All bacterial and phage strains used in this study are listed in Supplementary Table 1. E. coli strains were routinely grown at 37 °C in Luria broth (LB) medium for cloning and maintenance. Phages were propagated by infecting a culture of E. coli MG1655 at an optical density (OD600) of around 0.1–0.2 and multiplicity of infection of 0.1. Cleared cultures were pelleted by centrifugation to remove residual bacteria and filtered through a 0.2 μm filter. Chloroform was then added to phage lysates for prevention of bacterial growth. All phage-infection experiments were performed in LB medium at 25 °C. Antibiotics were used at the following concentrations (liquid, plates): carbenicillin (50, 100 μg ml−1); chloramphenicol (20, 30 μg ml−1).
All plasmids are listed in Supplementary Table 2, and all primers in Supplementary Table 3.
Wild-type or mutant variants of gp54Bas11 were PCR amplified from the corresponding wild-type Bas11 or escaping phage clones using primers TZ-3 and TZ-4, and inserted into pBAD33 linearized with TZ-1 and TZ-2 using Gibson assembly. To add a C-terminal HA-tag, primers TZ-9 and TZ-10 were used to PCR amplify pBAD33-gp54Bas11 followed by Gibson assembly. Mutations that produce the single amino acid substitutions D6E, A7V and I25V in Gp54Bas11 were generated by site-directed mutagenesis using primers TZ-38–43.
Mutations that produce the single amino acid substitutions N275D, D273K, K278E and S279P were generated by site-directed mutagenesis using primers TZ-11–18. Mutations that produce the triple substitution K278E/R314E/K316E were introduced by two-step, site-directed mutagenesis using primers TZ-23–24, then TZ-15 and TZ-16. To add a C-terminal FLAG-tag to CapRelSJ46, primers TZ-25 and TZ-26 were used to PCR amplify pBR322-capRelSJ46 followed by Gibson assembly.
The genes encoding Gp54Bas11 homologues (Gp57Bas10, Gp60Bas8, Gp57Bas5 and Gp19SECΦ27) were PCR amplified from the corresponding phage using primers TZ-30–37 and inserted into linearized pBAD33 by Gibson assembly. Mutations that produce the single amino acid substitutions V7A and V25I in Gp57Bas10 were generated by site-directed mutagenesis using primers TZ-44–47.
Either wild-type or the G24D variant of gp54Bas11 was PCR amplified from the corresponding phage using primers TZ-7 and TZ-8, and inserted into pET-His6 vector linearized with primers TZ-5 and TZ-6 using Gibson assembly.
capRelSJ46 was first PCR amplified from pBR322-capRelSJ46 using primers TZ-48 and TZ-49, and inserted into pET-His6 vector linearized with primers TZ-5 and TZ-6 using Gibson assembly. The gene encoding MBP was PCR amplified with TZ-52 and TZ-53, and inserted into pET-His6-capRelSJ46 linearized with primers TZ-50 and TZ-51 using Gibson assembly.
The gene encoding the MCP of Bas10 (Gp9Bas10) was PCR amplified from phage Bas10 using primers TZ-27 and TZ-28, and inserted into pBAD33 linearized with primers TZ-29 and TZ-1 using Gibson assembly.
gp54Bas11 with its native promoter was PCR amplified from phage Bas11 using primers TZ-58 and TZ-59, and inserted into pBR322-capRelSJ46-Flag linearized with primers TZ-60 and TZ-61 using Gibson assembly. Corresponding upstream mutations were introduced using site-directed mutagenesis.
Plasmids described above were introduced into E. coli MG1655 by TSS transformation or electroporation.
Bas11 mutant phage producing MCP(I115F) was generated using a CRISPR–Cas system for targeted mutagenesis as described previously34. In brief, sequences for RNA guides used to target Cas9-mediated cleavage were designed using the toolbox in Geneious Prime 2022.0.2 and selected for targeting of mcpBas11 (Gp8 in Bas11), but nowhere else in the Bas11 genome. Guides were inserted into the pCas9 plasmid and tested for their ability to restrict Bas11. An efficient guide was selected and the pCas9-guide plasmid was cotransformed into E. coli MG1655 with a high-copy-number repair plasmid containing mcpBas11(I115F), with the guide mutated synonymously to prevent self-cutting. The wild-type Bas11 phage was plated onto a strain containing both the pCas9-guide and the repair plasmid, and single plaques were screened by Sanger sequencing. Two clones that produce the I115F-substituted MCPBas11 were propagated on strains containing only pCas9-guide for further selection.
