Protein Expression and Purification
The mprF gene from R. tropici (RtMprF) was cloned into the pET21b vector and the recombinant plasmid was used to transform E. coli C41(DE3) cells. The cells were cultured in Terrific Broth media containing 50 mg·mL-1 ampicillin, and protein expression was induced with 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) overnight at 16 °C after OD600 reached 1.0. For purification of RtMprF, cells were harvested through centrifugation, resuspended in a buffer containing 25 mM Tris-HCl (pH 8.0) and 300 mM NaCl and then incubated with 1% lysozyme at 4 °C for 30 min. The suspension was sonicated and centrifuged at 11,000 g (JL-25.50 rotor, Beckman) for 30 min. The supernatant was ultracentrifuged at 158,000 g (Type 45 Ti rotor, Beckman) to collect membrane pellets. The pellets were resuspended in a buffer with 25 mM Tris-HCl (pH 8.0), 300 mM NaCl and 1.5 % b-DDM (Anatrace) and incubated at 4 °C for 30 min. After centrifugation at 40,000 g for 30 min, the supernatant was loaded onto a Ni-NTA column and the protein was purified at 4 °C by a step-wise elution method with three different buffers containing 25 mM Tris-HCl (pH 7.5), 300 mM NaCl, 20, 50 or 300 mM imidazole and 0.05% β-DDM for MprF(DDM) sample or 0.02% GDN for MprF(GDN) sample. The fraction eluted in the buffer with 300 mM imidazole was pooled and concentrated to 10 mg·mL-1 in 100 kDa molecular weight cutoff (MWCO) concentrator (Millipore) for further experiments.
To construct the RtPaMprF chimera, the cDNA sequence of RtMprF synthase domain (residues 542-869) on the pET21b-RtMprF vector was replaced by the coding sequence of PaMprF synthase domain (residues 554-881, AlaPG synthase) through two PCR reactions. In detail, the region encoding PaMprF synthase domain was amplified through the first PCR reaction by using a pair of primers with sequences of 5’-CCGGCAACGAAGCGGCCGGAGCCTGTCAGCGCGGAAGAGCTG-3’ and 5’-GTGGTGGTGCTCGAGTGCGGCCGCAAGCTTGCGTTTCACCAA-3’. The DNA product contains 30 bp nucleotides in the 5’- and 3’- terminal regions matching with the regions upstream and downstream of the coding sequence of RtMprF synthase domain on the vector pET-21b, respectively. The replacement was further accomplished through a second PCR reaction by using the DNA product of the first round PCR (with the cDNA encoding PaMprF synthase domain) as primers and the pET21b-RtMprF vector as template. The second PCR reaction adopts the protocol of the Quick Change method. After digestion of the template with DpnI, the product of the second round of PCR was used for transformation of DH5a E. coli competent cells for plasmid amplification. After transformation and antibiotics resistance screening, the clone with target plasmid was selected and verified through DNA sequencing. The protocols for RtPaMprF protein expression and purification were the same as the one used for wild-type RtMprF.
The DNA encoding the synthase domain of RtMprF (RtMprF-SD, residues 542-862) was cloned into the pET21b vector and transformed into the E. coli BL21(DE3) cells for protein expression. For purification of the recombinant RtMprF-SD protein expressed in BL21(DE3) cells, the cell pellets harvested through centrifugation were resuspended in a buffer with 25 mM Tris-HCl (pH 8.0) and 700 mM NaCl. After the cells were lysed through sonication, the suspension was centrifuged at 40,000 g (JL-25.50 rotor, Beckman) for 30 min. The protein was purified by using the Ni-NTA column through the step-wise elution protocol with buffers containing 25 mM Tris-HCl (pH 7.5), 700 mM NaCl and 20, 50 or 300 mM imidazole. The fraction eluted in the buffer with 300 mM imidazole was pooled and concentrated to 15 mg·mL-1 in 30 kDa MWCO concentrator. The protein was further purified through gel filtration on a Superdex 200 Increase 10/300 GL column (GE Healthcare) in a buffer with 25 mM Tris-HCl (pH 7.5) and 700 mM NaCl. The major peak fractions were collected and concentrated to 15 mg·mL-1 for crystallization. The Se-Met protein was purified through the same procedure as the one used for native protein purification except that 2 mM DTT and 0.2 mM EDTA are added to the buffers. The detailed informations about cell strains, plasmids and primers used for cloning and protein expression are included in Supplemental Table 1.
