Structural basis for antibacterial peptide self‐immunity by the bacterial ABC transporter McjD
2017; Springer Nature; Volume: 36; Issue: 20 Linguagem: Inglês
10.15252/embj.201797278
ISSN1460-2075
AutoresKiran Bountra, Gregor Hagelueken, Hassanul G. Choudhury, Valentina Corradi, Kamel El Omari, Armin Wagner, Indran Mathavan, Séverine Zirah, Weixiao Yuan Wahlgren, D. Peter Tieleman, Olav Schiemann, Sylvie Rebuffat, Konstantinos Beis,
Tópico(s)Pneumocystis jirovecii pneumonia detection and treatment
ResumoArticle1 September 2017Open Access Source DataTransparent process Structural basis for antibacterial peptide self-immunity by the bacterial ABC transporter McjD Kiran Bountra Kiran Bountra Department of Life Sciences, Imperial College London, London, UK Rutherford Appleton Laboratory, Research Complex at Harwell, Oxfordshire, UK Search for more papers by this author Gregor Hagelueken Gregor Hagelueken Institute for Physical and Theoretical Chemistry, University of Bonn, Bonn, Germany Search for more papers by this author Hassanul G Choudhury Hassanul G Choudhury Department of Life Sciences, Imperial College London, London, UK Rutherford Appleton Laboratory, Research Complex at Harwell, Oxfordshire, UK Search for more papers by this author Valentina Corradi Valentina Corradi Centre for Molecular Simulation and Department of Biological Sciences, University of Calgary, Calgary, AB, Canada Search for more papers by this author Kamel El Omari Kamel El Omari Rutherford Appleton Laboratory, Research Complex at Harwell, Oxfordshire, UK Diamond Light Source, Oxfordshire, UK Search for more papers by this author Armin Wagner Armin Wagner Rutherford Appleton Laboratory, Research Complex at Harwell, Oxfordshire, UK Diamond Light Source, Oxfordshire, UK Search for more papers by this author Indran Mathavan Indran Mathavan Department of Life Sciences, Imperial College London, London, UK Rutherford Appleton Laboratory, Research Complex at Harwell, Oxfordshire, UK Search for more papers by this author Séverine Zirah Séverine Zirah Communication Molecules and Adaptation of Microorganisms Laboratory (MCAM, UMR 7245 CNRS-MNHN), Muséum National d'Histoire Naturelle, Centre National de la Recherche Scientifique, Sorbonne Universités, Paris, France Search for more papers by this author Weixiao Yuan Wahlgren Weixiao Yuan Wahlgren Department of Life Sciences, Imperial College London, London, UK Rutherford Appleton Laboratory, Research Complex at Harwell, Oxfordshire, UK Chemistry & Molecular Biology, University of Gothenburg, Göteborg, Sweden Search for more papers by this author D Peter Tieleman D Peter Tieleman Centre for Molecular Simulation and Department of Biological Sciences, University of Calgary, Calgary, AB, Canada Search for more papers by this author Olav Schiemann Olav Schiemann Institute for Physical and Theoretical Chemistry, University of Bonn, Bonn, Germany Search for more papers by this author Sylvie Rebuffat Sylvie Rebuffat Communication Molecules and Adaptation of Microorganisms Laboratory (MCAM, UMR 7245 CNRS-MNHN), Muséum National d'Histoire Naturelle, Centre National de la Recherche Scientifique, Sorbonne Universités, Paris, France Search for more papers by this author Konstantinos Beis Corresponding Author Konstantinos Beis [email protected] orcid.org/0000-0001-5727-4721 Department of Life Sciences, Imperial College London, London, UK Rutherford Appleton Laboratory, Research Complex at Harwell, Oxfordshire, UK Search for more papers by this author Kiran Bountra Kiran Bountra Department of Life Sciences, Imperial College London, London, UK Rutherford Appleton Laboratory, Research Complex at Harwell, Oxfordshire, UK Search for more papers by this author Gregor Hagelueken Gregor Hagelueken Institute for Physical and Theoretical Chemistry, University of Bonn, Bonn, Germany Search for more papers by this author Hassanul G Choudhury Hassanul G Choudhury Department of Life Sciences, Imperial College London, London, UK Rutherford Appleton Laboratory, Research Complex at Harwell, Oxfordshire, UK Search for more papers by this author Valentina Corradi Valentina Corradi Centre for Molecular Simulation and Department of Biological Sciences, University of Calgary, Calgary, AB, Canada Search for more papers