SECΦ27 mutant phages producing Gp54Bas11 rather than its homologue in SECΦ27 (Gp19) were generated as described above. The guide was selected such that it targeted only gene 19 in SECΦ27, but not gene 54 from Bas11. The selected pCas9-guide plasmid was cotransformed into E. coli MG1655 with a high-copy-number repair plasmid containing the coding sequence of gene 54 from Bas11, flanked by the region that flanks gene 19 in SECΦ27 for homologous recombination. Either the wild-type SECΦ27 phage or the mutant producing MCP(L114P) was plated onto the strain containing pCas9 plasmid and the repair plasmid for selection. Two clones each were propagated and selected twice on strains containing only pCas9-guide.
Phage-spotting assays were conducted similarly to a method described previously11. Phage stocks isolated from single plaques were propagated in E. coli MG1655 at 37 °C in LB. To titre phage, dilutions of stocks were mixed with E. coli MG1655 and melted LB + 0.5% agar, spread on LB + 1.2% agar plates and incubated at 37 °C overnight. For phage-spotting assays, 80 μl of a bacterial strain of interest was mixed with 4 ml of LB + 0.5% agar and spread on an LB + 1.2% agar + antibiotic plate. Phage stocks were then serially diluted in 1× FM buffer (20 mM Tris-HCl pH 7.4, 100 mM NaCl, 10 mM MgSO4), and 2 μl of each dilution was spotted on the bacterial lawn. Plates were then incubated at 25 °C overnight before imaging. EOP was calculated by comparing the ability of the phage to form plaques on an experimental strain relative to the control strain. Experiments were replicated three times independently, and representative images are shown.
Bacterial toxicity assays were conducted similarly to a method described previously11. For coproduction of CapRelSJ46 with either Gp54 homologues or MCPs, single colonies of E. coli MG1655 harbouring pBR322-capRelSJ46 and pBAD33-gp54 homologue or pBAD33-mcp (wild-type or the corresponding variants) were grown for 6 h at 37 °C in LB-glucose to saturation. Next, 200 μl of each saturated culture was pelleted by centrifugation at 4,000g for 10 min, washed once in 1× PBS and resuspended in 400 μl of 1× PBS. Cultures were then serially diluted tenfold in 1× PBS and spotted on M9L plates supplemented with 0.4% glucose or 0.2% arabinose. M9L plates contain M9 medium (6.4 g l−1 Na2HPO4-7H2O, 1.5 g l−1 KH2PO4, 0.25 g l−1 NaCl, 0.5 g l−1 NH4Cl medium supplemented with 0.1% casamino acids, 0.4% glycerol, 2 mM MgSO4 and 0.1 mM CaCl2) supplemented with 5% LB (v/v). Plates were then incubated at 37 °C overnight before imaging.
Bas11 or SECΦ27 MCP(L114P) escape mutants were isolated by plating a population of phage onto CapRelSJ46-containing cells. Next, 20 µl of 1010 PFU ml−1 Bas11 or SECΦ27 MCP(L114P) phage, mixed with 40 µl of overnight culture of E. coli MG1655 pBR322-capRelSJ46, was added to 4 ml of LB + 0.5% agar and spread onto LB + 1.2% agar. Plates were incubated at 25 °C overnight. Single plaques were isolated and propagated using the same strain in LB at 25 °C. Amplified phage lysates were pelleted to remove bacteria, and sequenced by Illumina sequencing as described below to identify mutations.