Crystallization of RtMprF-SD and structure determination
The protein crystals of the RtMprF-SD were obtained through the hanging-drop vapor diffusion method at 16 °C with a well solution containing 0.1 M NaAc (pH 6.0) and 16% PEG3350. The Se-Met derivative crystals were grown with the well solution containing 0.1 M NaAc (pH 6.0) and 10% PEG3350. Both the native and the derivative crystals were cryo-protected in a solution with 0.1 M NaAc (pH 6.0), 10% PEG3350 and 20% glycerol before being flash frozen in liquid nitrogen. The native and Se-Met derivative datasets were collected at 0.96000 and 0.97909 Å wavelength respectively on BL1A and NW12A beamlines in the Photon Factory (Tsukuba, Japan), and were processed by using the HKL2000 program46. The phases were solved through the single-wavelength anomalous method using Phenix AutoSol program47. Model building and structure refinement were accomplished by using Coot48 and Phenix Refine. Structure figures were prepared with PyMOL (The PyMOL Molecular Graphics System, Version 2.0, Schrodinger, LLC).
Reconstitution of RtMprF in nanodiscs
The purified RtMprF protein was incorporated into lipid nanodiscs with a molar ratio of RtMprF protein : membrane-scaffold-protein 1E3D1 (MSP1E3D1) : POPG at 1 : 2 : 100. The mixture was incubated at 4 °C for 1 h on a sample rotator. Reconstitution was initiated by removing detergent through addition of Bio-beads (Bio-Rad) to the sample and incubation at 4 ℃ overnight with constant rotation. In the next day, the old Bio-beads were replaced by fresh Bio-beads and the sample was further incubated for 2 h. Subsequently, Bio-beads were removed from the sample and the nanodisc reconstitution mixture was incubated with 0.25 mL Ni-NTA resin for 1 h at 4 °C to enrich nanodiscs with the target protein and remove the empty ones. The resin was washed with 5 column volumes of wash buffer (20 mM Tris-HCl pH 7.5, 300 mM NaCl and 20 mM imidazole) followed by 4 column volumes of elution buffer (20 mM Tris-HCl pH 7.5, 300 mM NaCl and 300 mM imidazole). The eluted RtMprF protein in nanodiscs was further purified by loading the sample onto a Superdex 200 increase 10/300 GL size-exclusion column (GE Healthcare Life Sciences) and eluting it in the gel-filtration buffer with 20 mM Tris-HCl (pH 7.5) and 300 mM NaCl.
Cryo-EM sample preparation and data acquisition
The purified RtMprF protein in nanodics was concentrated to 6 mg·mL-1 for RtMprF(DDM)-nanodiscs or 13mg·ml-1 RtMprF(GDN)-nanodiscs using a 100-kDa MWCO Amicon concentrator (Millipore). The Quantifoil 1.2/1.3-μm holey carbon grids (300 mesh, copper) were glow discharged for 60 s firstly and then 3 μl of concentrated nanodisc sample was applied onto the grid, blotted for 8.0 s with a force level of 2, drained for 0.5 s for RtMprF(DDM)-nanodiscs and blotted for 4.0 s with a force level of 4 for RtMprF(GDN)-nanodiscs and plunge-frozen in liquid ethane cooled by liquid nitrogen, using Vitrobot Mark IV (Thermo Fisher) operated at 18 °C with 80% humidity for RtMprF(DDM)-nanodiscs and 4 °C with 100% humidity for MprF(GDN)-nanodiscs.
The grids containing RtMprF nanodisc samples were imaged with a 200 kV Talos Arctica microscope equipped with a Gatan K2 Summit direct detector camera. An energy filter with slit width of 20 eV was used during data collection at a nominal magnification of 130,000×, resulting in a super-resolution pixel size of 0.5 Å (physical pixel size of 1.0 Å). Movies (32 frames per movie file) were captured with a defocus value at a range of -1.5 to -2.0 μm in the super resolution mode using a dose rate of ~ 9.6 e-·pixel-1·Å-2 over 5.2 s yielding a cumulative dose of ~50 e-·Å-2.
