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

Nature volume 616, pages 190–198 (2023)Cite this article

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Abstract

The membrane-integrated synthase FKS is involved in the biosynthesis of β-1,3-glucan, the core component of the fungal cell wall1,2. FKS is the target of widely prescribed antifungal drugs, including echinocandin and ibrexafungerp3,4. Unfortunately, the mechanism of action of FKS remains enigmatic and this has hampered development of more effective medicines targeting the enzyme. Here we present the cryo-electron microscopy structures of Saccharomyces cerevisiae FKS1 and the echinocandin-resistant mutant FKS1(S643P). These structures reveal the active site of the enzyme at the membrane–cytoplasm interface and a glucan translocation path spanning the membrane bilayer. Multiple bound lipids and notable membrane distortions are observed in the FKS1 structures, suggesting active FKS1–membrane interactions. Echinocandin-resistant mutations are clustered at a region near TM5–6 and TM8 of FKS1. The structure of FKS1(S643P) reveals altered lipid arrangements in this region, suggesting a drug-resistant mechanism of the mutant enzyme. The structures, the catalytic mechanism and the molecular insights into drug-resistant mutations of FKS1 revealed in this study advance the mechanistic understanding of fungal β-1,3-glucan biosynthesis and establish a foundation for developing new antifungal drugs by targeting FKS.

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Fig. 1: Functional and structural characterization of S. cerevisiae FKS1.
Fig. 2: The FKS1 TM ___domain and structural comparison with the cellulose synthases BcsA, CesA and the hyaluronan synthase HAS.
Fig. 3: The active site of FKS1.
Fig. 4: Putative glucan translocation path.
Fig. 5: Structural interpretation of echinocandin-resistant or ibrexafungerp-resistant mutations.

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Data availability

EM density maps have been deposited in the Electron Microscopy Data Bank under the accession codes EMD-33154 (FKS1) and EMD-34115 (FKS1(S643P)). The coordinates have been deposited in the PDB under the accession codes 7XE4 (FKS1) and 7YUY (FKS1(S643P)). This study analysed several protein structures publicly available from the PDB under the accession codes 4HG6, 6WLB, 7SP7, 6YV8, 6iwr, 6h4m and 1qgq.

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Acknowledgements

We thank all staff members at the Cryo-EM Center, Southern University of Science and Technology for their assistance in data collection; and J. Zhao for advice on thin-layer chromatography. This work was supported by the National Natural Science Foundation of China (92053112 and 31971148 to H.Y., 32100575 to Min Zhang and 82188101 to Mingjie Zhang), ShenZhen Talent Program KQTD20210811090115021 (to Mingjie Zhang and X.L.), the Fundamental Research Funds for the Central Universities (5003510056 to H.Y. and 5003510112 to Min Zhang) and Program of HUST Academic Frontier Youth Team (2018QYTD02 to H.Y.).

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  1. These authors contributed equally: Xinlin Hu, Ping Yang

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    Xinlin Hu, Ping Yang, Changdong Chai, Jia Liu, Huanhuan Sun, Yanan Wu, Min Zhang & Hongjun Yu

  2. Department of Pathogen Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    Xinlin Hu, Jia Liu & Min Zhang

  3. School of Life Sciences, Southern University of Science and Technology, Shenzhen, China

    Mingjie Zhang & Xiaotian Liu

  4. Greater Bay Biomedical Innocenter, Shenzhen Bay Laboratory, Shenzhen, China

    Mingjie Zhang

  5. Cell Architecture Research Center, Huazhong University of Science and Technology, Wuhan, China

    Hongjun Yu

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Contributions

X.H., P.Y., Min Zhang, X.L. and H.Y. prepared the sample, collected the data and solved the structures. X.H., P.Y. and Min Zhang performed the functional experiments, assisted by C.C., J.L., H.S. and Y.W. Mingjie Zhang, Min Zhang, X.L. and H.Y. designed the experiments, analysed the data and wrote the manuscript.

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Correspondence to Min Zhang, Xiaotian Liu or Hongjun Yu.

