subtracted particles did not improve map quality, suggesting that it is the loss of BtuB from the complex which resulted in the increase in map resolution. Density for ColE9 was better resolved in the full compared to the partial translocon where only the T-domain was resolved. This is most likely due to the ColE9 R-domain providing some conformational rigidity in the fully assembled translocon through its interaction with BtuB. This contention is supported by the loss of density for the R-domain in the partial translocon map. By contrast, density for TolB was better resolved in the partial translocon map. The partial translocon map presented here is similar to a negative stain map of a proteolysed version of the translocon (comprising of OmpF, TolB and ColE9 residues 1–122), reported previously at a resolution ˜20 Å (Housden et al, 2013). However, the lower resolution of this earlier structure meant little information was forthcoming about ColE9 interactions with OmpF. Isolated crystallographic structures for the majority of ColE9 translocon components provided a starting point for model building into the full and partial translocon maps. Although the resolution of the full translocon map did not allow for local refinement of docked structures, rigid-body refinement were carried out to generate a molecular model. For the partial translocon model, OmpF was docked and refined locally along with the ColE9 IUTD (residues 3–67), which were built de novo into the map. The remaining ColE9 T-domain residues (85–316) and TolB-ColE9 (residues 32–47) were rigid-body refined into the partial translocon map due to the lower resolution associated with these parts of the map. In the fully assembled translocon model, the 22-stranded β-barrel of BtuB sits at a 35° angle relative to the trimeric β-barrel structure of OmpF (Fig 1C and D ). Unmodelled density, most likely that of a single molecule of LPS, is wedged between the two proteins which likely contributes to displacement of BtuB relative to OmpF. The region of unmodelled density in the full translocon map aligns with an LPS binding site (site A) observed in the structure of the OmpF orthologue from Enterobacter cloacae (PDB ID 5FVN), further supporting this as lipid density. Consistent with this interpretation, native state mass spectrometry has shown previously that a single LPS molecule remains associated with the purified translocon complex (Housden et al, 2013). Since there are no examples in the PDB of heterologous bacterial outer membrane proteins residing next to each other it is not clear if the 35° tilt reflects the natural position of BtuB in the OM or is a consequence of how the ColE9 translocon was assembled. We note however that if the β-barrels of OmpF and BtuB were flush in the membrane, the 45° trajectory of the ColE9 R-domain-BtuB complex, first shown for the ColE3 R-domain-BtuB complex (0.95 Å rmsd) (Kurisu et al, 2003), would project the T-domain beyond OmpF. The coiled-coil nature of the R-domain, with BtuB at its apex, means the cytotoxic DNase domain although absent from the current structure would sit ˜60 Å above OmpF. Finally, TolB is held on the periplasmic side of OmpF through its association with the ColE9 TBE but is not well-resolved in the full translocon model (Fig 1C and D ). The partial translocon is comprised of OmpF, the IUTD and T-domain of ColE9 and TolB (Fig 1E and F). Previous work on the translocation mechanism of group A colicins has demonstrated that these colicins contact their periplasmic binding partners (typically TolB and/or TolA) by a direct epitope delivery mechanism following binding to the cell surface receptor (Housden et al, 2010; Jansen et al, 2020). The colicin threads its IUTD through two of OmpF’s three pores thereby presenting a tethered protein-protein interaction epitope to the periplasm. In the case of ColE9, the TBE promotes contact between TolB and TolA in the inner membrane which is coupled to the PMF through the stator proteins, TolQ and TolR (Bonsor et al, 2009). The partial translocon structure of ColE9 shows how the IUTD (residues 3–67) navigates through the lumen of one OmpF subunit in order to thread back up into a neighbouring subunit. The end result is that each OBS is lodged within an individual subunit of OmpF: OBS1 (residues 2–18) is bound to subunit 1 while OBS2 (residues 54–63) is bound to subunit 2, leaving subunit 3 free of colicin. The relative positions of the two OBSs in the partial translocon model implies that subunit 2 is the entry port for the ColE9 IUTD following its initial binding to BtuB in the OM. The resolution of the full translocon