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Regulation of RAS palmitoyltransferases by accessory proteins and palmitoylation

Nature Structural & Molecular Biology volume 31, pages 436–446 (2024)Cite this article

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Abstract

Palmitoylation of cysteine residues at the C-terminal hypervariable regions in human HRAS and NRAS, which is necessary for RAS signaling, is catalyzed by the acyltransferase DHHC9 in complex with its accessory protein GCP16. The molecular basis for the acyltransferase activity and the regulation of DHHC9 by GCP16 is not clear. Here we report the cryo-electron microscopy structures of the human DHHC9–GCP16 complex and its yeast counterpart—the Erf2–Erf4 complex, demonstrating that GCP16 and Erf4 are not directly involved in the catalytic process but stabilize the architecture of DHHC9 and Erf2, respectively. We found that a phospholipid binding to an arginine-rich region of DHHC9 and palmitoylation on three residues (C24, C25 and C288) were essential for the catalytic activity of the DHHC9–GCP16 complex. Moreover, we showed that GCP16 also formed complexes with DHHC14 and DHHC18 to catalyze RAS palmitoylation. These findings provide insights into the regulatory mechanism of RAS palmitoyltransferases.

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Fig. 1: Substrate specificity of the RAS palmitoyltransferases.
Fig. 2: The overall structures of the RAS palmitoyltransferases.
Fig. 3: Organization of the active sites of the RAS palmitoyltransferases.
Fig. 4: Organization of the zinc finger motifs of the RAS palmitoyltransferases.
Fig. 5: Interactions between DHHC9 and GCP16.
Fig. 6: Cysteine palmitoylation regulates DHHC9 catalytic activity.
Fig. 7: GCP16 is an accessory protein for DHHC14 and DHHC18.

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Data availability

The cryo-EM structures have been deposited in the Protein Data Bank (www.rcsb.org) with the accession codes 8HFC (Erf2–Er4 complex) and 8HF3 (DHHC9–GCP16 complex). The cryo-EM maps have been deposited in the Electron Microscopy Data Bank with the accession codes EMD-34717 (Erf2–Er4 complex) and EMD-34711 (DHHC9–GCP16 complex). All other data are available in the manuscript or the supplementary materials. The crystal structure of the human DHHC20 palmitoyltransferase (PDB code: 6BMN) was used for structural analysis in this study. Source data are provided with this paper.

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Acknowledgements

We thank P. Lu and K. Sun for their help with structure prediction; the Cryo-EM Facility and HPC Center of Westlake University for providing cryo-EM and computation support; the Mass Spectrometry & Metabolomics Core Facility at the Center for Biomedical Research Core Facilities of Westlake University for sample analysis; Y. Chen and K. Wang at the Instrumentation and Service Center for Molecular Sciences at Westlake University for the ICP-MS analysis. This work was supported by Westlake Laboratory of Life Sciences and Biomedicine to Q.H. and Y.Zou, Central Guidance on Local Science and Technology Development Fund (2022ZY1006) and Westlake Education Foundation to Q.H., Zhejiang Provincial Natural Science Foundation of China (LR22C050003), Westlake University (1011103860222B1) and Westlake Education Foundation (101486021901) to J.W.

Author information

Author notes

  1. These authors contributed equally: Anlan Yang, Shengjie Liu, Yuqi Zhang.

Authors and Affiliations

  1. College of Life Sciences, Zhejiang University, Hangzhou, China

    Anlan Yang

  2. Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China

    Anlan Yang, Shengjie Liu, Yuqi Zhang, Jia Chen, Shan Feng, Jianping Wu & Qi Hu

  3. Fudan University, Shanghai, China

    Shengjie Liu

  4. Mass Spectrometry & Metabolomics Core Facility, Westlake University, Hangzhou, China

    Jia Chen & Shan Feng

  5. Westlake Four-Dimensional Dynamic Metabolomics (Meta4D) Laboratory, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China

    Yujing Fan, Fengxiang Wang & Yilong Zou

  6. School of Life Sciences, Westlake University, Hangzhou, China

    Fengxiang Wang & Yilong Zou

  7. Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China

    Jianping Wu

  8. Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, China

    Jianping Wu & Qi Hu

  9. Westlake AI Therapeutics Lab, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China

    Qi Hu

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Contributions

Q.H. conceived and supervised the project; A.Y. and S.L. purified the proteins, performed the biochemical assays, prepared the cryo-EM samples and collected the cryo-EM data; Y.Zhang and J.W. calculated the cryo-EM map and built the model; J.C. and S.F. performed the mass spectrum experiment to identify the acylation sites in the RAS palmitoyltransferases; Y.F., F.W. and Y.Zou performed the mass spectrum experiment to characterize lipids in the DHHC9–GCP16 samples; all authors contributed to data analysis; Q.H. and J.W. wrote the manuscript with inputs from other authors.

Corresponding authors

Correspondence to Jianping Wu or Qi Hu.

