Structural and mechanistic insights into fungal β-1,3-glucan synthase FKS1 – Nature

Expression and purification of FKS1

A 3× Flag tag was engineered to the C terminus of chromosomal FKS1 on S. cerevisiae strain BY4742, using a PCR-based tagging method⁴⁸. A yeast strain with the FKS1(S643P) chromosomal mutation was generated using the homologous recombination method⁴⁹. For cryo-EM analysis and product synthesis, FKS1 and the FKS1(S643P) mutant were purified with detergent GDN as described below. The strains were cultured in YPD medium at 30 °C for 20 h. Cells were collected by centrifugation and lysed by French Press in lysis buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl and 2 mM MgCl₂ supplemented with 1 mM phenylmethane sulphonylfluoride (PMSF). The lysate was centrifuged at 15,000g for 30 min. The supernatant was subject to ultracentrifugation at 100,000g for 1 h. The collected membrane pellet was solubilized in buffer 50 mM Tris-HCl pH 7.4, 500 mM NaCl, 2 mM MgCl₂, 10% (v/v) glycerol, 1.5% (w/v) n-dodecyl-β-d-maltopyranoside (DDM; Anatrace), 0.15% cholesteryl hemisuccinate Tris salt (CHS; Anatrace) and protease inhibitor (cOmplete protease inhibitor cocktail; Roche) by gentle agitation for 2 h. Supernatant was collected by centrifugation at 15,000g for 0.5 h and was applied to Anti-Flag M2 affinity gel (Sigma). The gel was then washed with washing buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM MgCl₂ and 0.04% GDN (Anatrace). The target proteins were eluted with washing buffer supplemented with 150 μg ml⁻¹ 3× Flag peptide. The eluate was concentrated and further purified using size-exclusion chromatography (Superose 6 10/300 GL column, GE Healthcare) with buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM MgCl₂ and 0.04% GDN (Anatrace). The central fractions of monodisperse peak were collected and concentrated for cryo-EM grid preparation and product synthesis.

Expression and purification of Rho1

The vector construction of N-terminal 6× His-SUMO-tagged S. cerevisiae Rho1 was transformed into Escherichia coli BL21(DE3). The strains were cultured in LB medium supplemented with 100 µg ml⁻¹ ampicillin at 37 °C till OD₆₀₀ reached approximately 0.6. Protein expression was induced with 0.5 mM isopropyl-β-d-1-thiogalactopyranoside at 18 °C for 20 h. The collected bacteria were resuspended and lysed by French Press in buffer A containing 50 mM Tris-HCl pH 7.4, 300 mM NaCl and 2 mM MgCl₂. After centrifugation, the protein was purified from the supernatant using nickel–nitrilotriacetic acid (Ni–NTA) affinity chromatography. When dialysing into buffer A, 6× His-tagged protease Ulp1 at a 1:200 Ulp1:Rho1 (w/w) ratio was added to remove the 6× His-SUMO tag. The cleaved 6× His-SUMO tag and the protease were removed by another run of Ni–NTA affinity chromatography, and the flow-through was collected, concentrated and subjected to a gel-filtration chromatography (Superdex 200, GE Healthcare) in buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl and 2 mM MgCl₂. The central fractions of the monodisperse peak at 15.6 ml were collected and concentrated to 10 mg ml⁻¹.

Cryo-EM data acquisition

For single-particle cryo-EM analysis, a 3 µl aliquot of the purified FKS1 at a concentration of 5 mg ml⁻¹ was applied to glow-discharged Quantifoil carbon grids (R1.2/1.3 Au, 300 mesh). The grids were blotted for 4 s at 100% humidity and flash frozen in liquid ethane using FEI Vitrobot IV. For the UDP-Glc-incubated FKS1(S643P) sample, FKS1(S643P) purified in GDN (7 mg ml⁻¹) was mixed with UDP-Glc (0.5 mM), supplemented with 0.7 mg ml⁻¹ Rho1, 10 μM GTPγS, 200 μM caspofungin, 0.1% CHAPS and 0.02% CHS. The mixture was incubated at 16 °C for 2 h before freezing the cryo-EM grids. Cryo-EM data were collected on a FEI Titan Krios electron microscopy operated at 300 kV equipped with a Gatan K3 Summit camera positioned after a GIF quantum energy filter. Automated data acquisition was performed with SerialEM or FEI EPU. Micrographs were recorded under super-resolution counting mode at a nominal magnification of ×130,000, in a physical pixel size of 0.92 Å per pixel. Defocus values ranged from −1.2 μm to −3 μm. A total exposure of 1.3 s was dose-fractionated into 32 frames, resulting in a total accumulated dose of 50 e⁻ per Å².