does not allow for detailed comparison of OBS binding with that of the partial translocon (details below); however, an overlay of the two models reveals that the ColE9 T-domain undergoes a large-scale movement, rotating by 15° and moving vertically by 12 Å along the rotation axis (Fig 2A–C, Movie EV1). In the full translocon, the orientation of the ColE9 T-domain is fixed by the R-domain’s contact with BtuB. Rotation of the ColE9 T-domain in the partial translocon to achieve the orientation adopted in the full translocon structure results in a poorer fit to map density. Importantly, rotation of the ColE9 T-domain from the full to the partial translocon repositions ColE9 from a central position above the OmpF trimer to become localised above subunit 2 within which OBS2 is bound (Fig 3A and C). Figure 2. Large-scale structural rearrangements accompany the loss of BtuB from the ColE9 translocon Superposition of the complete and partial ColE9 translocon structures (grey) aligned on OmpF. TolB in red and orange denote the full and partial translocons, respectively, ColE9 T-domain is presented in crimson and pale orange for the full and partial translocons, respectively. Side view comparison showing the relative positions of the ColE9 T-domain (residues 85–316) in the two structures and highlighting the 15° rotation that occurs transitioning from the full (crimson) to the partial (pale orange) translocon. Extracellular view of the ColE9 T-domain position, with the OmpF trimer shown in the background. The loss of BtuB from the translocon complex elicits a 12 Å movement along the axis of rotation (black arrow) that results in repositioning of the T-domain from a central location (crimson) to above subunit two of OmpF (pale orange). TolB undergoes both rotation and translation when transitioning from the full (red) to the partial translocon (orange). The C-terminal β-propeller domain of TolB, which binds the ColE9 TBE, moves towards OmpF in the OM by ˜8 Å along the rotation axis. View along the rotation axis from the periplasmic side of OmpF (grey surface) highlighting the 50° rotation that TolB undergoes upon loss of BtuB from the translocon complex. Download figure Download PowerPoint Figure 3. Distinct modes of ColE9 OBS1 and OBS2 recognition within the pores of OmpF enable threading and the tethered presentation of the TBE to the periplasm ColE9 residues 3–67 of the IUTD in the partial translocon structure pass through OmpF subunit 2 and then back up into subunit 1. As a result, OBS1 (gold) docks within subunit 1 and OBS2 (orange) within subunit 2. The ColE9 TBE motif (pink) interacts with TolB and is positioned below subunit 2, above which the ColE9 T-domain is located. Surface representation of OmpF subunit 1 (grey) and ColE9 residues 3–24 (gold) containing OBS1 sequence. ColE9 OBS1 binds the inner vestibule of OmpF subunit 1 such that it runs along the edge of the vestibule, stopping at the eyelet. ColE9 residues 45–67 (orange) encompassing OBS2 traverse the pore of OmpF subunit 2 (grey). After passing through the eyelet, OBS2 tracks more centrally in the inner vestibule unlike in panel C where residues 3–24 trail the side of the pore. OmpF subunit 1 displayed as an electrostatic surface (same cut-through as in B), revealing a patch of negative charge located on the extracellular side of the eyelet that interacts with OBS1, also shown as an electrostatic surface adjacent to the subunit. OmpF subunit 2 displayed as an electrostatic surface (same cut-through as in C), with OBS2 also shown as an electrostatic surface adjacent to the subunit. Projecting out of the page for OBS2 is a positively charged region flanked by negative charges. A zoom-in of grey box in panel A highlighting hydrogen bonding network between residues 3–7 of OBS1 (gold sticks) and nearby residues within OmpF subunit 1 (grey sticks). OBS1 residues 11–17 (gold sticks) also interact with the base of OmpF subunit 1 (grey sticks) in the periplasm. Region is a zoom-in of blue box in panel A. OmpF subunit 2 residues (grey sticks) form a hydrogen bond network with the backbone of residues of ColE9 OBS2. Region is a zoom-in of brown box in panel A. All electrostatic surfaces shown in panels D and E were calculated using the APBS plugin within Pymol. Download figure Download PowerPoint TolB also undergoes large-scale movements when transitioning from the full to partial translocon structures, which are likely linked to the re-arrangement of the ColE9 T-domain on the extracellular side of the porin (Fig 2D and E, Movie EV1). The position of TolB is rotated by ˜50° between the two structures and in the