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Nature Structural & Molecular Biology thanks Ping Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Purification and characterization of the Erf2-Erf4 complex and the DHHC9-GCP16 complex.

a,b, The Erf2-Erf4 complex (a) and the DHHC9-GCP16 complex (b) purified using affinity columns were further purified by size exclusion chromatography, then the peak fractions were analyzed by SDS-PAGE followed by Coomassie blue staining. The protein purification results from representative experiments are shown (a, b). c, Quantification of metal ions in the purified DHHC9-GCP16 complex. The metal ions in the purified DHHC9-GCP16 complex and those in the buffer control were analyzed using inductively coupled plasma mass spectrometry (ICP-MS). The data represent the mean ± SD of three independent measurements. d, Palmitoylation of NRAS catalyzed by the DHHC9-GCP16 complex. The wild-type or the catalytic-dead mutant (C169A) of DHHC9 in complex with GCP16 was incubated with wild-type NRAS or its C181S mutant and NBD-palmitoyl-CoA. At the indicated time points, the reactions were quenched with non-reducing SDS loading buffer and analyzed by SDS-PAGE. The SDS-PAGE gel was first imaged using Amersham Imager 680 (Cytiva) with a Cy2 filter (Excitation: 460 nm) (the upper panel), and then visualized by Coomassie blue staining (the lower panel). The results of a representative experiment from three independent experiments are shown (d). e, Quantification of the palmitoylation of NRAS catalyzed by the DHHC9-GCP16 complex. The data represent the mean ± SD of three independent measurements.

Source data

Extended Data Fig. 2 Effects of the C-termini of DHHC9, HRAS and RAS2 on the palmitoylation reaction.

a,b, A chimeric protein HRAS-RAS2(35aa) was designed by replacing the C-terminal 24 amino acids of HRAS with the C-terminal 35 amino acids of RAS2. Palmitoylation of this chimeric protein catalyzed by the Erf2-Erf4 complex was evaluated using the NBD assay. c,d, The catalytic activities of the full-length and the C-terminal truncated form (residues 1–305) of DHHC9 in complex with GCP16 were measured using HRAS and NRAS as the protein substrates and NBD-palmitoyl-CoA as the palmitoyl group donor. The data in (a) and (c) are the results of a representative experiment from three independent experiments. The data in (b) and (d) represent the mean ± SD of three independent measurements, and were analyzed using Prism.

Source data

Extended Data Fig. 3 Cryo-EM data analysis of Saccharomyces cerevisiae Erf2-Erf4 complex.

a, A representative cryo-EM. Scale bar: 50 nm. b, Representative two-dimensional class averages. Box size: 209 Å; Circle mask: 177 Å. Scale bar: 5 nm. c, Angular distribution of the particles of the final map reconstruction generated by cryoSPARC. d, Gold standard Fourier shell correlation (FSC) curves for the 3D reconstructions generated by cryoSPARC. The curves representing the corrected (random phase initialized), tight mask, loose mask and no mask are colored black, red, blue, and orange respectively. e, Validation of the final structure model. The curves indicate the FSC of the model versus the overall map/ half map 1/ half map 2 (FSC = 0.5). f, A flowchart of EM data processing. Please refer to ‘Cryo-EM image processing’ section in Methods for details. The map labelled ‘good reference’ was used to repick particle coordinates and further refinement. The map labelled ‘final map’ was applied to build and refine the atomic model subsequently. The 3D classes in grey dashed boxes were considered as good classes which were selected during the processing steps.

Extended Data Fig. 4 Cryo-EM data analysis of Homo Sapiens DHHC9-GCP16 complex.

a, A representative cryo-micrograph. Scale bar: 50 nm. b, Representative two-dimensional class averages. Box size: 215 Å; Circle mask: 183 Å. Scale bar: 5 nm. c, Angular distribution of the particles of the final map reconstruction generated by cryoSPARC. d, Gold standard Fourier shell correlation (FSC) curves for the 3D reconstructions generated by cryoSPARC. The curves representing the corrected (random phase initialized), tight mask, loose mask and no mask are colored black, red, blue, and orange respectively. e, Validation of the final structure model. The curves indicate the FSC of the model versus the overall map/ half map 1/ half map 2 (FSC = 0.5). f, A flowchart of EM data processing. Please refer to ‘Cryo-EM image processing’ section in Methods for details.

Extended Data Fig. 5 Docking of a 14-carbon acyl group into the catalytic sites of Erf2 and DHHC9.

a,d, The solvent-accessible surface of the Erf2-Erf4 complex (a) and that of the DHHC9-GCP16 complex (d) were generated in PyMOL. The surface transparency was set to 20% to show the acyl groups behind the surfaces. The 14-carbon acyl group in each structure is shown as sticks and colored magenta. b,c, Docking of a 14-carbon acyl group (b) or a 16-carbon acyl group (c) into the cryo-EM map of the Erf2-Erf4 complex. e,f, Docking of a 14-carbon acyl group (e) or a 16-carbon acyl group (f) into the cryo-EM map of the DHHC9-GCP16 complex.