Image processing and 3D reconstruction

The dose-fractionated movies recorded were first aligned and dose-weighted with MotionCor2 (ref. ⁵⁰). CTFFIND4 was then used to determine the contrast transfer function parameters for individual micrographs⁵¹. Low-quality micrographs revealed by manual inspection were excluded from further analysis. Subsequent image-processing steps were performed using RELION-3 (ref. ⁵²).

For the FKS1 dataset, a set of 2,000 particles were manually selected to generate 2D class templates for reference-based automatic particle picking. The automatic picking yielded in 2,321,905 particles from 11,606 micrographs. Two rounds of reference-free 2D classification were performed to remove particles with poor quality, resulting in a cleaned set of 1,335,014 particles. An ab initio map was generated with RELION and was used as the initial reference model for further 3D classification. After three rounds of 3D classification, a 3D class with high-resolution features (267,574 particles) was selected. Subsequent particle polishing, 3D refinement and post-processing generated a map with an overall resolution of 3.4 Å.

For the FKS1(S643P) dataset, a set of approximately 2,000 particles were manually selected to generate 2D class templates for reference-based automatic particle picking. The automatic picking yielded in 2,222,315 particles from 9,623 micrographs. Two rounds of reference-free 2D classification were performed to remove particles with poor quality, resulting in a cleaned set of 1,620,308 particles. The FKS1 model was used as the initial reference model for further 3D classification. A 3D class with high-resolution features (690,251 particles) was selected. Subsequent particle polishing, 3D refinement and post-processing generated a map with an overall resolution of 3.6 Å. This reconstruction revealed fragment density within the product translocation channel, indicating a mixed state with or without bound product. Therefore, we performed an additional round of focused 3D classification without particle realignment, using a focused mask around TM7–12 and the active site that surround the translocation channel. A 3D class with highest resolution features (176,682 particles) was selected. The selected particles were re-extracted for 3D refinement with full mask and subsequent post-processing. This generated a map with an overall resolution of 3.5 Å, which was used to build the FKS1(S643P) model.

The overall resolutions were estimated based on the gold-standard Fourier shell correlation 0.143 criterion⁵³. Local resolution distribution was estimated using ResMap⁵⁴.

Model building and refinement

A rough initial model of FKS1 was generated de novo using the map_to_model module in PHENIX⁵⁵. It was further improved by manual adjustments and rebuilding using Coot⁵⁶, which was facilitated by the good densities around the TM helices and bulky densities around residues such as Trp, Tyr, Phe and Arg. The refinement of the FKS1 model against the cryo-EM map in real space was carried out in PHENIX with secondary structure and geometry restraints⁵⁵. In the final FKS1 model, there are several unstructured regions, including N-terminal 145 residues, C-terminal 16 residues, six flexible segments of cytoplasmic domain (residues 244–278, 475–487, 798–805, 897–931, 1159–1167 and 1247–1266) and four loop segments to connect TM helices (residues 1419–1435, 1516–1554, 1627–1637 and 1698–1723). The model of FKS1(S643P) was built based on the model of FKS1. MOLPROBITY was used to assess the final model⁵⁷. The Fourier shell correlation between the model and the map was calculated by PHENIX.mtriage⁵⁸. The homologous structure search was performed using the DALI server⁵⁹. For structural comparison with FKS1, the AlphaFold-modelled structure of curdlan synthase was used^60,61. The illustrated figures were prepared using PyMOL (Schrödinger, LLC), Chimera and ChimeraX^62,63. Statistics of the 3D reconstructions and model refinements are shown in Extended Data Table 1.

Spot growth assay

Yeast strains with FKS1 chromosomal mutations and the FKS1-KO strain were generated using the homologous recombination method⁴⁹. The mutants were selected on SD-His (Yeast Synthetic Drop-out Medium without Histidine, cat no. S0020, Solarbio) and were confirmed by genomic PCR and DNA sequencing. For the analysis of growth phenotype, the spot growth assay of mutated strains was adapted from a previously described method²⁴. The strains were cultured in YPD media at 30 °C at 200 rpm to exponential phase until OD₆₀₀ was 0.8. The cells were diluted to an OD₆₀₀ of 0.1. Then, 5 μl portions of tenfold dilutions of the indicated strains were spotted on SD-His with or without FK506 (1 μg ml⁻¹). After incubation at 30 °C for indicated time, plates were photographed with the 5200CE Image System (TANON). Each assay was repeated three times with similar results.