by this author Kamel El Omari Kamel El Omari Rutherford Appleton Laboratory, Research Complex at Harwell, Oxfordshire, UK Diamond Light Source, Oxfordshire, UK Search for more papers by this author Armin Wagner Armin Wagner Rutherford Appleton Laboratory, Research Complex at Harwell, Oxfordshire, UK Diamond Light Source, Oxfordshire, UK Search for more papers by this author Indran Mathavan Indran Mathavan Department of Life Sciences, Imperial College London, London, UK Rutherford Appleton Laboratory, Research Complex at Harwell, Oxfordshire, UK Search for more papers by this author Séverine Zirah Séverine Zirah Communication Molecules and Adaptation of Microorganisms Laboratory (MCAM, UMR 7245 CNRS-MNHN), Muséum National d'Histoire Naturelle, Centre National de la Recherche Scientifique, Sorbonne Universités, Paris, France Search for more papers by this author Weixiao Yuan Wahlgren Weixiao Yuan Wahlgren Department of Life Sciences, Imperial College London, London, UK Rutherford Appleton Laboratory, Research Complex at Harwell, Oxfordshire, UK Chemistry & Molecular Biology, University of Gothenburg, Göteborg, Sweden Search for more papers by this author D Peter Tieleman D Peter Tieleman Centre for Molecular Simulation and Department of Biological Sciences, University of Calgary, Calgary, AB, Canada Search for more papers by this author Olav Schiemann Olav Schiemann Institute for Physical and Theoretical Chemistry, University of Bonn, Bonn, Germany Search for more papers by this author Sylvie Rebuffat Sylvie Rebuffat Communication Molecules and Adaptation of Microorganisms Laboratory (MCAM, UMR 7245 CNRS-MNHN), Muséum National d'Histoire Naturelle, Centre National de la Recherche Scientifique, Sorbonne Universités, Paris, France Search for more papers by this author Konstantinos Beis Corresponding Author Konstantinos Beis [email protected] orcid.org/0000-0001-5727-4721 Department of Life Sciences, Imperial College London, London, UK Rutherford Appleton Laboratory, Research Complex at Harwell, Oxfordshire, UK Search for more papers by this author Author Information Kiran Bountra1,2,‡, Gregor Hagelueken3,‡, Hassanul G Choudhury1,2,‡, Valentina Corradi4, Kamel El Omari2,5, Armin Wagner2,5, Indran Mathavan1,2, Séverine Zirah6, Weixiao Yuan Wahlgren1,2,7, D Peter Tieleman4, Olav Schiemann3, Sylvie Rebuffat6 and Konstantinos Beis *,1,2 1Department of Life Sciences, Imperial College London, London, UK 2Rutherford Appleton Laboratory, Research Complex at Harwell, Oxfordshire, UK 3Institute for Physical and Theoretical Chemistry, University of Bonn, Bonn, Germany 4Centre for Molecular Simulation and Department of Biological Sciences, University of Calgary, Calgary, AB, Canada 5Diamond Light Source, Oxfordshire, UK 6Communication Molecules and Adaptation of Microorganisms Laboratory (MCAM, UMR 7245 CNRS-MNHN), Muséum National d'Histoire Naturelle, Centre National de la Recherche Scientifique, Sorbonne Universités, Paris, France 7Chemistry & Molecular Biology, University of Gothenburg, Göteborg, Sweden ‡These authors contributed equally to this work *Corresponding author. Tel: +44 1235 567809; E-mail: [email protected] The EMBO Journal (2017)36:3062-3079https://doi.org/10.15252/embj.201797278 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 Certain pathogenic bacteria produce and release toxic peptides to ensure either nutrient availability or evasion from the immune system. These peptides are also toxic to the producing bacteria that utilize dedicated ABC transporters to provide self-immunity. The ABC transporter McjD exports the antibacterial peptide MccJ25 in Escherichia coli. Our previously determined McjD structure provided some mechanistic insights into antibacterial peptide efflux. In this study, we have determined its structure in a novel conformation, apo inward-occluded and a new nucleotide-bound state, high-energy outward-occluded intermediate state, with a defined ligand binding cavity. Predictive cysteine cross-linking in E. coli membranes and PELDOR measurements along the transport cycle indicate that McjD does not undergo major conformational changes as previously proposed for multi-drug ABC exporters. Combined with transport assays and molecular dynamics simulations, we propose a