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Activity-based directed evolution of a membrane editor in mammalian cells

Nature Chemistry volume 15, pages 1030–1039 (2023)Cite this article

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Abstract

Cellular membranes contain numerous lipid species, and efforts to understand the biological functions of individual lipids have been stymied by a lack of approaches for controlled modulation of membrane composition in situ. Here we present a strategy for editing phospholipids, the most abundant lipids in biological membranes. Our membrane editor is based on a bacterial phospholipase D (PLD), which exchanges phospholipid head groups through hydrolysis or transphosphatidylation of phosphatidylcholine with water or exogenous alcohols. Exploiting activity-dependent directed enzyme evolution in mammalian cells, we have developed and structurally characterized a family of ‘superPLDs’ with up to a 100-fold enhancement in intracellular activity. We demonstrate the utility of superPLDs for both optogenetics-enabled editing of phospholipids within specific organelle membranes in live cells and biocatalytic synthesis of natural and unnatural designer phospholipids in vitro. Beyond the superPLDs, activity-based directed enzyme evolution in mammalian cells is a generalizable approach to engineer additional chemoenzymatic biomolecule editors.

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Fig. 1: Design of an activity-dependent directed enzyme evolution strategy to create a phospholipase D (PLD)-based membrane editor in mammalian cells.
Fig. 2: Directed evolution yields PLD mutants with greatly enhanced activities.
Fig. 3: Evaluation of superPLD activity in cells.
Fig. 4: Synthesis of designer phospholipids by in vitro reaction.
Fig. 5: Application of superPLD to manipulate PA signalling.
Fig. 6: Structural comparison of superPLD and PLDWT.

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Data availability

Structural models and structure factors of superPLD (2–48) and superPLD (2–23) have been deposited in the RCSB PDB under accession nos. 8CTQ and 8CTP, respectively. The superPLD plasmids used for this study have been deposited in Addgene (https://www.addgene.org/browse/article/28229113/). Other data supporting the findings of this study are available within the Article and the Supplementary Information. Source data are provided with this paper.

Code availability

Python code used for sequencing data processing, systematic mutational analysis and flow cytometry data processing are available within the Supplementary Information.

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Acknowledgements

J.M.B. acknowledges support from a Beckman Young Investigator award, a Sloan Research Fellowship and the NSF (CAREER CHE-1749919). R.T. was supported by Honjo International, Funai Overseas and Cornell fellowships. J.C.F. and S.R.B were supported by NIH/NIGMS grant no. R35GM136358. Beamline 24-ID-E of APS is funded by the National Institute of General Medical Sciences from the National Institutes of Health (P30 GM124165). The Eiger 16M detector on the 24-ID-E beamline is funded by an NIH-ORIP HEI grant (S10OD021527). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. Beamline ID7B2 of CHESS is supported by the NSF under award no. DMR-1829070, and by award no. 1-P30-GM124166-01A1 from the National Institute of General Medical Sciences, National Institutes of Health, and by New York State’s Empire State Development Corporation (NYSTAR).

Author information

Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA

    Reika Tei & Jeremy M. Baskin

  2. Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA

    Reika Tei, Saket R. Bagde, J. Christopher Fromme & Jeremy M. Baskin

  3. Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA

    Saket R. Bagde & J. Christopher Fromme

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Contributions

R.T. and J.M.B. designed the study and analysed data. R.T. carried out directed evolution, molecular cloning, in cellular and in vitro activity assays, protein production and purification. S.R.B. and R.T. performed protein crystallization and X-ray data collection. S.R.B. analysed structural data. J.C.F. supervised X-ray crystallography analysis. R.T. and J.M.B. wrote the manuscript, with input from all authors.

Corresponding author

Correspondence to Jeremy M. Baskin.

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Competing interests

R.T. and J.M.B. are co-inventors on a patent application (PCT/US2022/080999) filed by Cornell University for inventions related to super-active phospholipase Ds. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 A plasma membrane-targeted optoPLD library.

a, Schematic depicting the design of plasma membrane-targeted optoPLD, which consists of CRY2-mCherry-PLD and CIBN-CAAX. OptoPLD-targeting membrane can be controlled by swapping the plasma membrane-targeted domain, CAAX, with another membrane-targeting domain. b–c, Confocal microscopy images of mCherry fluorescence showing localization of optoPLD library (after FACS-based selection) in HEK 293T cells before (b) and after (c) illumination with 488 nm light. The experiment was repeated for at least three times with similar results, and the representative images are shown. Scale bar: 10 µm.