partial translocon is located closer to OmpF through a ˜8 Å upward movement along the axis of rotation. The positioning of TolB closer to OmpF in the partial translocon structure is stabilised by additional interactions between the periplasmic region of OmpF subunit 1 and the IUTD sequence immediately preceding the TBE (Fig 3G). Irrespective of the observed movements between the two cryo-EM models, TolB remains located beneath subunit 2 within which OBS2 is bound. In summary, we have solved the first cryo-EM structure of a colicin OM translocon. The ColE9 translocon includes the OMPs BtuB and OmpF, as well as the periplasmic target TolB. We have also solved a partial translocon structure in which the primary receptor BtuB is lost from the complex, which leads to substantial rearrangements of ColE9 and TolB above and below the membrane, respectively. As we demonstrate below, these structural changes within the translocon are integral to the mechanism by which ColE9 translocates across the OM. ColE9 IUTD interactions with the subunits of OmpF The higher resolution structure of the partial ColE9 translocon provides a molecular explanation as to how bacteriocins exploit porins to establish their translocon complexes at the OM. The lumen of an OmpF monomer is not a straight channel but akin to an hourglass (Fig 3A–C). The outer (extracellular side) and inner (periplasmic side) vestibules of OmpF have diameters of 25 and 30 Å, respectively, constricting at the eyelet of the channel to ˜7 Å. After the IUTD region passes through OmpF subunit 2, OBS1 threads back up into subunit 1 so that it snakes along the bottom of the lumen at its widest point and finishes up at the eyelet (Fig 3A and B). By contrast, OBS2 spans the eyelet of OmpF subunit 2 via a kink created at Gly61, which is enforced by the hydrogen bonding of neighbouring residues, Ser60 and Arg62 (Fig 3C). After OBS2 traverses the eyelet, the remaining ColE9 sequence does not track the OmpF interior as observed for OBS1, instead remaining more centrally located in the inner vestibule. The bound conformations of the two OBSs are stabilised by electrostatic and hydrogen bonding interactions (Fig 3D–H). The vestibules and eyelet of OmpF are highly charged environments; the porin exhibits slight cation selectivity in solute diffusion studies (Basle et al, 2006). Charged patches within the eyelet of OmpF are matched by opposing charges of the OBS sequences (Fig 3D and E). In addition to these interactions, networks of hydrogen bonds lock OBS1 and OBS2 of ColE9 into defined conformations within their respective OmpF subunits (Fig 3F and H). Key OBS1 residues that interact with subunit 1 include Asp5, Arg7, His9, Thr11 and Ser15, (Fig 3F and G). Previous studies have shown that mutation of Asp5, Arg7 or Thr11 to alanine substantially weakens OmpF binding by OBS1 while an Asp5Ala/Arg7Ala double mutant abolishes OmpF binding, consistent with their essential role in stabilising the OBS1-OmpF subunit 1 complex (Housden et al, 2005, 2010). We have reported previously the 3 Å crystal structure of an OBS1 peptide bound to OmpF (Housden et al, 2010). At the time, there was uncertainty as to the orientation of the peptide, which was refined with the N-terminus facing the periplasm. We re-processed these data and rebuilt the model based on the orientation observed in the cryo-EM data for the partial translocon, validating this re-processed model (Appendix Fig S2K–M). The new model shows conclusively that OBS1 has its N-terminus pointing towards the extracellular environment (Fig 3A) and in both models the conformation adopted by OBS1 is identical. This orientation agrees with recent electrophysiological data and live-cell imaging experiments all of which show OBS1 binding subunit 1 of OmpF from the periplasm (Housden et al, 2018; Lee et al, 2020). In contrast to OBS1, the hydrogen bond network that stabilises OBS2 within subunit 2 of OmpF primarily involves OBS2 backbone atoms and OmpF side-chains (Fig 3H), likely explained by the high glycine content within the OBS2 sequence (6/11 residues are glycine, compared to 6/16 for OBS1). The high g" @default.
• W3199685002 created "2021-09-27" @default.
• W3199685002 creator A5013271893 @default.
• W3199685002 creator A5025024523 @default.
• W3199685002 creator A5030512848 @default.
• W3199685002 creator A5033107091 @default.
• W3199685002 creator A5049764579 @default.
• W3199685002 creator A5050798434 @default.
• W3199685002 creator A5052498496 @default.
• W3199685002 creator A5080002799 @default.
• W3199685002 date "2021-09-13" @default.
• W3199685002 modified "2023-10-11" @default.
