The cyclic octapeptide antibiotic argyrin B inhibits translation by trapping EF-G on the ribosome during translocation

Significance The increase in multidrug-resistant bacteria highlights the urgent need for compounds with novel target sites that can be developed as antibiotics. The argyrins represent a family of naturally produced octapeptides that display promising activity against Pseudomonas aeruginosa by inhibiting protein synthesis. Our structural and kinetic analyses reveal that argyrins inhibit protein synthesis by interacting with, and trapping, the translation elongation factor G (EF-G) on the ribosome, analogous to that reported previously for the unrelated antibiotic fusidic acid. However, the binding site of argyrin on EF-G is distinct from that of fusidic acid, indicating that intramolecular movements at the domain III/V interface of EF-G are also essential for facilitating late events in the translocation mechanism.

but now in presence of the test synergistic partner compound (Appendix SI, Fig. S1A-F). The partner compound (0.5 μL) was added at a fixed final concentration that corresponded to its previously established IC50. The IC50 of each compound alone was then compared with the IC50 measured in the presence of the partner compound (Appendix SI, Fig. S1G-H). Since the in vivo (4) and in vitro (5) synergy between virginiamycin M1 (VrgM1) and virginiamycin S1 (VrgS1) is welldocumented, this combination was used as positive control for synergy. In addition, we considered the use of the erythromycin (Ery) as a negative control since macrolides such as erythromycin have a different mechanism of action, bind to the ribosome (and not EF-G), and the binding site is located far from ArgB-EF-G (6). Moreover, Ery and ArgB display IC50 values within the same order of magnitude. Collectively, the results reveal that the combination of VrgM1 and S1 leads to a 6-fold increase in inhibitory activity compared to when these compounds are used separately, whereas no significant increase was observed for all other combinations of compounds tested (Appendix SI, Fig. S1H).
Ribosome purification for co-sedimentation assays and the EF-G-ArgB-70S cryo-EM studies. Ribosomes were purified as previously described (7). In short, E. coli BL21 (2 L in LB media) was incubated at 37°C and harvested 5,000 x g and 4°C for 25 min in mid exponential phase. Cell pellets were resuspended in 20 mL of buffer A (25 mM HEPES-KOH, pH 7.4, 20 mM Mg(OAc)2, 100 mM KOAc, 1 mM DTT) and disrupted utilising a microfluidizer. The crude cell lysate was centrifuged at 21,000 x g for 20 min at 4 °C. Subsequently, the cleared lysate was layered on a high salt (800 mM KCl) 25% sucrose cushion in buffer A and centrifuged for 17.5 h at 39,000 rpm and 4°C in a Ti45 rotor (Beckmann coulter). The obtained ribosome pellet was resuspended in buffer A. 120 OD260 of the purified ribosomes were layered onto six 10-40% sucrose density gradients in buffer A and centrifuged for 3.5 h at 30,000 rpm and 4°C in a SW32 rotor (Beckmann coulter). Ribosome profiles were recorded at a wavelength of 254 nm using a gradient station (Biocomp). The 70S peaks were collected and subsequently pelleted for 14.5 h at 43,000 rpm and 4°C using a Ti45 rotor. The obtained pellets were resuspended in buffer A and following A260 concentration measurements was stored at -80°C.
Co-sedimentation assays. Co-sedimentation assays were performed as previously described (8). In short, 0.4 μM of purified E. coli 70S were mixed in buffer A (25 mM HEPES-KOH, pH 7.4, 20 mM Mg(OAc)2, 100 mM KOAc, 1 mM DTT) with 1 μM EF-G, 500 µM GTP and 50 μM of either argyrin B or fusidic acid. Reactions were incubated for 15 min at 37 °C and subsequently loaded on a 10% (w/v) sucrose cushion in buffer A and centrifuged for 35 min at 70,000 rpm in a TLA100 rotor at 4 °C. The pre-centrifugation reaction (R) and supernatant (S), as well as 10 pmol of the pellet (P), were applied to an 18% SDS-PAGE and stained with instant blue (Expedeon).