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Extended data figures and tables

Extended Data Fig. 1 Sample preparation, functional characterization and in vitro product synthesis.

a, Elution profile of Gel-filtration chromatography (Superose 6 Increase 10/300 GL) of FKS1 purified in detergent GDN. Green dashed box marks the fractions pooled for cryoEM analysis. b, SDS-PAGE (left) and western blot (right) analysis of the fractions of the monodisperse elution peak in (a). Representative gel and western-blot image among 3 replicates are shown. c, Elution profile of Gel-filtration chromatography (Superose 6 Increase 10/300 GL) of FKS1 S643 purified in detergent GDN. Green dashed box marks the fractions pooled for cryoEM analysis and product synthesis. d, SDS-PAGE analysis of the fractions of the monodisperse elution peak in (c). A representative gel among 3 replicates is shown. e, Elution profile of Gel-filtration chromatography (Superdex 200 Increase 10/300 GL) of Rho1. Green dashed box marks the fractions pooled for activity assay and product synthesis. f, SDS-PAGE analysis of the fractions of the monodisperse elution peak in (e). Two peaks in (e) shows similar three-band pattern (f), and all three bands were identified as Rho1 by Mass Spec analysis. A representative gel among 3 replicates is shown. g, Western blot analyses of wild-type FKS1 and different FKS1 variants purified in detergent CHAPS. A representative image among 3 replicates is shown. h, SDS-PAGE analysis of GDN-purified flag-tagged FKS1 variants, which were used for the assays of activity (i-j) and product synthesis (k). First lane, FKS1 WT; second lane, FKS1 S643P (the echinocandin-resistant mutant); third lane, purified FKS1 S643P is immunodepleted by anti-flag beads; fourth lane, FKS1 S643P/K1261A. A representative gel among 3 replicates is shown. For (b,d,f,g,h), their full scans are provided in Supplementary Fig. 1. i, In vitro activity of FKS1 variants purified in GDN. The activity was assayed by monitoring UDP generated (1-h reaction). Data are mean ± SEM, n = 3 independent experiments. j–k, In vitro product synthesis assay by FKS1 variants purified in GDN. The assay was performed either by staining the synthesized products with dye aniline blue (j; 12-h reaction) or by visualization of the products synthesized (k; 48-h reaction). These experiments were repeated three times with similar results. l, Donor specificity of product synthesis by GDN-purified FKS1 S643P. Water-insoluble polymers (indicated by white arrow) appear only in the presence of UDP-Glc instead of UDP-GlcNAc. This experiment was repeated three times with similar results. m, The effects of Mg2+ on the catalytic activity of GDN-purified FKS1 S643P. Activity was assayed in the presence of 1 mM EDTA or 200 μM Mg2+. Data are mean ± SEM, n = 3 independent experiments. Inset, product synthesis in the presence of 1 mM EDTA or 200 μM Mg2+. This experiment was repeated three times with similar results.

Extended Data Fig. 2 Enzymatic hydrolysis and glycosyl linkage (methylation) analysis of FKS1-synthesized products.

a, Quantification of the released reducing sugars during enzymatic digestion of the water-insoluble polymer synthesized by GDN-purified FKS1 S643P. Two specific glucanases were used: specific endo-1,3-β-glucanase and endo-1,4-β-glucanase. Data are mean ± SEM, n = 3 independent experiments. b–c, The hydrolysis specificity of endo-1,3-β-Glucanase and endo-1,4-β-Glucanase was tested against standard polysaccharide β-1,3-glucan curdlan (b) and cellulose (c). A mixture of glucose (G1), laminaribiose (G2), laminaritriose (G3) and laminarihexaose (G6) was used as the standards (lane M). d, Mass spectrum of GC peak (11.64min) in (Fig. 1f) reveals the identity of this PMAA as 1,3,5-tri-O-acetyl-2,4,6-tri-O-methyl glucitol, confirming 1,3-Glcp linkage.