• W3199685002 title "Porin threading drives receptor disengagement and establishes active colicin transport through <i>Escherichia</i> <i>coli</i> OmpF" @default.
• W3199685002 cites W1538296334 @default.
• W3199685002 cites W1554612905 @default.
• W3199685002 cites W1555559786 @default.
• W3199685002 cites W1603332843 @default.
• W3199685002 cites W1963676019 @default.
• W3199685002 cites W1966688540 @default.
• W3199685002 cites W1970918777 @default.
• W3199685002 cites W1992092427 @default.
• W3199685002 cites W1994890449 @default.
• W3199685002 cites W2005986251 @default.
• W3199685002 cites W2006006403 @default.
• W3199685002 cites W2008023389 @default.
• W3199685002 cites W2010264737 @default.
• W3199685002 cites W2011665065 @default.
• W3199685002 cites W2012727169 @default.
• W3199685002 cites W2015940571 @default.
• W3199685002 cites W2016158361 @default.
• W3199685002 cites W2026210802 @default.
• W3199685002 cites W2026840192 @default.
• W3199685002 cites W2035213224 @default.
• W3199685002 cites W2064389237 @default.
• W3199685002 cites W2094643746 @default.
• W3199685002 cites W2097399082 @default.
• W3199685002 cites W2099540110 @default.
• W3199685002 cites W2100584256 @default.
• W3199685002 cites W2104234755 @default.
• W3199685002 cites W2107716680 @default.
• W3199685002 cites W2110054502 @default.
• W3199685002 cites W2120061656 @default.
• W3199685002 cites W2132629607 @default.
• W3199685002 cites W2134141380 @default.
• W3199685002 cites W2138440215 @default.
• W3199685002 cites W2141235024 @default.
• W3199685002 cites W2142230661 @default.
• W3199685002 cites W2144081223 @default.
• W3199685002 cites W2148501500 @default.
• W3199685002 cites W2148708097 @default.
• W3199685002 cites W2151635778 @default.
• W3199685002 cites W2152967503 @default.
• W3199685002 cites W2163388241 @default.
• W3199685002 cites W2164023475 @default.
• W3199685002 cites W2166650607 @default.
• W3199685002 cites W2167819403 @default.
• W3199685002 cites W2168686586 @default.
• W3199685002 cites W2297006149 @default.
• W3199685002 cites W2416771819 @default.
• W3199685002 cites W2471198927 @default.
• W3199685002 cites W2587625522 @default.
• W3199685002 cites W2593511418 @default.
• W3199685002 cites W2599151123 @default.
• W3199685002 cites W2734356283 @default.
• W3199685002 cites W2735764554 @default.
• W3199685002 cites W2766274239 @default.
• W3199685002 cites W2766328249 @default.
• W3199685002 cites W2786390877 @default.
• W3199685002 cites W2791670278 @default.
• W3199685002 cites W2810158180 @default.
• W3199685002 cites W2899714098 @default.
• W3199685002 cites W2905118631 @default.
• W3199685002 cites W2948849558 @default.
• W3199685002 cites W2950967305 @default.
• W3199685002 cites W2973457155 @default.
• W3199685002 cites W2991574658 @default.
• W3199685002 cites W3005655837 @default.
• W3199685002 cites W3010760226 @default.
• W3199685002 cites W3011998109 @default.
• W3199685002 cites W3020288999 @default.
• W3199685002 cites W3025199443 @default.
• W3199685002 cites W3034312851 @default.
• W3199685002 cites W3039812751 @default.
• W3199685002 cites W3043584664 @default.
• W3199685002 cites W3083406432 @default.
• W3199685002 cites W3084848063 @default.
• W3199685002 cites W3108495343 @default.
• W3199685002 cites W3137122686 @default.
• W3199685002 cites W3182767159 @default.
• W3199685002 cites W3199685002 @default.
• W3199685002 cites W4210359611 @default.
• W3199685002 doi "https://doi.org/10.15252/embj.2021108610" @default.
• W3199685002 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/8561637" @default.
• W3199685002 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/34515361" @default.
• W3199685002 hasPublicationYear "2021" @default.
• W3199685002 type Work @default.
• W3199685002 sameAs 3199685002 @default.
• W3199685002 citedByCount "9" @default.
• W3199685002 countsByYear W31996850022021 @default.
• W3199685002 countsByYear W31996850022022 @default.

• next