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

Nature Structural & Molecular Biology volume 29, pages 653–664 (2022)Cite this article

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Abstract

Chitin is an essential component of the fungal cell wall. Chitin synthases (Chss) catalyze chitin formation and translocation across the membrane and are targets of antifungal agents, including nikkomycin Z and polyoxin D. Lack of structural insights into the action of these inhibitors on Chs has hampered their further development to the clinic. We present the cryo-EM structures of Chs2 from Candida albicans (CaChs2) in the apo, substrate-bound, nikkomycin Z-bound, and polyoxin D-bound states. CaChs2 adopts a unique domain-swapped dimer configuration where a conserved motif in the domain-swapped region controls enzyme activity. CaChs2 has a dual regulation mechanism where the chitin translocation tunnel is closed by the extracellular gate and plugged by a lipid molecule in the apo state to prevent non-specific leak. Analyses of substrate and inhibitor binding provide insights into the chemical logic of Chs inhibition, which can guide Chs-targeted antifungal development.

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Fig. 1: Biochemical characterization and overall architecture of CaChs2.
Fig. 2: Translocation tunnel for chitin.
Fig. 3: The UDP-GlcNAc binding site.
Fig. 4: Domain-swapped region.
Fig. 5: CaChs2 structures bound to nikkomycin Z and polyoxin D.
Fig. 6: Overlay of the substrate binding sites of UDP-GlcNAc-bound, nikkomycin Z-bound, and polyoxin D-bound CaChs2.
Fig. 7: Working models of lipid modulation and drug inhibition on CaChs2 function as well as of higher-order assembly of chitin chains.

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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.

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Acknowledgments

Cryo-EM data were screened and collected at the Duke University Shared Materials Instrumentation Facility (SMIF), the Pacific Northwest Center for Cryo-EM (PNCC) in OHSU, and the UNC CryoEM core facility. We thank N. Bhattacharya at SMIF, J. Myers at PNCC, and J. Peck and J. Strauss of the UNC CryoEM Core Facility for assistance with the microscope operation. We thank A. Stelling for assistance with the FTIR measurements, M. M. Draelos for preparation of nikkomycin Z and J. Fedor for his critical reading of the manuscript. This research was supported by a National Institutes of Health grant R01GM115729 (K.Y) and Duke startup funds (S.-Y.L). A portion of this research was supported by NIH grant U24GM129547 and performed at the PNCC at OHSU and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. Duke SMIF is affiliated with the North Carolina Research Triangle Nanotechnology Network, which is in part supported by the NSF (ECCS-2025064). The UNC CryoEM core facility is supported by NIH grant P30CA016086.

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Author notes

  1. These authors contributed equally: Zhenning Ren, Abhishek Chhetri.

Authors and Affiliations

  1. Department of Biochemistry, Duke University School of Medicine, Durham, NC, USA

    Zhenning Ren, Abhishek Chhetri, Ziqiang Guan, Yang Suo, Kenichi Yokoyama & Seok-Yong Lee

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  1. Zhenning Ren
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Contributions

Z.R. conducted biochemical preparation, sample freezing, single-particle 3D reconstruction of the enzyme, crosslinking experiment and ITC experiments under the guidance of S.-Y.L. A.C. carried out enzymatic assays, and the FTIR measurement under the guidance of K.Y. Y.S. and Z.R. collected cryo-EM data. Z.R. and S.-Y.L. performed model building and refinement. Z.G. performed the lipid MS analysis. S.-Y.L, Z.R., K.Y. and A.C. wrote the paper.

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Correspondence to Kenichi Yokoyama or Seok-Yong Lee.

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Extended data

Extended Data Fig. 1 Purification and enzymatic reaction of CaChs2 and isothermal titration calorimetry (ITC) analysis.

a, Representative gel-filtration profile and SDS-PAGE of purified CaChs2. The experiments have been repeated more than ten times independently with essentially the same results. b, Enzymatic reaction of CaChs2. c-e. Representative ITC data of CaChs2 WT (c), W647A (d) and Q643A (e) vs Nikkomycin. The Kd is the average of three independent repeats. The uncropped gel image for a is available as source data.

Source data

Extended Data Fig. 2 Cryo-EM analysis of CaChs2 in apo, UDP-GlcNAc bound, nikkomycin Z bound, and polyoxin D bound states.

a, General flowchart for data processing. b, Representative micrographs for each dataset. The number of total micrographs in each dataset is shown in Extended Data Fig.3. c, Representative 2D classes. d, Local resolution maps. e, Euler angle distribution of the final reconstructions. f, The gold-standard Fourier shell correlation (FSC) curves for the final reconstructions. g, FSC curves of the refined models versus the corresponding full map (red) and half maps (green and blue).

Extended Data Fig. 3 Data processing flowchart.

Data processing for CaChs2 in apo (a), UDP-GlcNAc bound (b), nikkomycin Z bound (c), and polyoxin D bound (d) states.

Extended Data Fig. 4 Representative EM densities.

Representative densities for CaChs2 in apo (a), UDP-GlcNAc bound (b), nikkomycin Z bound (c), and polyoxin D bound (d) states.