For RtMprF(DDM)-nanodiscs, A total of 2921 cryo-EM movies were aligned with dose-weighting using MotionCor2 program49 with 5 by 5 patches and a B-factor of 250. Micrograph contrast transfer function (CTF) estimations were performed by using CTFFIND4 program50. Particle picking, 2D classification and ab initio 3D reference generation were performed in cryoSPARC v2 program51. After manual inspection of the micrographs, 2549 were selected and ~100 particles were picked manually from the micrograph and sorted into 2D classes. The best classes were selected and used as references for subsequent autopicking procedure. After the process, 887,196 particles were auto-picked and extracted using a box size of 200 pixels. 2D classification was performed to remove ice spots, contaminants and aggregates, yielding 529,371 particles. The particles were exported from cryoSPARC v2 using the UCSF pyem v0.5 script (https://doi.org/10.5281/zenodo.3576630) and re-extracted in RELION-3 program52 from the original micrographs for 3D classification. Consequently, 247,321 particles were selected for further refinement. Per-particle CTF refinement, with estimation of the beam tilt and Bayesian polishing, was performed in RELION-3. Particles with resolution lower than 4 Å resolution were discarded, and the refinement with C2 symmetry imposed resulted in a 3.7 Å cryo-EM density map from a major class of 160,417 particles. For the minor classes of asymmetric shapes, four classes with tilted synthase domains are subject to a second round of 3D classification and the three classes with particle numbers over 10,000 are chosen for auto-refine with C1 symmetry individually. To improve the local map quality around LysPG in cavity C, a mask covering Subdomain 1 was generated by Chimera and applied for local refinement in Relion 3. The local refinement procedure with the mask and solvent-flattened Fourier Shell Correlations (FSCs) yielded a reconstruction for Subdomain 1 at 3.4 Å.
For RtMprF(GDN)-nanodiscs, a total of 2579 cryo-EM movies were aligned with dose-weighting using MotionCor2 program with 5 by 5 patches and a B-factor of 250. Micrograph contrast transfer function (CTF) estimations were performed by using CTFFIND4 program. Particle picking, 2D classification and ab initio 3D reference generation were performed in cryoSPARC v2 program. After manual inspection of the micrographs, 2317 were selected and ~200 particles were picked manually from the micrograph and sorted into 2D classes. The best classes were selected and used as references for subsequent autopicking procedure. After the process, 1,232,621 particles were auto-picked and extracted using a box size of 200 pixels. 2D classification was performed to remove ice spots, contaminants and aggregates, yielding 343,117 particles. The particles were exported from cryoSPARC v2 using the UCSF pyem v0.5 script and re-extracted in RELION-3 program from the original micrographs for 3D classification. Consequently, 276,824 particles were selected, removed duplicates and re-extracted with boxsize 320 instead of 200 for further refinement. Per-particle CTF refinement, with estimation of the beam tilt and Bayesian polishing, was performed in RELION-3. A tight mask for TM domain was generated by Chimera and RELION-3, followed by 3D classification by skipping alignment. Finally, 144,479 particles were selected and the refinement with C2 symmetry imposed resulted in a 2.96 Å cryo-EM density map.
Model building and refinement
The structural model of the flippase domain of RtMprF was built manually in Coot program48, guided mainly by the cryo-EM map. The secondary structure prediction from PSIPRED program53 and the transmembrane helix prediction from TMHMM54 were used as references during model building. While most of the transmembrane helices are identified in the map and the models are registered with amino acid sequences, the density for TM14 is too weak for MprF(DDM)-nanodiscs and it is tentatively interpreted with a poly-alanine a-helix model. For the synthase domain, the crystal structure was docked manually into the corresponding region of the cryo-EM map of the full-length RtMprF, subject to rigid body refinement and local adjustment in Coot, and then merged with the flippase domain. The structural model of RtMprF was refined against the cryo-EM map by using phenix.real_space_refine program followed by manual adjustment in Coot. The program refinement and manual adjustment were carried out iteratively till the model-to-map fitting is optimal and the model geometric parameters are within reasonable range (Extended Data Fig. 4). The final model covers 793 or 820 out of 869 amino acid residues of the full-length RtMprF protein for MprF(DDM)-nanodiscs or MprF(GDN)-nanodiscs respectively, while several regions in the loops or near the amino- and carboxyl- termini (1-22 region, 326-333 region, 375-384 region, 492-510 region, 531-538 region and 861-869 region) for MprF(DDM)-nanodiscs or (1-23 region, 326-333 region, 531-539 region, 861-869 region) for MprF(GDN) nanodiscs are unobserved in the map due to high flexibility.