Extended Data Fig. 3 Cryo-EM structural determination of S. cerevisiae FKS1 and structural features of FKS1 cytoplasmic region.

a, A representative cryo-EM micrograph of FKS1 from 11606 collected micrographs. b, Representative 2D class averages. c, Flow chart of cryo-EM data acquisition and data processing of FKS1. See Methods for more details. d, The gold-standard Fourier shell correlation (FSC) curve of the reconstructed map. e, The FSC curve between model and map, which is calculated by PHENIX.mtriage58. f, Cutaway views of the angular distribution of all particles used in the final 3D reconstruction. g, Local resolution distribution of the final cryo-EM map of FKS1, calculated with ResMap54. h, Topological diagram of FKS1 GT-___domain. Flexible segments invisible in the 3D reconstruction are depicted by dashed lines. i, Interactions between AC and GT ___domain. Labeled are central β strands and the structural elements involved in the extensive contacting interface: α2-5, α8-11, loops Lα9-α10 and Lα11-α12 of AC ___domain; α31, α34-35, loops Lβ5-β6, Lβ7-β8; Lβ8-β9 of GT ___domain). The FKS1 R319, a previously identified essential residue for FKS124, is marked by its Cα atom as sphere and is embedded in this interface. j, Zoomed in view of the boxed region in (i), showing the interaction between R319 and N1087 to maintain the interface between AC and GT ___domain.

Extended Data Fig. 4 Fit of cryo-EM map with FKS1 model in example regions.

a, Cryo-EM densities of TM1-8, 11–17. b-c, Cryo-EM densities of the continuous central β-sheet of 9 strands featured in FKS1 GT ___domain. d, Cryo-EM density of the N-glycan on N1849. e, Cryo-EM densities of active site. f, Cutaway view of the cryo-EM densities near TM9 and TM10. The map is segmented and colored as in Fig. 2a. The densities corresponding to TM helices TM7-12, TM14-16 are denoted by numbers. Only TM9-10 main chain can be traced from this map, though they exhibit weaker densities than other TM helices. g–h, Two views of FKS1 model with bound lipids. FKS1 model is colored as Fig. 1h and ordered lipids are shown as yellow spheres. The cryo-EM densities of the lipids are illustrated in the dashed boxes. The position and the density of the phospholipid embedded within the transmembrane ___domain is shown in the solid box.

Extended Data Fig. 5 Structure and sequence comparison of the conserved cellulose-synthase-like folds between FKS1 and BcsA.

a, The conserved fold of FKS1 (residues 615–1510) was superposed with that of BcsA (PDB ID: 4P00; residues 63–584), with an RMSD value of 4.3 Å over 308 aligned residues. BcsA is colored in grey. FKS1 core is colored in orange while FKS1-exclusive elements in its GT ___domain are in color-coded display as labeled in (a) and (b). Corresponding TM helices are denoted as labels while TM9 and TM10 are exclusive for FKS1. b, Close-up view of the superimposed GT domains in (a). The central β-sheet featured in GT ___domain gains extension in FKS1. The extended stands β6 and β7 in FKS1 and the elongated loop Lβ8-β9 contribute to a substantial portion of the interacting interface with AC ___domain in FKS1 (Fig. 1j). Moreover, two FKS1-specific sub-regions inserts to two sides of β3, the first strand of central β-sheet. The sub-region preceding β3 (residues 719–840) contains six helices and a pair of antiparallel β-strands (β1, β2); the sub-region ensuing β3 (residues 848–998) is α-helical, composed of 9 helices. These two sub-regions interact to pack the central β-sheet from one side. c, Back view of the superimposed transmembrane domains. d, Close-up view of superimposed membrane-cytosol interfaces. Interface helix IF1 is preserved in FKS1, which shows dramatic rearrangement relative to that of BcsA. Interface helix IF2 connecting a beta-sheet and transmembrane ___domain is preserved in FKS1 but disordered in the structure, which is also indicated in (c). Interface helix IF3 is gone in FKS1 and it is replaced by transmembrane helices TM9 and TM10 in FKS1. e, Structure-based sequence alignment of the conserved cellulose-synthase-like folds between FKS1 and BcsA. The alignment was first generated using PROMALS3D and then illustrated using ESPript3 server. Residues and motifs with important functions are labeled. Shown above and below the alignment are secondary structural elements derived FKS1 structure (blue symbol) and BcsA structure (PDB ID: 4P00) (orange symbol), respectively. The symbols are TM helices (filled bar), helices (empty bar) and β strands (arrow).

