Structural basis for inhibition and regulation of a chitin synthase from Candida albicans.

Ca Chs2 protein expression and purification. The full-length CHS2 gene from C. albicans was synthesized with codon-optimization for Spodoptera frugiperda 9 (Sf9) cells and cloned into a modified pFastBac vector (Invitrogen), in frame with C-terminal PreScission protease cite, FLAG-tag, and 10x His-tag. Baculovirus was generated according to the manufacturer’s protocol (Bac-to-Bac, Invitrogen). All mutants were made on wild-type Chs2 construct by site-directed mutagenesis using KOD hot start DNA polymerase (Novagen). For Ca Chs2 expression, Sf9 insect cells were infected with a baculovirus at a density of 2.5–3 M cells ml −1 and grown at 27 °C for ~48 h in an orbital shaker. Cells were then harvested by centrifugation at 2,500 g for 10 min at 4 °C. Cell pellets were resuspended in buffer A (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, 2 mM β-mercaptoethanol (BME), 12 μg ml −1 leupeptin, 12 μg ml −1 pepstatin, 12 μg ml −1 aprotinin, 1 mM PMSF, and DNaseI) and lysed by sonication (5 × 30 pulses). All Ca Chs2 purification steps were performed at 4 °C. To solubilize Ca Chs2, 30 mM n-dodecyl-β-D-maltopyranoside (DDM, Anatrace) and 3 mM cholesteryl hemisuccinate tris salt (CHS, Anatrace) was added to the lysate and stirred at 4 °C for 1.5 h. Insoluble material was removed by centrifugation (16,000 g , 30 min), and anti-FLAG M2 resin (Sigma-Aldrich) was added to the supernatant and incubated for 1 h at 4 °C. The resin was then washed with 10 column volumes of buffer B (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, 0.04% glyco-diosgenin (GDN, Anatrace), 2 mM BME, 5 mM ATP-Mg), followed by 10 column volumes of buffer C (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, 0.04% GDN, 2 mM BME). Ca Chs2 was eluted with five column volumes of elution buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, 0.04% GDN, 2 mM BME, 0.2 mg ml −1 FLAG peptide (GenScript)). The FLAG and 10x His affinity tags were removed by incubation with PreScission protease at room temperature for 15 min. Ca Chs2 was further purified by size-exclusion chromatography (Superose 6 Increase 10/300 GL column (Cytiva)) with SEC buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl, 0.015% GDN, 2 mM DTT. Ca Chs2 mutants were purified similarly but without tag cleavage by PreScission protease. The size-exclusion chromatography buffer for Ca Chs2 mutants is 20 mM HEPES pH 7.5, 150 mM NaCl, 0.015% GDN, 0.2 mM tris(2-carboxyethyl) phosphine (TCEP, Thermo Scientific). Enzymes for activity assays were flash-frozen in liquid nitrogen with the addition of 30% glycerol. Cryo-electron microscopy sample preparation and data collection. Peak fractions containing Ca Chs2 from the size-exclusion chromatography were concentrated to 3–5 mg ml −1 . All cryo-EM samples in this study were prepared on freshly glow-discharged UltrAuFoil R1.2/1.3 300 mesh grids (Quantifoil), using a Leica EM GP2 plunge freezer with the chamber set at 4 °C and 80% humidity. For the apo- Ca Chs2 sample, 3 μl of Ca Chs2 was applied to the grid, incubated in the chamber for 90 s, and then blotted for 1.5–2 s, followed by plunge-freezing in liquid ethane cooled by liquid nitrogen. For the Ca Chs2-UDP-GlcNAc sample, concentrated Ca Chs2 was mixed with 20 mM MgCl 2 and 20 mM UDP-GlcNAc. Immediately after mixing, 3 μl of the mixture was then applied to the grid, incubated for 60 s, and blotted for 1.5–2 s, followed by plunge-freezing in liquid ethane cooled by liquid nitrogen. For the Ca Chs2-nikkomycin sample, concentrated Ca Chs2 was mixed with 5 mM MgCl 2 and 4 mM nikkomycin Z on ice for 30 min. Then 3 μl of the mixture was applied to the grid, incubated for 90 s, and blotted for 1.5–2 s, followed by plunge-freezing in liquid ethane cooled by liquid nitrogen. For the Ca Chs2-polyoxin sample, concentrated Ca Chs2 was mixed with 5 mM MgCl 2 and 1.5 mM polyoxin D on ice for 1 min. Then 3 μl of the mixture was applied to the grid, incubated for 90 s, and blotted for 1.5–2 s, followed by plunge-freezing in liquid ethane cooled by liquid nitrogen. The apo-Chs2, Chs2-UDP-GlcNAc, Chs2-nikkomycin, and Chs2-polyoxin datasets were collected with a Titan Krios microscope (Thermo Fisher) operating at 300 kV and equipped with a K3 detector (Gatan) in counting mode, using Serial EM or the Latitude-S automated data-acquisition program. Movie datasets were collected at a nominal magnification of ×81,000 with a pixel size of 1.08 Å per pixel at specimen level. Each movie contained 60 frames over a 4.6-s exposure time, using a dose rate of ~15 e − pixel −1 s −1 , resulting in the total accumulated dose of ~60 e − /Å 2 . The nominal defocus range was set from −1 to −2 μm. Cryo-electron microscopy data processing. All datasets were processed using similar procedures. Beam-induced motion