Bas10 or Bas11 phage with gene 54 deleted was evolved to completely overcome CapRelSJ46 defence using an experimental evolution protocol described previously35. In brief, five independent populations were evolved in a 96-well plate containing a sensitive host E. coli MG1655 pBR322-EV and a resistant host E. coli MG1655 pBR322-capRelSJ46. One control population was evolved with only the sensitive host. Overnight bacterial cultures were back-diluted to an OD600 of 0.01 in LB, and 100 μl was seeded into each well. Cells were infected with tenfold serial dilutions of Bas10 or Bas11 phage with gene 54 deleted, at multiplicity of infection 100–10−4, with one well uninfected to monitor for contamination. Plates were sealed with breatheable plate seals and incubated at 25 °C for either 14 h (for Bas10) or 17 h (for Bas11) in a plate shaker at 1,000 rpm. Cleared wells from each population were pooled, pelleted at 4,000g for 20 min to remove bacteria and supernatant lysates were transferred to a 96-deep-well block with 40 µl of chloroform added to prevent bacterial growth. Lysates were spotted onto both sensitive and resistant hosts to check the defence phenotype. Three rounds of evolution were performed for Bas10, and four populations were able to overcome CapRelSJ46 defence. Two rounds of evolution were performed for Bas11 phage with gene 54 deleted. Evolved clones from each evolved population were isolated by plating to single plaques on lawns of resistant host, and control clones from the control population were isolated on a lawn of the sensitive host. Two clones from each population were propagated using the corresponding host and sequenced as described below.
Phage DNA extraction and sequencing were conducted as described previously11. To extract phage DNA, high-titre phage lysates (over 106 PFU µl−1) were treated with DNase I (0.001 U µl−1) and RNase A (0.05 mg ml−1) at 37 °C for 30 min, then 10 mM EDTA was used to inactivate the nucleases. Lysates were then incubated with Proteinase K at 50 °C for 30 min to disrupt capsids and release phage DNA, which was isolated by ethanol precipitation. In brief, sodium acetate pH 5.2 was added to 300 mM followed by 100% ethanol to yield a final volume fraction of 70%. Samples were incubated at −80 °C overnight, pelleted at 21,000g for 20 min and supernatant removed. Pellets were washed with 100 µl of isopropanol and 200 µl of 70% (v/v) ethanol, then air-dried at room temperature and resuspended in 25 µl of 1× TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0). Concentrations of extracted DNA were measured by NanoDrop (Thermo Fisher Scientific).
For preparation of Illumina sequencing libraries, 100–200 ng of genomic DNA was sheared in a Diagenode Bioruptor 300 sonicator water bath for 20 × 30 s cycles at maximum intensity. Sheared gDNA was purified using AMPure XP beads, followed by end repair, 3′ adenylation and adaptor ligation. Barcodes were added to both 5′ and 3′ ends by PCR with primers that anneal to the Illumina adaptors. The libraries were cleaned by AMPure XP beads using a double cut to elute fragment sizes matching the read lengths of the sequencing run. Libraries were sequenced on an Illumina MiSeq at the MIT BioMicro Center. Illumina reads were assembled to the reference genomes using Geneious Prime 2022.0.2.
Coimmunoprecipitation experiments were conducted similarly to those described previously11. For coproduction of CapRelSJ46 and Gp54Bas11 or with MCPSECΦ27, E. coli MG1655 containing pBR322-capRelSJ46 or pBR322-capRelSJ46-Flag (wild-type or mutant variants) and pBAD33-gp54Bas11-HA (wild-type or mutant variants) or pBAD33-mcpSECΦ27-HA were grown overnight in M9-glucose. Overnight cultures were back-diluted to an OD600 of 0.05 in 50 ml of M9 (no glucose) and grown to an approximate OD600 of 0.3 at 37 °C. Cells were induced with 0.2% arabinose for 30 min at 37 °C, then OD600 was measured and cells pelleted at 4,000g for 10 min at 4 °C. Supernatant was removed and cells resuspended in 800 μl of lysis buffer (25 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 5% glycerol) supplemented with protease inhibitor (Roche), 1 μl ml−1 Ready-Lyse Lysozyme Solution (Lucigen) and 1 μl ml−1 benzonase nuclease (Sigma). Samples were lysed by two freeze–thaw cycles, and lysates normalized by OD600. Lysates were pelleted at 21,000g for 10 min at 4 °C, and 750 μl of supernatant was incubated with prewashed anti-Flag magnetic agarose beads (Pierce) for 1 h at 4 °C with end-over-end rotation. Beads were then washed three times with 500 μl of lysis buffer, followed by the direct addition of 1× Laemmli sample buffer (Bio-Rad) supplemented with 2-mercaptoethanol to beads to elute proteins. Samples were boiled at 95 °C, analysed by 4–20% SDS–PAGE and transferred to a 0.2 μm polyvinylidene difluoride membrane. Anti-Flag and anti-HA antibodies (Cell Signaling Technology) were used at a final concentration of 1:1,000, and SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher) was used to develop blots. Blots were imaged by the ChemiDoc Imaging system (Bio-Rad). Images shown are representatives of two independent biological replicates.