Extended Data Fig. 6 Overlay of the GT catalytic domains of the structural matches to FKS1 by DALI analysis.

The analyzed GT catalytic domains are overlaid and shown separately as cartoon. Residues involved in substrate binding or catalysis are indicated as sticks. The bound nucleotide is shown as thin magenta sticks. The conserved residues are highlighted as yellow when using the active site of bacterial cellulose synthase BcsA (a) as reference. a–f, Overlay of GT catalytic domains of membrane-bound glycosyltransferases: bacterial cellulose synthase BcsA (a; PDB ID: 4hg6), β-1,3-glucan synthase FKS1 (b), plant cellulose synthase CesA (c; PDB ID: 6wlb), virus hyaluronan synthase (d; PDB ID: 7sp7), Agrobacterium curdlan synthase (e; model from AlphaFold Database; Uniprot ID: Q9X2V0), archaeal mannosyltransferase PcManGT (f; PDB ID: 6YV8). The panels outlined by blue box (a–e) are membrane-bound polysaccharide synthases containing cellulose-synthase-like fold as shown in Fig. 2f–m. PcManGT (panel f) contains half membrane tunnel of BcsA, and it was proposed to have a minimal cellulose-synthase-like fold35. g–i, Overlay of GT catalytic domains of soluble glycosyltransferases: human GalNAc-T7 (g; PDB ID: 6iwr), S. aureus TarP (h; PDB ID:6h4m), B. subtilis SpsA (i; PDB ID: 1qgq).

Extended Data Fig. 7 Cryo-EM analysis of FKS1 S643P, a drug-resistant mutant of the enzyme.

a, A representative cryo-EM micrograph of FKS1 S643P from 9623 collected micrographs. b, Representative 2D class averages. c, Flow chart of cryo-EM data acquisition and data processing of FKS1 S643P. See Methods for more details. d, The gold-standard Fourier shell correlation (FSC) curve of the reconstructed map. e, The FSC curve between model and map, which is calculated by PHENIX.mtriage58. f, Cutaway views of the angular distribution of all particles used in the final 3D reconstruction. g, Local resolution distribution of the final cryo-EM map of FKS1 S643P, calculated with ResMap. h, Fit of cryo-EM map with FKS1 S643P model in example regions.

Extended Data Fig. 8 Analysis of caspofungin-resistant phenotype and chemical structure of echinocandin drugs.

a, Analysis of caspofungin-resistant phenotype of S. cerevisiae strain with indicated FKS1 mutations. The concentrations of caspofungin tested are indicated on the top. b, Chemical structure of four types of echinocandin drugs. They all feature a lipid tail as highlighted by the box. c, Chemical structure of the lipid alkyl chains (left panel) and phospholipid (right panel) identified from FKS1 structure.

Extended Data Fig. 9 Sequence conservation of fungi FKS illustrated on S. cerevisiae FKS1 structure.

a—c, FKS1 structure is colored according to level of sequence conservation (red, high; blue, low). a–b, Back and front view parallel to the membrane, in cartoon display. c, The same view as (b) with surface representation.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
Full size table

Supplementary information

Supplementary Figures

This file contains the uncropped blots (Supplementary Fig. 1) and the multiple sequence alignment of FKS orthologs from various pathogenic fungi and Saccharomyces cerevisiae (Supplementary Fig. 2).

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Hu, X., Yang, P., Chai, C. et al. Structural and mechanistic insights into fungal β-1,3-glucan synthase FKS1. Nature 616, 190–198 (2023). https://doi.org/10.1038/s41586-023-05856-5

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