novel mechanism for toxic peptide ABC exporters that only requires the transient opening of the cavity for release of the peptide. We propose that shielding of the cavity ensures that the transporter is available to export the newly synthesized peptides, preventing toxic-level build-up. Synopsis Bacteria employ dedicated ABC transporters to secrete antibacterial peptides. X-ray structure analysis shows that the antibacterial lasso peptide transporter McjD, in contrast to other ABC transporters, lacks a stable open cavity conformation, thus preventing the influx of the exported toxic peptide. The structure of the ABC transporter McjD was determined in two distinct conformations, apo and nucleotide-bound. Both conformations show an occluded cavity at both sides of the membrane. Pulsed electron-electron double resonance (PELDOR) measurements in the presence of the antibacterial peptide MccJ25 did not identify a stable open-conformation. Cross-linking studies in proteoliposomes show that the cavity opens transiently to release the bound substrate. Introduction ATP-binding cassette (ABC) transporters (exporters) are one of the largest superfamilies of membrane transporters and are found in both bacteria and eukaryotes (Holland et al, 2003); they are usually involved in efflux or detoxification pathways including multi-drug resistance in both bacterial and eukaryotic cells. Bacteria, under nutrient starvation, produce and release antibacterial peptides, microcins, which can kill microcin-sensitive cells and therefore provide more nutrients for the surviving bacteria (Duquesne et al, 2007a). The microcin J25 (MccJ25) is a plasmid-encoded, ribosomally synthesized and post-translationally modified antibacterial peptide consisting of 21 amino acids and adopting a lasso structure (Rosengren et al, 2003); its C-terminal tail threads through an N-terminal eight-residue macrolactam ring, where it is locked by bulky amino acid side chains, forming a compact interlocked structure. Four genes are required for the biosynthesis and export of MccJ25 (Duquesne et al, 2007b): mcjA encodes the linear precursor of MccJ25; mcjB and mcjC encode enzymes involved in the post-translational modification of McjA to the lasso structure; and mcjD encodes an ABC exporter that is required for both MccJ25 secretion and self-immunity of the producing strain towards MccJ25 (Choudhury et al, 2014). Since the genes are under the same operon, production and export from the cell are very efficient and toxic levels of MccJ25 are prevented from building up. Inside the target cell (Runti et al, 2013; Mathavan et al, 2014), MccJ25 inhibits the bacterial RNA polymerase (Semenova et al, 2005). We have previously functionally characterized and determined the high-resolution structure of McjD from Escherichia coli (Choudhury et al, 2014; Fig 1A). Its core architecture is composed of a dimeric transmembrane domain (TMD) of 12 transmembrane (TM) helices, which forms the translocation pathway across the membrane bilayer and contains the ligand binding site, and a dimeric nucleotide binding domain (NBD) where ATP binds and is hydrolysed. ABC exporters either use the alternating access mechanism or outward-only mechanism (Perez et al, 2015) to transport their substrates. ABC exporters that use the alternating access mechanism switch between inward- and outward-facing states, which exposes the ligand binding site alternatively to the inside or outside of the membrane, coupled to ATP binding and hydrolysis (Beis, 2015). The previous structure of McjD was determined in a nucleotide-bound outward-occluded conformation (occluded at both sides of the membrane), representing an intermediate state between the outward- and the inward-facing conformations (Choudhury et al, 2014). Figure 1. Crystal structures of McjD in three distinct conformations A–C. McjD is shown in cartoon and nucleotides in red sticks. Each half transporter is coloured in blue and green. Top panel is a view along the plane of the membrane, and bottom panel shows the NBDs for each state. The membrane is depicted in grey. (A) AMPPNP-bound outward-occluded conformation (PDB ID: 4PL0; Choudhury et al, 2014), (B) high-energy intermediate outward-occluded conformation (ADP-VO4) and (C) apo