Crosslinking of RtMprF
The oligomeric state of the RtMprF protein was analyzed through chemical crosslinking experiment by using the membrane preparation from the E. coli cells expressing the full-length RtMprF. The cells were resuspended in a buffer consisting of 20 mM HEPES (pH 7.5) and 300 mM NaCl (buffer A). After the cells were lysed by passing through a high-pressure homogenizer (ATS Engineering), the cell debris was removed through low-speed centrifugation at 11,000 g for 15 min and the membrane fraction was further collected through ultracentrifugation at 100,000 g for 30 min at 4 °C. The membrane pellets were re-suspended in buffer A and sonicated with 1 s on, 5 s off for 2 min to homogenize the sample. The membrane suspension was aliquoted and then treated with disuccinimidyl suberate (DSS) at 0-5 mM final concentration for 1 h at 30 °C with constant mixing on a shaker. The reactions were quenched by adding 100 mM Tris-HCl (pH 7.5). The cross-linked samples were solubilized by adding 1% b-DDM for 1 h in the shaker. Subsequently, the samples were centrifugated at 18,000 g for 10 min and the supernatant was mixed with 5 ´ SDS-PAGE loading buffer, and then loaded on the SDS-PAGE gel for electrophoresis. The protein bands on the gel were transferred to polyvinylidene difluoride (PVDF) membrane and then detected through western blot by using the Anti-His Mouse monoclonal antibody and Goat anti-Mouse IgG (H+L)–HRP. After being developed with the western lightning Ultra ECL horseradish peroxidase substrate (Perkin–Elmer), the blots were imaged on a chemiluminescence CCD system (ChemiScope 3500 mini imager, Clinx Science Instruments).
Thin-layer chromatography and mass spectrometry
The lipids from E. coli membrane expressing recombinant RtMprF/RtPaMprF or from the purified RtMprF protein samples were extracted according to Bligh and Dyer procedure55. In detail, 12 mL chloroform:methanol (1:2, v:v) mixture was added to 3.2 mL sample and mixed well through vortex. Subsequently, 4 mL chloroform was added to the sample and vortexed again to mix. Finally, 4 mL water was added to the sample and vortexed well. The mixture was centrifuged at 1000 rpm for 5 min to get a two-phase system with aqueous phase at the top and organic phase at the bottom. The bottom phase was washed twice with an aqueous upper phase solution (freshly made by mixing chloroform, methanol and water at 2:2:1.8 (v:v:v) ratio and centrifuging the mixture). Finally, the bottom phase was recovered, dried under vacuum and dissolved in 100 mL chloroform. The lipid samples were separated on the HPTLC silica gel 60 F254 plates (Merck) in a mobile phase of chloroform:methanol:water mixture (65:25:4). Lipid spots were visualized through staining with iodine or ninhydrin. For separation of AlaPG and PE, a mobile phase of chloroform:methanol:acetic acid:water (80:12:15:4) mixture was used.
The liquid chromatography mass spectrometry (LC-MS)/MS analysis was performed by using a Thermo Scientific Dionex Ultimate 3000 LC system coupled to a TripleTOF 5600 quadrupole time-of-flight tandem mass spectrometer. An ACQUITY UPLC C18 reversed-phase column (1.7 μm, 2.1 × 100 mm, Waters) was used in LC. Mobile phase A consisted of methanol/acetonitrile/aqueous 15 mM ammonium acetate (1:1:1, vol/vol/vol), and mobile phase B consisted of 80% 2-propanol and 20% methanol containing 5 mM ammonium acetate. The LC process was operated at a flow rate of 250 μL/min with a linear gradient as follows: 10% B was held constantly for 1 min and then increased linearly to 60% B over 5 min, further to 100% B over 12 min and finally held at 100% B for 2 min. The conditions for MS were set with the following parameters: electrospray voltages, +5,500 V (positive ion mode) and -4,400 V (negative ion mode); declustering potential, 100 V; GS1 and GS2, 60 psi. The collision-induced dissociation tandem mass spectra were obtained with collision energy of +35 V in the positive ion mode or -35 V in the negative ion mode. Nitrogen was used as the collision gas.