Extended Data Fig. 6 Point mutations decreased the palmitoyltransferase activity of the DHHC9-GCP16 complex.

a,c, Effects of disease-causing mutations R148W and P150S in DHHC9 on the catalytic activity of the DHHC9-GCP16 complex. b,d,e,f,g,h, Effects of point mutations in DHHC9 and GCP16 on the acyltransferase activity of the DHHC9-GCP16 complex. The catalytic activities of the DHHC9-GCP16 complexes were measured using HRAS as the protein substrate and NBD-palmitoyl-CoA as the palmitoyl group donor. The upper panels in (a), (b), (e) and (f) show the SDS-PAGE gels imaged by fluorescence imaging, representing the NBD-palmitoylation levels, while the lower panels show the same gels visualized by Coomassie blue staining. The data in (a), (b), (e) and (f) are the results of a representative experiment from three independent experiments. The data in (c), (d), (g) and (h), which represent the mean ± SD of three independent measurements, were analyzed using the unpaired t-test in Prism to calculate the two-tailed P-values: ****, P < 0.0001; ***, P < 0.001.

Source data

Extended Data Fig. 7 Characterization of the bound-lipid in the DHHC9-GCP16 complex.

a, Brief scheme summarizing the experimental procedure for lipid extraction (see also Methods). b, Extracted ion chromatography (EIC) of PA (36:3e), PA (38:4e) and PA (36:4e). c, Data-dependent tandem mass spectrum of PA (36:3e), PA (38:4e) and PA (36:4e). Abbreviation: NL, neutral loss. d, Dot and bar plot showing the normalized intensities (in 71.5 µg protein samples) of detected phosphatidic acid (PA) species from three extraction and mass spectrometry experiments. e,f, Evaluation of the role of different phospholipids on the catalytic activity of the DHHC9-GCP16 complex. The DHHC9-GCP16 complex was immobilized on anti-FLAG beads and subsequently washed with a buffer containing 0.1% Triton X100 in order to remove the bound lipids. The complex was then eluted from the beads by FLAG peptide and incubated with various phosphatidic acids (PA), phosphatidylcholines (PC), or a buffer control. The catalytic activity was evaluated using the NBD assay. The data in (e) are the results of a representative experiment from three independent experiments. The data in (d) and (f) represent the mean ± SD of three independent measurements. The data in (f) were analyzed using the unpaired t test in Prism to calculate the two-tailed P values: **, P < 0.01; ns, P ≥ 0.05.

Source data

Extended Data Fig. 8 Interactions between Erf2 and Erf4.

a, The overall structure of the Erf2-Erf4 complex. b, Residues R126, R216 and S329 in TM2, TM3 and the PPII helix of Erf2 interact with residues G176, S179, N164 and R222 in α4′-α8′ helices of Erf4 through hydrogen bonding. P325 and P328 in the PPII helix of Erf2 dock into to hydrophobic pockets in Erf4. c, Residues T162, H163, S165 and I166 in Erf2 form hydrogen bonds with residues R17 and R83 in Erf4. d, e, The linker between the TM2 and β1 strand of Erf2 interacts with the N-terminal region and the loop between β2′ and β3′ of Erf4 through hydrogen bonding, π–π stacking and CH-π interactions. The hydrogen bonds are represented by yellow dashed lines.

Extended Data Fig. 9 Identification of S-palmitoylation sites in DHHC9 by MS/MS.

a, The MS/MS spectra of tryptic peptides NTFCCDGR containing C24 and C25 from DHHC9. b, The MS/MS spectra of tryptic peptides NCCEVLCGPLPPSVLDR containing C283, C284 and C288 from DHHC9. The delta mass labeled in (a) and (b) is equal to the molecular weight of palmitoylated C25 and C288, respectively.

Source data

Extended Data Fig. 10 Alignment of the protein sequence of DHHC9 with that of its human homologs.

a, The distance tree of human DHHC acyltransferases was generated by alignment of the protein sequences using the Basic Local Alignment Search Tool (BLAST)40. The UniProt ID and the corresponding name of each DHHC acyltransferase are shown. b, Alignment of the protein sequences of DHHC9 with DHHCs that share the highest homology with DHHC9 using BLAST.

Supplementary information

Supplementary Information

Supplementary Figs 1–4.

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Supplementary Table 1

Lipidomic analysis of the purified DHHC9–GCP16 complex.

Source data

Source Data Fig. 1

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Unprocessed SDS–PAGE gels.

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Unprocessed SDS–PAGE gels.

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Gel filtration curves, unprocessed SDS–PAGE gels, western blots and confocal images.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 1

Gel filtration curves and unprocessed SDS–PAGE gels.

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Statistical source data.

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Unprocessed SDS–PAGE gels.

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Unprocessed SDS–PAGE gels.

Source Data Extended Data Fig. 9

Raw mass spectrometry data.

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Yang, A., Liu, S., Zhang, Y. et al. Regulation of RAS palmitoyltransferases by accessory proteins and palmitoylation. Nat Struct Mol Biol 31, 436–446 (2024). https://doi.org/10.1038/s41594-023-01183-5

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