inward-occluded. In all three transport steps represented by the crystal structures, the TMDs are in an occluded conformation without access to the periplasmic (nucleotide-bound) or cytoplasmic side (apo) of the membrane. In the presence of nucleotides, the NBDs dimerize similar to other ABC exporters. In the apo state, the NBDs of McjD disengage but with a smaller degree of separation compared to other ABC exporters. The two NBDs have separated by 7.9 Å as indicated by a black dashed line. Download figure Download PowerPoint In order to understand the detailed mechanism of toxic peptide export by bacterial cells, it is important to trap the transporter in different conformations. Here, we have determined the structure of McjD in a novel conformation, apo inward-occluded and an additional nucleotide-bound state, high-energy outward-occluded intermediate with bound ATP-vanadate (ADP-VO4). We further characterized these new states in E. coli membranes using predictive cysteine cross-linking in inside-out vesicles (ISOVs). Using a spin-labelled McjD mutant and pulsed electron-electron double resonance (PELDOR), also known as DEER (double electron-electron resonance), the conformation of McjD was investigated in bicelles. In addition, we applied molecular dynamics (MD) simulations to study the dynamics of the outward-occluded conformation of McjD in the presence of ATP or ADP molecules bound at the NDBs. The new conformations in combination with the PELDOR data, transport assays in proteoliposomes and molecular dynamics simulations have significant mechanistic implications in understanding the detailed mechanism of the antibacterial peptide exporter McjD. Results Structures of McjD in different conformations In order to gain a detailed understanding of the conformations that McjD adopts during the transport cycle, we have determined its structure in the presence of the transition state analogue ADP-VO4 (mimic of ATP hydrolysis) and in the absence of nucleotides, that is in the apo form, at 3.4 Å and 4.7 Å resolution, respectively (Fig 1B and C). We determined the structure of McjD in complex with ADP-VO4 by molecular replacement using the McjD-AMPPNP (adenosine 5′-(β,γ-imido)triphosphate) (PDB ID: 4PL0) structure as a search model (Choudhury et al, 2014). The structure was refined with an Rwork of 25.7% and Rfree of 26.0%. Clear electron density was observed for ADP-VO4 and Mg2+ (Fig EV1A–D). The presence of vanadate was verified by collecting the data close to the vanadium K-edge, 2.26 Å wavelength and calculating anomalous difference electron density maps (see Materials and Methods; Fig EV1C). At both NBDs, two strong electron density peaks of 14 and 20 sigma, respectively, confirmed the presence of two VO4 groups. McjD-ADP-VO4 is almost identical to the McjD-AMPPNP, as the two structures can be superimposed with a root-mean-square deviation (rmsd) of 0.7 Å over 560 Cα atoms (Fig EV2A and B). In contrast to the MsbA-ADP-VO4 structure that adopts an outward-open conformation as a result of domain intertwining (Ward et al, 2007), the McjD-ADP-VO4 structure is outward-occluded without domain intertwining (Fig 2A). We call it here high-energy intermediate outward-occluded. The TMD dimer interface in McjD-ADP-VO4 is formed between TM2 and TM5/TM6 from one subunit with the equivalent TMs from the opposite subunit similar to the McjD-AMPPNP structure. In the presence of ADP-VO4, the NBDs are dimerized with the P-loop and ABC signature motifs involved in the binding of the nucleotide. The McjD-ADP-VO4 structure represents the transition state of a water molecule making a nucleophilic attack on the γ-phosphate of ATP. Click here to expand this figure. Figure EV1. Electron density maps Clear |Fo|-|Fc| electron density map (green mesh contoured at 3 σ) could be observed around the ADV-VO4 molecule after molecular replacement. ADV-VO4 is only shown for clarity but it was not included in the refinement. Final 2|Fo|-|Fc| electron density map (blue mesh contoured at 1 σ) after including the ADV-VO4 in the refinement. No negative or positive electron density peaks are observed. Anomalous difference electron density map (purple mesh) around the vanadate, from data collected close to the vanadium edge, 2.26 Å. The map is contoured at 10 σ. McjD and ADV-VO4 are shown as sticks. The magnesium ion is shown as green sphere. McjD is coloured as in Fig 1. The ADV-VO4 carbons are coloured grey, oxygens red, phosphate orange and vanadate dark grey. Colouring scheme as in Fig 1. Positive |Fo|-|Fc| electron density map (purple mesh contoured at 3 σ) around Mg2+ after excluding it from the refinement. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Comparison of McjD-ADP-VO4 and McjD-apo Stereo figure of McjD-ADP-VO4 (red ribbon) superimposed on McjD-apo (green ribbon) at the TMDs. View along the membrane. No significant changes are observed along the TMDs, whereas the NBDs do not align. Stereo figure of McjD-ADP-VO4 (red ribbon) and McjD-apo (green ribbon) NBDs. View from the cytoplasmic side. The NBDs of the McjD-apo have disengaged in the absence of nucleotides. Download figure Download PowerPoint Figure 2. Structural comparison of McjD and MsbA MsbA-ADP-VO4 adopts an outward-open conformation with subunit intertwining (right panel), whereas McjD-ADP-VO4 is in an occluded conformation (left panel). MsbA-apo adopts an inward-open conformation with subunit intertwining (right panel). McjD-apo adopts an inward-occluded conformation as a result of movement of TMs 3–5 from one subunit towards the equivalent TMs of the opposite subunit, and subsequent loss of subunit intertwining (left panel). Data information: MsbA and McjD are shown in cartoon and nucleotides in sticks. Each half transporter is coloured as in Fig 1. The transmembrane helices of one subunit are numbered. The membrane is depicted in grey. Download figure Download PowerPoint The McjD-apo structure was also determined by molecular replacement using the McjD-AMPPNP structure but only after splitting the McjD monomer into two domains, TMD and NBD (see Materials and Methods). The structure was refined to an Rwork of 31.3% and Rfree of 33.4%. Clear electron density could be observed for both the TMD and NBDs (Fig 3A and B). Inspection of the NBDs did not reveal any electron density for nucleotides that may have been co-purified with McjD (Fig 3A). In the absence of nucleotides, ABC transporters adopt an inward conformation with their TMDs separated and NBDs disengaged (Ward et al, 2007; Fig 2B). A striking difference between McjD-apo and other apo ABC exporters, such as MsbA (Ward et al, 2007) and PglK (Perez et al, 2015), is that its TMD is in an occluded conformation, similar to the McjD-AMPPNP structure, whereas the NBDs have disengaged in the absence of nucleotides. This conformation is called here apo inward-occluded. Inward-facing ABC exporters display domain intertwining of TMs 1–3 and 6 from one subunit and TMs 4–5 from the opposite subunit, which results in opening of the TMD to the cytoplasmic side of the inner membrane (Ward et al, 2007; Perez et al, 2015). In the inward-occluded McjD, TMs 3–5 from one subunit have moved towards the equivalent TMs of the opposite subunit, resulting in the loss of intertwining and occlusion of the cytoplasmic opening (Figs 2B and EV2). The McjD-apo structure can be superimposed with the McjD-AMPPNP structure with an rmsd of 2.1 Å over 569 Cα atoms; their TMDs can be superimposed with an rmsd of 0.7 Å over 290 Cα atoms. The heterodimeric human sterol apo-ABCG5/8 (Lee et al, 2016) and Pseudomonas aeruginosa lipopolysaccharide apo-LptB2FG (Luo et al, 2017) do not display intertwining either. Figure 3. Electron density maps Composite omit map (blue mesh) covering the McjD molecule (left panel). Right panel shows the composite omit map for the NBDs. Both maps are contoured at 1 σ. Final 2|Fo|-|Fc| electron density map (grey mesh contoured at 1 σ) around McjD after refinement. Good-quality electron density could be observed around the TMD and NBDs. 2|Fo|-|Fc| electron density (grey mesh contoured at 1 σ) around TM 1 (right panel) and the NBDs after refinement (bottom panel). No electron density is present for nucleotides at the NBDs; the NBDs are in the same orientation as in panel (A). Download figure Download PowerPoint In the absence of nucleotides, the NBDs of ABC exporters disengage (Ward et al, 2007; Perez et al, 2015). The NBDs of the McjD-apo have separated by 7.9 Å (measured between S509 and