For quantification of lipid:protein molar ratio, the lipids were extracted from 47.5 nmol of purified RtMprF protein according to Bligh and Dyer procedure and were dissolved in 100 μL chloroform. 2 μL of the lipid solution were applied on the HPTLC silica gel 60 F254 plates (Merck) and separated in the solvent of chloroform:methanol:water mixture (65:25:4). As the standard samples, 0.25, 0.5, 1.0, 2.0, 4.0 nmol LysPG (Avanti) were also loaded on the same plate. Lipid spots were visualized through the iodine staining procedure. It is noteworthy that the data obtained through iodine staining generates a linear standard curve better than those stained with ninhydrin. For quantifying the relative amount of LysPG/AlaPG co-purified with RtMprF/RtPaMprF mutants, lipids extracted from same amount of purified RtMprF/RtPaMprF mutant protein were separated on HPTLC silica gel 60 F254 plates (Merck) and stained by ninhydrin. The protein concentration was measured through the Bicinchoninic Acid (BCA) method (TransGen Biotech, Beijing). The mobile phase chloroform:methanol:water mixture (65:25:4) and chloroform:methanol:acetic acid:water (80:12:15:4) were utilized to separate lipids extracted from RtMprF and RtPaMprF mutant protein respectively. The image of the iodine- or ninhydrin-stained TLC plate was processed by Image J and analyzed by GraphPad program. For the data presented in Fig. 3e, 3f, 4c and Extended Data Fig. 9b, three aliquots of the sample of the same type are loaded on the TLC plates and the measurements of the three parallel spots are used for statistical analysis.
Relative quantification of total LysPG and fluorescamine labeled LysPG
The cells were cultured in Terrific Broth media containing 50 mg·mL-1 ampicillin, and protein expression was induced with 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) for 2 h at 37℃ after OD600 reached 1.0. After the cells were harvested through centrifugation, the pellets were washed and suspended in a solution containing 25 mM HEPES-Na (pH 8.5) and 300 mM NaCl (Buffer B), and then adjusted to a concentration of 4 × 109 cell/mL.
For total lipid extraction, 5 mL cell suspension was centrifuged and resuspended in 1.2 mL Buffer B. Subsequently, 4.5 mL chloroform:methanol (1:2, v:v) mixture was added to the suspension and vortexed for 30 s. Afterwards, 1.5 mL chloroform and 1.5 mL HEPES buffer were added to the sample sequentially and vortexed for 10 s after each step. After the mixture was centrifuged at 1000 rpm for 5 min, the bottom phase was recovered, dried under vacuum and dissolved in 150 mL chloroform. To separate LysPG from other lipids, 4.5 μL total lipid samples were loaded on the HPTLC plate and the plate was developed in a solvent of chloroform:methanol:water (65:25:4) mixture, dried and then stained with ninhydrin.
For detection of LysPG in the outer leaflet of the membrane, 50 μL fluorescamine (50 mM stock solution in DMSO) was added to 5 mL cell suspension and incubated at room temperature for 20 min. Afterwards, 500 μL Tris-HCl (pH = 8.0, 1 M) was added to the mixture and incubated for 5 min to stop the reaction. Finally, the cells were collected through centrifugation, washed once in 5 mL Buffer B and resuspended in 1.2 mL Buffer B. The following procedures of lipid extraction and TLC experiments were the same with the above protocols used for the total lipid extraction sample, except that the lipid spots were visualized under UV light after they were separated on HPTLC plates. Image analysis was accomplished by using Image J and GraphPad program. For the data presented in Fig. 4d-k, the aliquots of three repeats of distinct samples are loaded on the TLC plates and the measurements of the three parallel spots are used for statistical analysis. For Extended Data Fig. 9e, the data presented are mean values of three independent repeats of TLC experiments with distinct samples.
Computational modeling analysis
The model of SaMprF is constructed through the Modeller 9.23 program38 by using the cryo-EM structure of RtMprF as the template and the amino acid sequence alignment data of the two homologs as the other input. Virtual docking of daptomycin molecule on SaMprF was carried out through the Autodock Vina program (v1.1.2)56 by providing the homologous model of SaMprF and the structure of daptomycin downloaded from the Protein Data Bank (PDB code: 1T5M). A cubic box with 60 ´ 60 ´ 60 grid points (in the x, y and z dimensions) and 0.375 Å spacing was applied to define the search region on the SaMprF model during the Autodock analysis.