correction and dose-weighting were performed using MotionCor2 (ref. 43 ). The motion-corrected micrographs were then subjected to contrast transfer function (CTF) estimation using Gctf 44 . Micrographs were subsequently selected based on CTF fit quality and CTF estimated resolution and then imported into cryoSPARC 45 . An initial set of ~5,000 particles was manually picked and subjected to a reference-free two-dimensional classification, from which the classes with clear protein features were selected as reference for template-based particle picking. Picked particles were extracted with the 216-pixel box size. Iterative two-dimensional classification was performed in cryoSPARC, and classes showing clear secondary structure features of Ca Chs2 were selected for ab initio reconstruction with three classes. Heterogeneous refinement was performed with the models from ab initio reconstruction. The only class that shows clear protein density was selected for non-uniform refinement 46 . After non-uniform refinement, the particles were transferred to RELION 3.1 47 and subject to several rounds of Bayesian polishing and CTF refinement, which improved resolution and map quality. Model building, refinement, and alignment. The apo- Ca Chs2 structure was built de novo in Coot 48 . During model building, residue registration was guided by the presence of large aromatic side chains. The structure was manually refined in Coot with ideal geometry restraints. The ligand-bound Ca Chs2 models used the apo- Ca Chs2 structure as a reference. The restraints for lipids and ligands, including PE, UDP-GlcNAc, nikkomycin Z, and polyoxin D, were generated in eLBOW (as implemented in Phenix 49 ) from isomeric SMILES strings and optimized using the REEL QM2 method. Ligand restraints were then inspected and adjusted manually to ensure correct stereochemistry before being fitted into the cryo-EM maps in Coot. The MolProbity 50 server was utilized to identify problematic regions in the models, which were then manually adjusted in Coot. The final refinement was performed using the phenix-real_space_refine function with global minimization and secondary structure restraints as implemented in the Phenix suite 49 . The Fourier shell correlation of the half- and full-maps against the model, calculated in Phenix, were in good agreement, indicating that the models were not over-refined. Structural analyses and illustrations were performed using PYMOL (Schrödinger) and UCSF Chimera and ChimeraX 51 . Radius calculation was performed using the HOLE program 52 . Structure alignments and cryo-EM density map alignments were performed using Fit-In-Map in UCSF Chimera. Based on the aligned map, each structural model was aligned to its corresponding map by Fit-In-Map. Isothermal titration calorimetry. Protein samples were purified as described above and concentrated to around 7–10 μM. Ca Chs2 was in the ITC buffer that contains 20 mM HEPES pH 7.5, 150 mM NaCl, 0.015% GDN, and 0.2 mM TCEP. The ITC measurements were performed with a Nano ITC microcalorimeter (TA Instruments) at 25 °C. For Ca Chs2 WT and Q643A, nikkomycin Z stock at 100 μM was prepared in the same ITC buffer and injected 19 times (0.4 μl for injection 1 and 2 μl for injections 2–19), with 150–250 s intervals between injections. For Ca Chs2 W647A, 300 μM nikkomycin Z was used with the same titration protocol. The background data obtained from injecting nikkomycin into the ITC buffer were subtracted before the data analysis. The data were fitted using NanoAnalyze v3.11.0 (TA Instruments). Cysteine crosslinking. Cysteine mutations are engineered and expressed as described above. After the cells were harvested, cell membrane was prepared under non-reducing conditions. Cells were resuspended in low salt buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 12 μg ml −1 leupeptin, 12 μg ml −1 pepstatin, 12 μg ml −1 aprotinin, 1 mM PMSF, and DNaseI) and lysed by osmosis pressure. Cell membrane was collected by centrifugation and homogenized in high salt buffer (50 mM Tris-HCl, pH 8.0, 1 M NaCl, 12 μg ml −1 leupeptin, 12 μg ml −1 pepstatin, 12 μg ml −1 aprotinin, 1 mM PMSF, and DNaseI). Then cell membrane was collected by centrifugation and resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol). The membrane was first incubated with 20 mM N-ethylmaleimide for 10 min at room temperature and then purified as described above in the absence of reducing reagent and subjected to SDS–PAGE analysis. Ca Chs2 activity assay. Aliquots of the flash-frozen enzyme were thawed on ice immediately before the assay. Typical reactions were performed by adding one volume of enzyme to one volume of 2x assay mixture (4 mM UDP-[U- 14 C]GlcNAc (specific activity ~1,000 c.p.m. nmol −1 ), 100 mM Tris-HCl pH 7.5, 64 mM GlcNAc, 10 mM MgCl 2 ). Reactions were incubated at 30 °C, and at each time point, an aliquot (5 