Extended Data Fig. 5 Comparison of architectures of chitin synthase CaChs2 and cellulose synthases RsBcsA and PttCesA8.

a, Domain arrangement of CaChs2, RsBcsA and PttCesA8. b-c, Topology diagrams of RsBcsA (b) and PttCesA8 (c).

Extended Data Fig. 6 The dimer interface of CaChs2.

a, Cartoon representation of a CaChs2 dimer. The dimer interface is highlighted in the rectangle. b-c, Close-up view of the dimer interface in two orientations.

Extended Data Fig. 7 Structural comparison of polymer translocation tunnels and substrate binding sites of CaChs2, RsBcsA and PttCesA8.

a, Left: Ribbon representation of RsBcsA in the product bound state (PDB code: 5EJZ) with the tunnel highlighted in surface representation and the cellulose polymer in sticks. Trp383, which defines the intracellular entry of the tunnel is highlighted in sticks. Right: Length and radius of the tunnel calculated by the HOLE program. b. Ribbon representation of PttCesA (PDB code: 6WLB) with the tunnel highlighted in surface representation and the cellulose polymer in sticks. Trp718, which defines the intracellular entry of the tunnel is highlighted in sticks. Right: Length and radius of the tunnel calculated by the HOLE program. c-d. Overlay of the substrate binding sites of CaChs2 (green) and substrate-bound RsBcsA (orange) (PDB code: 5EIY). Conserved residues that interact with the substrates and the catalytic residues are shown as sticks. e-f. Overlay of binding sites of CaChs2 (green) and product-bound PttCesA8 (purple) (PDB code: 6WLB). Conserved residues that interact with the substrates and the catalytic residues are shown as sticks.

Extended Data Fig. 8 Conservation mapping of CaChs2 and EM density of UDP-GlcNAc and Mg2+ at the substrate binding site.

a, Cross-section surface representation of a CaChs2 monomer bound to nikkomycin Z (yellow spheres). b, Detailed interactions between CaChs2 and nikkomycin Z. In both a and b, CaChs2 is colored based on the conservation score calculated by the Consurf Server (https://consurf.tau.ac.il/) from 150 Chs homologs whose sequence identity varies from 35% to 99%. Conservation scores: 1, ~15% conserved; 5, ~60% conserved; 9, 100% conserved. c-d, Density (Threshold = 0.007) of within 6 Å of UDP-GlcNAc, Mg2+, and residues interacting with the substrate, viewed from two orientations, is shown as grey surfaces. The protein model is shown as cartoon and the substrate and residues at the binding site are shown as sticks. The map and model of residues obstructing the view are hidden for clarity.

Extended Data Fig. 9 Identification of lipids copurified with CaChs2 by LC/MS.

a, Total ion chromatogram (TIC) of normal phase LC/MS of lipids extracted from the CaChs2 protein sample. b, Representative chemical structure of ceramide and mass spectrum of the 2.88 min TIC peak in a showing the chloride adduct [M + Cl]- ions of ceramide species. c, Representative chemical structure of and mass spectrum of the 16.20 min TIC peak in a showing the [M-H]- ions of PE molecular species. This identification of ceramide and PE was supported by exact mass measurement and MS/MS. The TIC peaks at 14.09, 21.16 and 27.18 min are consistent with glycodiosgenins (GDN) containing two, three and four sugars whose [M + Cl]- ions are observed at m/z 875.5, 1037.5 and 1199.5, respectively.

Extended Data Fig. 10 Map and model of the domain-swapped region and crosslinking experiments to validate the domain-swapped assembly.

a-b, Unsharpened map of the domain-swapped region of UDP-GlcNAc bound CaChs2 shown at threshold 0.00475, viewed from two orientations. c. Representative density of the domain-swapped region. The protein model is shown as cartoon and side chains are shown as sticks. Some of the side chains are not built due to lack of signal. d. CaChs2 viewed from the intracellular side. The Cα atom of P906 and N917, which define the N and C-terminus of the loop between H10 and TM5, are shown as spheres. The distances from the Cα of P906 to that of N917 within the same monomer or in different monomers are labeled with dashed lines. It is noteworthy that the distance between P906 and N917 is closer in the domain swapped arrangement (30 Å) than the non-domain swapped arrangement (46.5 Å). Because the length of an amino acid in a linear chain is ~3.5 Å, non-domain swapped configuration is physically impossible. e. Model of CaChs2 dimer with one protomer in blue and the other in salmon. Cα atoms of residues where cysteine was introduced are shown as spheres. The crosslinking cysteine pairs are labeled with dashed lines. f. SDS-PAGE analysis of single and double cysteine mutants of CaChs2. The experiment has been repeated four times from two biological repeats with essentially the same results. Uncropped gel images for f are available as source data.

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Uncropped gel for 1a.

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Uncropped gels for 10f.

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Ren, Z., Chhetri, A., Guan, Z. et al. Structural basis for inhibition and regulation of a chitin synthase from Candida albicans. Nat Struct Mol Biol 29, 653–664 (2022). https://doi.org/10.1038/s41594-022-00791-x

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