Single colonies of E. coli MG1655 pBR322-gp54Bas11-Flag with its wild-type or mutant native promoter were grown overnight in LB. Overnight cultures were back-diluted to OD600 of 0.05 in 10 ml of fresh LB and grown to OD600 of 0.4 at 37 °C. OD600 was measured, and 5 ml of cells pelleted at 4,000g for 5 min with OD600 normalized. Supernatant was removed and pellets resuspended in 1× Laemmli sample buffer (Bio-Rad) supplemented with 2-mercaptoethanol. Samples were then boiled at 95 °C and analysed using 4–20% SDS–PAGE and transferred to a 0.2 μm polyvinylidene difluoride membrane. Anti-Flag antibody (Cell Signaling Technology) and anti-RpoA antibody (BioLegend) were used at a final concentration of 1:1,000, and SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher) was used to develop blots. Blots were imaged using a ChemiDoc Imaging system (Bio-Rad). Images shown are representative of two independent biological replicates.
The C terminus of CapRelSJ46 was mutagenized using error-prone PCR-based mutagenesis as described previously11. In brief, primers TZ-54 and TZ-55 were used to amplify the C terminus of CapRelSJ46 using Taq polymerase (NEB), with 0.5 mM MnCl2 added to the reaction as the mutagenic agent. PCR products were treated with Dpn I, column purified and inserted into a pBR322-capRelSJ46 backbone amplified with primers TZ-56 and TZ-57 using Gibson assembly. Gibson products were transformed into DH5α and grown overnight in LB at 37 °C. Overnight cultures were miniprepped to obtain the mutagenized library, and individual colonies were Sanger sequenced to assess the number of mutations. To perform selection, the mutagenized library was electroporated into E. coli MG1655 pBAD33-gp54Bas11 and plated onto M9L plates containing 0.2% arabinose to select for survivors. Colonies were picked and sequenced to identify mutations in CapRelSJ46.
For the production of His6-MBP-tagged CapRelSJ46, E. coli BL21(DE3) cells were transformed with pET-His6-MBP-capRelSJ46 and grown in LB medium to OD600 = 0.5. Protein expression was induced by the addition of 0.3 mM isopropyl-β-d-thiogalactopyranoside, and cells grown for 3 h at 30 °C. The culture was centrifuged at 4,000g for 10 min at 4 °C and the cell pellet resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 500 mM KCl, 2 mM MgCl2, 1 mM DTT) supplemented with 0.4 mM phenylmethanesulfonyl fluoride, 10 μg ml−1 lysozyme and 7.5 U ml−1 benzonase nuclease (Millipore). Cells were disrupted using sonication (Qsonica), and glycerol was added to the lysate at a final 10% concentration following sonication. The supernatant was separated from the pellet by centrifugation (15,000 rpm for 30 min, JA-25.50 rotor, Beckman Coulter). The clarified supernatant was loaded onto a gravity-flow chromatography column (Bio-Rad) packed with 2 ml of Ni-NTA agarose resin (Qiagen) pre-equilibrated with 15 ml of lysis buffer. The resin was washed with ten column volumes of wash buffer 1 (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 500 mM KCl, 2 mM MgCl2, 10 mM imidazole, 10% glycerol, 1 mM DTT) and then with ten column volumes of wash buffer 2 (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 500 mM KCl, 2 mM MgCl2, 50 mM imidazole, 10% glycerol, 1 mM DTT). Proteins were eluted in 4 ml of elution buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 500 mM KCl, 2 mM MgCl2, 300 mM imidazole, 10% glycerol, 1 mM DTT). For removal of any remaining contaminants, the eluted protein sample was loaded onto a size exclusion chromatography (SEC) Superdex 200 Increase 10/300 GL column (Cytiva) pre-equilibrated in SEC buffer (50 mM Tris-HCl pH 8.0, 250 mM NaCl, 250 mM KCl, 2 mM MgCl2, 1 mM DTT). Fractions containing the protein of interest were pooled and concentrated to around 1 mg ml−1. Purity of protein samples was assessed both spectrophotometrically and by SDS–PAGE.