μl) was quenched in 10% TCA (w/v). The insoluble product was filtered through a glass filter and washed five times with 70:30 ethanol:1 M acetic acid in water. The radioactivity retained on the filter was analyzed on a liquid scintillation counter with 3 ml of scintillation fluid. Steady-state kinetic analyses were performed in triplicate from two biological replicates. Ca Chs2 inhibition assays. Ca Chs2 assays were performed as described above, except that the assay contained specified concentrations of nikkomycin Z or polyoxin D. The antifungal stock solutions were prepared in 10 mM ammonium acetate pH 6.0 to prevent hydrolysis of the amide bond and were diluted five- or tenfold in the final assay mixture. Fractional activities of each reaction were plotted and fit to the four-parameter logistic equation to determine the IC 50 values. Lipid mass spectrometry. Extraction of lipids copurified with Chs2 protein. Lipids copurified with the Ca Chs2 protein sample were extracted using the method of Bligh and Dyer as previously described 53 . PBS was added to the Chs2 protein solution in a glass tube to a final volume of 1.6 ml. Subsequently, 2 ml of chloroform and 4 ml of methanol were added to make the single-phase Bligh-Dyer mixture comprising chloroform/methanol/PBS (1:2:0.8, v/v/v). This mixture solution was subjected to sonic irradiation in a bath apparatus for 5 min. The subsequent single-phase extraction mixture was then centrifuged at 500 g for 10 min using a clinical centrifuge and the protein precipitate was pelleted. The supernatant was transferred to a fresh glass tube where chloroform (2 ml) and PBS (2 ml) were added to generate the two-phase Bligh-Dyer mixture composed of chloroform/methanol/PBS (2:2:1.8, v/v/v) 53 . After mixing and centrifugation (500 g ) for 10 min, the upper phase was removed, and the lower phase was dried under a nitrogen stream. The dried lipid extract was stored at −20 °C until LC–MS analysis. Lipid identification by liquid chromatography–mass spectrometry/mass spectrometry. Normal phase LC was performed on an Agilent 1200 Quaternary LC system equipped with an Ascentis Silica HPLC column, 5 μm, 25 cm × 2.1 mm (Sigma-Aldrich, St Louis, MO) as described previously 54 . Mobile phase A comprised chloroform/methanol/aqueous ammonium hydroxide (800:195:5, v/v/v); mobile phase B comprised chloroform/methanol/water/aqueous ammonium hydroxide (600:340:50:5, v/v/v); mobile phase C comprised chloroform/methanol/water/aqueous ammonium hydroxide (450:450:95:5, v/v/v). The elution program was as follows: 100% mobile phase A was held for 2 min, followed by a linear increase to 100% mobile phase B over 14 min, and then held at 100% B for 11 min. The LC gradient was changed to 100% mobile phase C over 3 min, held at 100% C for 3 min, returned to 100% A over 0.5 min, and held at 100% A for 5 min (ref. 54 ). The LC eluent (with a total flow rate of 300 μl/min) was introduced into the ESI source of a high resolution TripleTOF5600 mass spectrometer (Sciex, Framingham, MA). Instrumental settings for negative ion ESI and MS/MS analysis of lipid species were as follows: IS = −4500 V; CUR = 20 psi; GSI = 20 psi; DP = −55 V; and FP = −150 V as described 54 . The MS/MS analysis used nitrogen as the collision gas. Data analysis was performed using Analyst TF1.5 software (Sciex, Framingham, MA). Fourier-transform infrared spectroscopy. Chitin from Ca Chs2 was prepared as described in the Ca Chs2 activity assay section in a 100 μl scale with 10 mM UDP-GlcNAc for 20 h. The cloudy reaction mixture was quenched by adding 100 μl of a 5% (w/v) SDS solution, and the insoluble chitin was collected by centrifuging for 15 minutes at 20,000 g . The pellet was washed with 5 × 200 μl of 2% (w/v) SDS, water, and ethanol. The pellet was then dried by speedvac for 30 min. The IR spectra were collected (128 scans, 4 cm −1 resolution) on a Perkin-Elmer Frontier instrument with a Pike MIRacle attenuated total reflectance (ATR) optic and a Mercury-Cadmium-Telluride (MCT) detector. Before collecting sample spectra, baseline scans of air were taken and subtracted automatically from the sample spectra in the Spectrum software (Perkin-Elmer). Sample spectra were collected by placing the dried chitin samples directly on the ATR crystal. Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The coordinates are deposited in the Protein Data Bank with the PDB IDs 7STL (apo), 7STM (UDP-GlcNAc-bound), 7STN (nikkomycin Z-bound state) and 7STO (polyoxin D-bound state), respectively. The cryo-EM density maps are deposited in EMDB with the IDs EMD-25432 (apo), EMD-25433 (UDP-GlcNAc-bound), EMD-25434 (nikkomycin Z-bound state) and EMD-25435 (polyoxin D-bound state), respectively. Source data are provided with this paper.

Supplementary Material Structural basis for inhibition and regulation of a chitin synthase from Candida albicans SI