S509') relative to the McjD-AMPPNP structure, a distance much shorter compared to MsbA-apo with a distance of 35 Å (Ward et al, 2007). The NBDs have moved in a "scissors-like" motion similar to the transition between inward and outward-open MsbA. A small degree of disengagement between the NBDs has also been reported for the heterodimeric ABC exporter TM287/288 (Hohl et al, 2012; Hohl et al, 2014), ABCG5/8 (Lee et al, 2016) and LptB2FG (Luo et al, 2017). Cysteine cross-linking in ISOVs The presence of apo inward-occluded and high-energy intermediate outward-occluded states have not been observed or characterized in other ABC exporters. We selected specific amino acids along the periplasmic, cytoplasmic and NBD sides to perform predictive cysteine cross-linking in ISOVs to further characterize them in their native lipid environment. We have previously shown that the occluded McjD is a result of the movement of TMs 1–2 and TMs 1′–2′ and we characterized this conformation by cysteine cross-linking L53C in the presence and absence of nucleotides, ATP/Mg2+ and AMPPNP (Choudhury et al, 2014). Since the McjD-ADP-VO4 structure is also in an outward-occluded conformation, this mutant was further characterized in the presence of ADP-VO4. McjD-L53C is capable of forming cross-linked dimers in the presence of ADP-VO4, suggesting that the high-energy post-hydrolysis McjD also adopts an occluded conformation in the E. coli membrane similar to our crystal structure (Figs 4A and EV3A). Figure 4. Predictive cysteine cross-linking along McjD in ISOVs Cross-linking of L53C at the periplasmic side of the McjD TMD (left panel); L53 from each monomer is shown as sticks. L53C can be effectively cross-linked in the presence or absence of nucleotides (right panel). Cross-linking of A122C at the cytoplasmic side of McjD (left panel); A122 from each monomer is shown as sticks. Cross-linking of the A122C is less effective in the absence of nucleotides compared to the nucleotide ones (right panel). MccJ25 induces small enhancement of cross-linking in the absence of nucleotides (bottom right panel and Fig EV3); the incubation with MccJ25 was performed in duplicate. Cross-linking of S509C at the NBDs of McjD (left panel); S509 from each monomer is shown as sticks. Preformed cross-links can be observed even in the absence of CuCl2 and DTT treatment, suggesting that the NBDs are in very close proximity (right panel). Inclusion of the nucleotides enhances the cross-linking dimer formation. The formation of the cross-linking dimers along the McjD transport cycle verifies that these conformations also exist in the E. coli inner membrane. Data information: The reaction conditions for each lane are indicated above and below the gels (see Materials and Methods). All cross-linking experiments were visualized by Western blot. "CLD" denotes the formation of the cross-linking dimer and "M" the monomer in the absence of CuCl2. Asterisk (*) denotes SDS-stable dimers. All gels contain the same amount of total membrane proteins and have not been normalized for McjD expression. Densitometry analysis of the gel band intensities are shown in Fig EV3. Source data are available online for this figure. Source Data for Figure 4 [embj201797278-sup-0002-SDataFig4.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Densitometry analysis of Western blots Densitometry analysis of the L53C Western blot from Fig 4A. Densitometry analysis of the A122C Western blot from Fig 4B. The degree of cross-linking of McjD A122C in ISOVs in the absence and presence of MccJ25 was estimated by densitometry of the Western blot from Fig 4B (bottom panel). In the presence of MccJ25, a small enhancement of cross-linking was observed. Densitometry analysis of the S509C Western blot from Fig 4C. The degree of mPEG-10k to modify the cavity mutants in the absence and presence of MccJ25 was estimated by densitometry of the Western blot from Fig 7C. N134C can be PEGylated in the presence of MccJ25, whereas T298C shows abolishment of PEGylation. I138C shows very reduced PEGylation in the presence of MccJ25. The sampling of an inward-open conformation is not affected by pH. The PEGylation is as effective at pH 9 as at pH 7.5 (Fig 7), suggesting that the pH does not induce an