To produce His6-tagged Gp54Bas11 or the G24D variant, E. coli BL21(DE3) cells were transformed with pET-His6-gp54Bas11 (wild-type or G24D) and grown in LB medium to an OD600 of 0.5. Protein expression was induced by the addition of 0.3 mM isopropyl-β-d-thiogalactopyranoside, and cells were grown for 3 h at 30 °C. Purification steps were performed similarly to those described above, with the following buffers. Lysis buffer contained 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM MgCl2 and 1 mM DTT supplemented with 0.4 mM phenylmethanesulfonyl fluoride, 10 μg ml−1 lysozyme and 7.5 U ml−1 benzonase nuclease (Millipore). Wash buffer 1 contained 50 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM MgCl2, 10 mM imidazole, 10% glycerol and 1 mM DTT. Wash buffer 2 contained 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM MgCl2, 50 mM imidazole, 10% glycerol and 1 mM DTT. Elution buffer contained 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM MgCl2, 300 mM imidazole, 10% glycerol and 1 mM DTT. To remove any remaining contaminants, the eluted protein sample was loaded onto a SEC Superose 6 Increase 10/300 GL column (Cytiva) pre-equilibrated in SEC buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM MgCl2, 1 mM DTT). Fractions containing the protein of interest were pooled and concentrated to around 5 mg ml−1. Purity of protein samples were assessed both spectrophotometrically and by SDS–PAGE.
Experiments using the PURExpress in vitro protein synthesis kit (NEB, E6800) were performed as per the manufacturer’s instructions. All reactions were supplemented with 0.8 U µl−1 RNase Inhibitor Murine (NEB, M0314S). Purified His6-MBP-tagged CapRelSJ46 protein was added to the reaction at a final concentration of 500 nM, and either purified His6-tagged Gp54Bas11 or the G24D variant was used at a final concentration of 4 µM. A template plasmid encoding the control protein DHFR (provided by the kit) was used at 6 ng µl−1. The reactions were incubated at 37 °C for 2 h, and 2 µl of each reaction was mixed with 10 µl of 1× Laemmli sample buffer (Bio-Rad) supplemented with 2-mercaptoethanol. Mixtures were boiled for 5 min at 95 °C and analysed by 12% SDS–PAGE. Gels were stained with Coomassie stain and imaged using the ChemiDoc Imaging system (Bio-Rad). Images shown are representative of three independent biological replicates.
His6-tagged Gp54Bas11 was purified as described above and concentrated to 5 mg ml−1 for crystallization. Crystallization conditions for His6–Gp54Bas11 were screened by sitting-drop vapour diffusion using a Formulatrix NT8 drop setter and commercial screening kits. Each drop, consisting of 100 nl of protein solution plus 100 nl of reservoir solution, was equilibrated against 70 µl of reservoir solution. Crystals appeared in Index HT (Hampton Research) condition B12 (2.8 M sodium acetate trihydrate pH 7.0). These conditions were optimized, and the final crystals were grown by hanging-drop vapour diffusion, with drops consisting of 2 µl of protein plus 2 µl of well solution (3.2 M potassium acetate pH 7.0) at room temperature. After 8 days, a crystal was harvested and directly vitrified in a nitrogen gas stream at 100 K (Oxford Cryostream). X-ray diffraction data were collected on a Rigaku Micromax-007 rotating anode with Osmic VariMax-HF mirrors and a Rigaku Saturn 944 detector. Diffraction data were processed with the XDS suite36. Phaser37 was used to solve the structure by molecular replacement using an AlphaFold38 model. The molecular replacement solution was refined in PHENIX39 with manual model building done with Coot40. The model was refined to a final Rwork/Rfree of 0.211/0.252. X-ray data collection and refinement statistics are summarized in Extended Data Table 1.