Growth curve analysis

To profile the cell growth, the WT strain and the FKS1-KO strain were grown in YPD media at 30 °C overnight to exponential phase. Then, cultured cells were inoculated into the fresh YPD medium with an initial OD₆₀₀ of 0.01, which was supplemented with or without 1 μg ml⁻¹ FK506. The new cultures were then grown at 30 °C till stationary phase. During the cultivation, the optical densities (OD₆₀₀) were measured at 8 h interval for 48 h. The experiments were done in triplicate to make the cell growth curves.

FKS1 activity assay

The yeast strains carrying different FKS1 variants with the C-terminal 3× Flag tag were generated, cultured and lysed as described above. The collected membrane was solubilized in buffer containing 50 mM Tris-HCl pH 7.4, 1 mM EDTA, 33% glycerol, 0.5% CHAPS (Anatrace), 0.1% CHS (Anatrace), 4 µM GTPγS and protease inhibitor (cOmplete protease inhibitor cocktail, Roche). FKS1 and its variants were purified using anti-Flag M2 affinity gel (Sigma) and were eluted in buffer containing 50 mM Tris-HCl pH 7.4, 1 mM EDTA, 33% glycerol, 0.2% CHAPS and 0.04% CHS, supplemented with 150 μg ml⁻¹ 3× Flag peptide. The proteins purified in detergent CHAPS or GDN were assayed by using the UDP-Glo Glycosyltransferase Assay kit (Promega) to monitor the UDP released. Of FKS variants, 2.5 µl was added to 30 μl reaction mixture in total containing 50 mM Tris-HCl pH 7.4, 33% glycerol, 1 mM EDTA, 6 μg ml⁻¹ Rho1, 0.2% CHAPS, 0.04% CHS, 4 µM GTPγS and 20 mM potassium fluoride (KF). The reaction was initiated by adding UDP-Glc to a final concentration of 2.5 mM. The reaction was carried out at 30 °C for 1 h. Luminescence was recorded using a Pherastar FS system (BMG Labtech). The final activity was normalized to the FKS1 amount that was measured by immunoblotting against the 3× Flag tag, using the DYKDDDDK tag monoclonal antibody (1:1,000 dilution; cat no. 66008-3-Ig, clone no. 2B3C4, Proteintech). In the case of inhibitor profiling, serial dilutions of echinocandin drugs were first incubated with FKS1 variants for 10 min at room temperature before adding other components to react.

In vitro synthesis of β-1,3-glucan and aniline blue staining

For in vitro β-1,3-glucan synthesis, the enzyme purified in detergent GDN (FKS1 or the FKS1(S643P) mutant; 0.02 mg ml⁻¹) and donor UDP-Glc (2.5 mM) were mixed in reaction buffer (50 mM Tris-HCl pH 7.4, 33% glycerol, 1 mM EDTA, 6 μg ml⁻¹ Rho1, 0.2% CHAPS, 0.04% CHS, 4 µM GTPγS and 20 mM KF), in the presence or absence of 200 μM caspofungin. The reaction volume was set up as 100 μl (for aniline blue staining) or 10 ml (for product enrichment and subsequent enzymatic degradation and glycosyl linkage analysis). The reaction was carried out at 30 °C for indicated time period.

For the staining of the synthesized products by aniline blue, a 100 μl aliquot of reactants or 0.1% (w/v) of S. cerevisiae β-glucan (Sigma) was taken and added to equal volume of aniline blue (0.03%; Sigma). The mixture was incubated in the dark for 20 min for complete dye binding. Each sample was then loaded into a capillary and was observed under a fluorescence microscope with an excitation wavelength of 365 nm and an emission wavelength of 433 nm. Each assay was repeated three times with similar results.

Enzymatic degradation analysis of the synthesized polymer

The in vitro synthesized polymer is water-insoluble and was collected by centrifugation. It was washed twice with buffer: 50 mM Tris-HCl pH 7.4, 33% glycerol, 1 mM EDTA, 0.2% CHAPS, 0.04% CHS, 20 mM KF and twice with deionized water. Two enzymes were used for the degradation analysis of the synthesized polymer: endo-β-1,3-glucanase (Trichoderma sp.; product code: E-LAMSE, Megazyme) or endo-β-1,4-glucanase (Aspergillus niger; product code: E-CELAN, Megazyme). These two enzymes were first dialysed into the buffer (100 mM NaAc pH 4.5). Then, 0.5 U of each enzyme was added to 50 µl 1% (w/v) of synthesized polymer in 200 mM NaAc (pH 4.5). Degradation of standard controls cellulose (C6288, Sigma) or curdlan (C7821, Sigma) was also performed with the same condition^64,65. The mixtures were incubated at 40 °C, and samples were withdrawn at various time intervals and were boiled for 5 min for reaction termination. The enzymatic degradation products were then analysed by thin-layer chromatography. In brief, a 2 µl aliquot of the withdrawn samples was spotted on a silica gel plate (Merck Silica gel 60 F254) and developed in a solvent system containing n-butanol:acetic acid:water (2:1:1 v/v/v). The plates were visualized by immersing in methanol:sulfuric acid (95:5 v/v) and subsequent heating at 95 °C. A mixture of glucose (G5767, Sigma), laminaribiose (O-LAM2, Megazyme), laminaritriose (O-LAM3, Megazyme) and laminarihexaose (O-LAM6, Megazyme) was used as the thin-layer chromatography standards.