Data collection was performed using a FEI Titan Krios transmission electron microscope equipped with a Falcon II direct electron detector at 300 kV with a dose of 2.5 e -/Å 2 per frame, using a pixel size of 1.084 Å and a defocus range of -0.5 to -3.5 μm, resulting in 7,891 micrographs. The first ten frames (28 e -/Å 2 in total) were aligned using MotionCor2 (9). Power spectra, defocus values, astigmatism and estimation of micrograph resolution were determined by CTFFIND4 software (10). A resolution cut-off of 3.5 Å and defocus cut-off of 30,000 were applied. Following further visual inspection of micrographs for thon-rings and ice quality, 3,466 micrographs were used for automated particle picking by GAUTOMATCH (http://www.mrc-lmb.cam.ac.uk/kzhang). 610,946 particles were extracted using RELION 3.0 (11) and subjected to 2D-classification using 130 classes. 557,434 particles were focus-sorted with a soft-mask around EF-G into four classes for 300 rounds. Class II to IV contained density for EF-G, whereas class I appeared to contain a vacant 70S ribosome and was not further pursued. Contrast transfer function (CTF) and beam tilt refinement with subsequent Bayesian polishing was performed on class II to IV. Using the gold standard criterion (Fourier shell correlation of 0.143), post-processed maps showed an improved resolution by roughly 0.5 Å, resulting in average resolution of 3 Å for class II to IV. Class IV was subjected multi-body refinement with three masks, consisting of large subunit with EF-G, small subunit body and platform, and small subunit head. Resolutions were improved to 2.76 Å, 2.77 Å and 3 Å, respectively, reaching 2.6 Å in the large subunit core. The refined maps were sharpened by applying an automatically determined B factor using RELION 3.0 (11). Local resolution and maps filtered according to local resolution were computed using SPHIRE 1.3 (12). For detailed information on data collection parameters see Fig. S5 and Table S1.
Modelling for the EF-G-ArgB-70S complex. The INT2-FA-translocation intermediate structure (PBD-ID 7N2C), which was based on PDB-ID 4YBB (13), was used as a starting model (14), fit into the EM map and refined through iterative rounds of manual model building in Coot (v.0.9.4.1) (15,16), and real-space refinement using PHENIX (v.1.19-4092) (17,18). A 3D atomic model for Argyrin B was generated de novo from the chemical 2D structure (PubChem CID 9940787) using the Chemdraw-integrated Chem3D software. Restraints for Argyrin B were generated using PHENIX eLBOW (19). Molecular models were manually adjusted and refined using Coot (15,16). The atomic models were combined and refined using real space-refined with secondary structure restraints calculated by PHENIX (v1.14) (17,18). For detailed information on model-building statistics see Table S1. smFRET experiments. 70S ribosomes labelled with LD550 on uL1 and LD650 on uS13, programmed with 5′-biotinylated mRNA, containing P-site bound fMet-tRNA fMet and displaying the codon UUC in the A site were immobilized on passivated coverslips as described previously (21)(22)(23)(24). The ribosomes were then incubated for 2 minutes with EF-Tu ternary complex containing Phe-tRNA Phe , leading to stoichiometric formation of PRE ribosomes containing A-site fMet-Phe-tRNA Phe , P-site tRNA fMet . To initiate translocation, 5 µM EF-G with 1 mM GTP, with or without 10 µM ArgB or 10 µM FA, was delivered to the flow cell by stopped-flow injection. All smFRET experiments were carried out at 25°C and ribosomes, tRNA, EF-Tu and EF-Ts were prepared as previously described (23,24). The time-evolution of the FRET signal was then recorded using a home-built total internal reflection-based fluorescence microscope (25) at ~0.1 kW/cm 2 laser (532 nm) illumination at a time resolution of 40 or 400 ms. Donor and acceptor fluorescence intensities were extracted from the recorded movies and FRET efficiency traces were calculated using the SPARTAN software implemented in MatlabR2015b (25). FRET traces were selected for further analysis according to the following criteria: a single catastrophic photobleaching event, at least 8:1 signal/background-noise ratio and 6:1 signal/signal-noise ratio, less than four donor-fluorophore blinking events, a correlation coefficient between donor and acceptor <0.5. To accurately estimate the FRET efficiencies of the major states occupied by the ribosome during translocation histograms were constructed using the first 50 frames of all FRET efficiency traces for each condition. Three gaussian functions were then fit to each histogram to estimate the mean FRET intensity of the PRE, intermediate and POST (either EF-G-bound in the presence of the drugs or unbound in their absence) states.