For the CapRelSJ46–Gp54Bas11 complex, CapRelSJ46 and Gp54Bas11 were purified as described above and mixed in a 1:1 ratio at a concentration of 2 mg ml−1. The complex was then further purified by SEC (in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM MgCl2 and 1 mM DTT) and the resulting sample concentrated to 10 mg ml−1 for crystallization. Crystallization conditions for the CapRelSJ46–Gp54Bas11 complex were either screened as such or supplemented with 5 mM ATP. Crystals grew within 1 week in 25% PEG 1500 in a malic acid, MES, Tris buffer system (pH 8.0). Before data collection, crystals were cryoprotected by soaking in the mother liquor solution supplemented with 25% glycerol and flash-frozen in liquid nitrogen for storage. X-ray diffraction data were collected at the I24 beamline of the Diamond Light Source synchrotron (UK) on a CdTe Eiger2 9M detector, then processed using the XDS suite36 and scaled with Aimless. The structure was solved by molecular replacement performed with Phaser37 using the coordinates of the toxSYNTH domain of CapRelSJ46 (PDB: 7ZTB). Initial automated model building was performed with Buccaneer41, which partially completed Gp54Bas11 and further improved with the MR-Rosetta suite from the Phenix package42. Following several iterations of manual building with Coot40 and maximum-likelihood refinement as implemented in Buster/TNT43, the model was refined to Rwork/Rfree of 0.193/0.236. X-ray data collection and refinement statistics are summarized in Extended Data Table 1.
CapRelSJ46 homologues were identified, aligned and used as input for ConSurf analysis as described previously11. Homologues of the MCPs or Gp54Bas11 in BASEL phages were identified by BLASTp44 searches against each phage genome, and aligned by MUSCLE45. Whole genomes of phages were aligned using Mauve46 in Geneious Prime 2022.0.2.
The structure prediction of CapRelSJ46-MCPSECΦ27 was reported previously11 and was calculated using AlphaFold2. The structure of CapRelSJ46 in the closed state for comparison with the experimental SAXS curve was also calculated using AlphaFold2 using default parameters (as implemented in ColabFold38) and running the calculations for ten recycles. Both models are deposited in the ModelArchive Database (https://www.modelarchive.org) with the accession codes ma-zblch (https://doi.org/10.5452/ma-zblch) and ma-9z23e (https://doi.org/10.5452/ma-9z23e).
Circular dichroism measurements were performed on a MOS-500 spectropolarimeter (BioLogic) using a cuvette of 0.1 cm path length. Spectra were collected between 200 and 250 nm with a data interval of 0.25 nm at 25 °C. Measurements were recorded in 15 mM K2HPO4, 0.05 mM KH2PO4 pH 7.5, 300 mM KCl, 300 mM NaCl and 1 mM tris(2-carboxyethyl)phosphine (TCEP). Protein concentration used in measurements was 0.6 mg ml−1. Molar residue ellipticities (θ, mdeg cm2 dmol−1) were obtained from the raw data (θ, ellipticity) following buffer correction, according to the relation [θ] = θMw(ncl), where Mw is weight-averaged molecular mass, c mass concentration, l optical path length and n the number of amino acid residues.