During enzymatic digestion of synthesized polymer, the reducing sugars released were measured using the DNS reagents (Micro Reducing Sugar Assay Kit, Abbkine). In brief, 175 μl of the diluted hydrolysis sample was mixed with 125 μl of DNS reagent. Standard from the kit at the concentration range of 0.2–0.6 mg ml⁻¹ was used for the generation of the standard curve. Reaction mixtures were boiled in a water bath for 5 min. After cooling to room temperature in a water-ice bath, the absorbance of a 0.2 ml sample was measured at 540 nm.

Glycosyl linkage (methylation) analysis

The in vitro synthesized polymer by FKS1 was washed twice with buffer: 50 mM Tris-HCl pH 7.4, 33% glycerol, 1 mM EDTA, 0.2% CHAPS, 0.04% CHS, 20 mM KF and two times with deionized water. The washed sample was then dialysed into deionized water and freeze-dried. Methylation analysis was performed following a previous study⁶⁶. The dried sample (approximately 1 mg) was dissolved in DMSO (500 μl). The methylation was performed in DMSO/NaOH with iodomethane for 1 h. The methylated products were hydrolysed with 2 M trifluoroacetic acid at 121 °C for 90 min, reduced by NaBD4 and acetylated with acetic anhydride at 100 °C for 2.5 h. The resulting partially methylated alditol acetates were analysed using the GC-MS system (6890A-5975C, Agilent Technology) equipped with an Agilent BPX70 chromatographic column (30 m × 0.25 mm × 0.25 µm; SGE). The temperature program was set as follows: 140 °C for 2 min, 140–230 °C at 3 °C per minute and 230 °C for 3 min.

Quantitative determination of β-1,3-glucan in cell walls

The levels of β-1,3-glucan were determined using the aniline blue assay, as previously described^{23,26,67,68,69,70,71,72}. Testing strains were grown in YPD media to exponential phase until OD₆₀₀ was 0.5. The same amount of cells for each strain was collected by centrifugation at 5,000g. The collected cells were washed twice with TE buffer (10 mM Tris-HCl pH 8.0, and 1 mM EDTA). The pelleted cells were suspended with 0.5 ml TE buffer and mixed with 0.1 ml of 6 M NaOH. The mixture was incubated in an 80 °C water bath for 30 min to solubilize the glucan. Then, 2.1 ml of aniline blue (0.03% aniline blue, 0.18 M HCl and 0.49 M glycine-NaOH pH 9.5) was added to each sample. The samples were briefly vortexed and incubated at 50 °C for 30 min and an additional 30 min at 24 °C. Fluorescence was quantified using a fluorescence plate reader (CLARIOstar Plus, BMG Labtech) under an excitation wavelength of 400 nm and an emission wavelength of 460 nm with a cut-off of 455 nm. All samples were measured in triplicates.

Protein identification with mass spectrometry analysis

The protein samples were separated by SDS–PAGE gel and were stained with Coomassie. The gel band was manually excised and destained. The proteins were reduced with DTT, alkylated with IAA and digested with proteomics-grade trypsin in 20 mM ammonium bicarbonate⁷³. The digestion was performed overnight at 37 °C and stopped by adding 2% formic acid. The peptides in gel were extracted using solution containing 2% formic acid and 67% acetonitrile. The peptides were vacuum-dried, resuspended in 0.1% formic acid, loaded onto the trap column nanoViper C18 (3 μm, 100 Å) and separated on an analytic column (Acclaim PepMap RSLC, 75 μm × 25 cm; C18 2 μm, 100 Å) using the EASY nLC 1200 HPLC system (Thermo Fisher). The elution gradient was 5–38% buffer A (0.1% formic acid and 80% acetonitrile) over 30 min. The mass spectrometry analysis was performed on a Q Exactive mass spectrometer (Thermo Fisher). The resulting data were converted to mgf file with ProteoWizard and analysed using the Mascot search engine for protein identification against a UniProt Saccharomyces cerevisiae database with a false discovery rate of less than 1%.

Reporting summary

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