Buffers and reagents for smFRET and EF-G-
Purification of reagents for EF-G-ArgB-INT2 cryo-EM structure determination. Wild-type ribosomal subunits were purified from E. coli MRE600 for cryo-EM experiments as previously described (20). EF-Tu/EF-Ts (22) and EF-G (21) were purified as previously described. E. coli tRNA fMet and tRNA Phe were purified as previously described (23). To prepare Phe-tRNA Phe ternary complex, phenylalanine (1 mM Cryo-EM grid preparation for EF-G-ArgB-INT2 structure. Cryo-EM grids were prepared using a Vitrobot Mark IV plunge-freezing device (Thermo Fisher Scientific). For each experiment, 3 μl of sample was applied to UltrAuFoil R 1.2/1.3 gold 300 mesh grids that had been glow-discharged (Ar/O2) for 20 s using a Solarus II Plasma Cleaning system (Gatan). Grids immediately blotted in the Vitrobot chamber (4°C, 100%, 4.5-5.5 s blot time; -5 blot force) and plunged into liquid ethane.

Data collection and single particle reconstruction for EF-G-ArgB-INT2 cryo-EM studies.
Cryo-EM data for the translocation intermediate structure were collected using a Titan Krios G3i (Thermo Fisher Scientific) transmission electron microscope equipped with a K3 direct electron detector and post column GIF (energy filter). K3 gain references were acquired just before data collection. Data collection was performed using an image shift protocol (nine images were collected with one defocus measurement per nine holes). Movies were recorded at defocus values from −0.5 μm to −2.0 μm at a magnification of 130,000×, which corresponds to the pixel size of 0.648 Å per pixel at the specimen level (super-resolution 0.324 Å per pixel). During the 2-s exposure, 70 frames (0.03 s per frame, dose/frame 1 e − per frame per Å 2 ) were collected with a total dose of around 70 e − per Å 2 . Motion correction was performed on raw super-resolution movie stacks and binned twofold using MotionCor2 software (9). CTF parameters were determined using CTFFind4.1 (10) and refined later in Relion 4.0 (26). All pre-processing and particle sorting were conducted in Relion 4.0 (26). Sharpened and locally filtered maps were used to aid in model building. Pixel size was calibrated against high resolution 70S crystal structure PDB-ID 4YBB. Cryo-EM density map values were normalized to mean = 0 and standard deviation (σ) = 1 in UCSF Chimera (27) using the vop scale function. For detailed information on data collection parameters see Fig. S5 and Table S1. The INT2-FA-translocation intermediate structure  (PDB-ID 7N2C), which was based on PDB-ID 4YBB (13), was used as a starting model (14), fit into the EM map and refined through iterative rounds of manual model building in Coot (v.0.9.4.1) (15,16), and real-space refinement using PHENIX (v.1.19-4092) (17,18). mRNA nucleotide 37 corresponds to the +1 position. Restraints for refinement were generated using phenix.elbow (19). Models were validated using phenix.validation_cryoem (28) with the built-in MolProbity (29) scoring. See Table S1 for more information.       (26). Particles from two datasets were refined separately for initial reconstructions. Subsequently, a focus ligand mask containing EF-G, P-site tRNA, and E-site tRNA was applied for sorting each reconstruction into ten classes. Class X of dataset 1 and Class VIII of dataset 2 contained EF-G. Classes containing EF-G were combined, refined, and subjected to another round of classification with a ligand mask into five classes. The EF-G-containing class was refined and subjected to a final round of unmasked classification into eight classes. Class II containing two tRNAs and EF-G was refined (undecimated) followed by CTF-refinement, Bayesian polishing, and Post Processing.       Global alignment (top) and of EF-G from the EF-G-ArgB-INT2 complex (coloured by domain) to S. cerevisiae eEF2 (grey) bound to Sordarin (yellow) and zoom-in of drug binding site between domains III and V of EF-G/eEF2.