Hydrogen deuterium exchange mass spectrometry (HDX–MS) experiments were performed on an HDX platform comprising a Synapt G2 mass spectrometer (Waters Corporation) connected to a nanoAcquity ultra-performance liquid chromatography (UPLC) system following the protocol previously described11. Samples of CapRelSJ46, Gp54Bas11 and CapRelSJ46–Gp54Bas11 were prepared at a concentration of 100 µM (the integrity of the complex was confirmed by SEC before the HDX–MS experiment). For each experiment, 8 µl of sample was incubated for 1, 5, 15 and 60 min in 72 µl of labelling buffer L (50 mM HEPES, 500 mM KCl, 500 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 0.002% mellitic acid, pD 7.5) at 20 °C. Non-deuterated reference points were prepared by replacement of buffer L with equilibration buffer E (50 mM HEPES, 500 mM KCl, 500 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 0.002% mellitic acid, pH 7.5). After labelling, samples were quenched by mixing with 80 µl of prechilled quench buffer Q (50 mM K2PO4, 1 mM TCEP, 1.2% formic acid, pH 2.4). Samples were then directly flash-frozen in liquid nitrogen and stocked at −80 °C until injection. For injection, samples were thawed at room temperature and 150 µl of quench samples directly transferred to a Enzymate BEH Pepsin Column (Waters Corporation) at 200 µl min−1 and 20 °C, with a pressure 3,000 pounds per square inch. Peptic peptides were trapped for 3 min on an Acquity UPLC BEH C18 VanGuard Pre-column (Waters Corporation) at a flow rate of 200 µl min−1 in water (0.1% formic acid in high-performance liquid chromatography water, pH 2.5) before elution on an Acquity UPLC BEH C18 Column for chromatographic separation. Separation was performed with a linear gradient buffer (3–45% gradient of 0.1% formic acid in acetonitrile) at a flow rate of 40 µl min−1. Peptide identification and deuteration uptake analysis were performed on a Synapt G2, using positive electrospray ionization, data independent acquisition, and triwave ion-mobility for improved resolution and identification. Leucine enkephalin was applied for mass accuracy correction, and sodium formate was used as calibration for the mass spectrometer. MSE data were collected with a 20–30 V transfer collision energy ramp. The pepsin column was washed between injections using pepsin wash buffer (1.5 M guanidinium HCl, 4% (v/v) acetonitrile, 0.8% (v/v) formic acid). A cleaning run was performed on every third sample to prevent peptide carryover. Optimized peptide identification and peptide coverage for all samples were performed from undeuterated controls (five replicates). All deuterium time points were performed in triplicate. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium by the PRIDE47 partner repository with the dataset identifier PXD050526.
Samples for small angle X-ray scattering (SAXS) were concentrated to 10 mg ml−1, flash-frozen and stored at −80 °C. SAXS data were collected at the SWING beamline (Soleil and ESRF synchrotrons, France) on a Pilatus 2M detector using the standard beamline set-up in SEC mode. Samples were prepared in 500 mM NaCl, 500 mM KCl, 2 mM TCEP and 30 mM HEPES pH 7.5. SEC–SAXS was performed with a Shodex KW404–4 F column coupled to a high-performance liquid chromatography system, in front of the SAXS data collection capillary. Samples were flowed at 0.2 ml min−1 and data collected at 10 °C. Radiation-damaged frames were removed before data analysis. Data were analysed with the ATSAS suite48. SAXS-based models were derived from the coordinates of the X-ray structure of the CapRelSJ46–Gp54Bas11 complex and an AlphaFold model of unbound CapRelSJ46. Calculation of ab initio shapes was carried out with the program DAMMIF from the ATSAS package.
All titrations were performed with an Affinity ITC (TA instruments) at 30 °C. For titration, CapRelSJ46 was loaded in the instrument syringe at 200 µM and Gp54Bas11 used in the cell at 10 µM. Titrations were performed in 50 mM HEPES pH 7.5, 500 mM KCl, 500 mM NaCl, 2 mM MgCl2 and 1 mM TCEP. Final concentrations were verified by absorption using a Nanodrop One (ThermoScientific). All isothermal titration calorimetry (ITC) measurements were performed by titrating 2 µl of CapRelSJ46 into Gp54Bas11 (Gp54Bas11(G24D) was used at 260 µM) at a constant stirring rate of 75 rpm. All data were processed, buffer corrected and analysed using the NanoAnalyse and Origin software packages.
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