inward-occluded conformation. Asterisk (*) denotes SDS-stable dimers. Data information: Error bars are shown for all densitometry measurements (mean ± SEM; n = 3 with the exception of the competition assay that is n = 2). Download figure Download PowerPoint Since an inward-occluded conformation without subunit intertwining has only been observed for McjD and not for other ABC exporters, we also characterized this new conformation by predictive cysteine cross-linking in ISOVs. We designed the A122C mutant at the beginning of TM3 on the cytoplasmic side of McjD, with a Cα-Cα′ distance of 7.3 Å. The McjD-A122C can form cross-linked dimers in ISOVs in the absence of nucleotides but less efficiently compared to the AMPPNP or ADP-VO4 cross-linking experiments (Figs 4B and EV3B). This is the first time that an apo inward-occluded conformation has been reported in E. coli membranes. A possible explanation for the reduced A122C cross-linking in ISOVs in the absence of nucleotides is that McjD alternates between inward-open and inward-occluded conformations. Nevertheless, the transporter is capable of adopting this conformation within the membrane. In the presence of MccJ25, we observed small enhancement of A122C cross-linking (Figs 4B and EV3C), suggesting that binding of the substrate brings the TM helices closer. In the absence of nucleotides, the NBDs of ABC exporters disengage. There are conflicting reports regarding the degree of NBDs disengagement in bacterial ABC exporters (Borbat et al, 2007; Zou et al, 2009; Zoghbi et al, 2016). We sought to characterize the degree of McjD-apo NBD disengagement by cysteine cross-linking of the S509C mutant. This mutant was capable of forming cross-linked dimers even in the absence of CuCl2, even after the ISOVs had been treated with 1 mM reducing agent, suggesting that the NBDs are also close within the E. coli membrane (Fig 4C). In the presence of AMPPNP and ADP-VO4, the McjD-S509C can also form cross-linked dimers that are in agreement with our crystal structures (Figs 4C and EV3D). The predictive cysteine cross-linking data both in the presence and in the absence of nucleotides indicate that McjD mostly exists in an occluded conformation with its NBDs closely engaged in the E. coli membrane. Pulsed electron-electron double resonance Since our crystal structures and predictive cross-linking experiments revealed that McjD exists mostly in an occluded conformation, we performed Q-band PELDOR distance measurements on spin-labelled McjD mutants. We aimed to investigate the conformational changes associated with the export of the antibacterial peptide MccJ25 from the cytoplasmic to the periplasmic leaflet of the inner membrane. The data discussed below were recorded on McjD reconstituted in bicelles (Ward et al, 2014). Based on our crystal structures, a pair of R1 spin labels was introduced at the periplasmic side of the TMD at the loop connecting TMs 1 and 2 (L52R1), and another at the NBDs at position C547 (C547R1) since they could provide us with distances along the transport cycle and reveal stable states (Fig 5A). The ATPase activity of the labelled mutants was similar to the wild-type, unlabelled McjD. The mtsslWizard software (Hagelueken et al, 2012) was used to predict the interspin distances. Figure 5. PELDOR time traces and distance distributions of McjD L52R1 and 547R1 in bicelles A. The McjD cartoon figure shows the position of the labelled side chains (blue lines) with respect to the structure. B–G. For each measurement, the uncorrected PELDOR time trace is shown on the left as a black line and the fitted intermolecular background as a red line. On the right of time traces (B–E), L52R1, is the calculated distance distribution (black trace) with error bars as calculated by DeerAnalysis2016 (red). Grey shades are mtsslWizard predictions for the corresponding structure. The condition under which the time trace was recorded is indicated in each case. Download figure Download PowerPoint The L52R1 mutant produced PELDOR time traces of good quality with clearly visible oscillations. In the apo state, McjD L52R1 produces a sharp interatomic distance peak at 28 Å and a broad distribution of smaller peaks
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