Extended Data Fig. 2 FACS plot and gating strategy for each sorting of directed evolution and superPLD characterization by IMPACT.

a–c, Gating strategy for sorting and analysis of HEK 293T cells expressing optoPLD mutants labelled by IMPACT. Plots of FSC-A vs. BSC-A, population B selected (a), FSC-A vs. FSC-H, population C selected (b), and BSC-A vs. BSC-H, population D selected (c) were used to gate for live, singlet cells. d–k, FACS plot of PLD library from each sorting in three rounds of selection with mutagenesis. l–s, FACS plot of PLD library from each sorting in five rounds of selection without mutagenesis. The round number, sort number within that round, concentration of azidopropanol (AzPr) used for IMPACT labelling, and the percentage of cells collected are indicated in each plot. t, Gating strategy for quantitative comparison of IMPACT labelling. The average BODIPY signal of cells with similar amount of mCherry signal (population shown in red, which is gated for an mCherry fluorescence range of 5×103–1×104), was used. u–v, mCherry (u) and IMPACT labelling (v) histograms of cells expressing PLDWT (black), 1-4 (blue), 1-12 (green) and 2-48 (magenta), demonstrating that optoPLD mutants with different activity have similar expression levels. w–x, The effect of light stimuli on optoPLD activity. Cells expressing optoPLD were treated with 0.5 mM azidopropanol with or without intermittent blue light illumination, followed by treatment with 1 µM BCN-BODIPY, and IMPACT fluorescence intensity normalized to optoPLD expression was determined by flow cytometry. Horizontal lines indicate average (n = 3 independent transfection replicates) of mean intensities of IMPACT fluorescence. PLDdead, a catalytically dead PLD bearing the H167A mutation; PLDWT, wild-type PLD; superPLDlow, superPLD mutant clone 1-4; superPLDmed, superPLD mutant clone 1-12; superPLDmed/high, superPLD mutant clone 1-27; superPLDhigh, superPLD mutant clone 2-48.

Source data

Extended Data Fig. 3 Characterization of superPLD activity in cells.

a–b, Quantification of substrate conversion by superPLD in cells. HEK 293T cells expressing plasma membrane-targeted optoPLD (superPLDmed) were treated with 0.5–2% ethanol (Et) for 30 min with intermittent blue light illumination. As a control, cells expressing PLDdead were treated with 2% ethanol accordingly. The relative levels of the most abundant PLD substrate (16:0/18:1 PC; POPC) and its transphosphatidylation product (16:0/18:1 PEt; POPEt) were quantified by LC–MS. Relative PC levels compared to PC levels in control samples (PLDdead-expressing cells) (a) and relative PEt levels compared to PC levels (b) are plotted. Horizontal lines indicate average, and vertical error bars indicate standard deviation (n = 4 for superPLDmed with 2% ethanol and n = 5 for the others, and n refers to the number of biologically independent samples). Statistical significance was determined by one-way ANOVA followed by Sidak’s multiple comparisons test. ***, p < 0.001. The p values for all the indicated pairwise comparisons in b are <0.0001. c–d, Confocal microscopy images of HEK 293T cells co-expressing a PA probe (GFP-PASS) and optoPLD targeted to the plasma membrane (c) or lysosomes (d) before (0 min), immediately after (1 min), and 30 min after incubation with 488 nm light. e, Confocal microscopy images of cells co-expressing the PA probe and the indicated optoPLD construct (WT or superPLDmed) targeted to lysosomes, stained with LysoView 633. The experiment was repeated at least three times with similar results, and representative images are shown. Scale bars: 10 µm.

Source data

Extended Data Fig. 4 Purification and in vitro characterization of superPLD and PLDWT.

a, SDS-PAGE showing His-NusA-superPLD (shown here is clone 2-48) (113 kDa) in cell lysate, His-NusA (58 kDa) being retained on the TALON beads, and superPLD (55 kDa) eluting from the beads and exhibiting high purify after size-exclusion chromatography (SEC). 6xHis-NusA-superPLD was expressed in E.coli Rosetta 2 and purified using TALON resin. HRV 3C protease was used to cleave between NusA and PLD. Black arrows indicate the bands derived from PLD. b, Ponceau stain with PLDWT and superPLDhigh purified from Rosetta 2 vs. Gami-2 (Rosetta-gami 2) strains. When expressed in the Rosetta strain, PLDWT showed substantial degradation and lower yield. c, SDS-PAGE showing general correlation between the activity of superPLD mutants in mammalian cells and the robustness of purification from E. coli. The numbers in parentheses indicate relative PLD activity in mammalian cells as determined by IMPACT (Fig. 2d). Black arrow indicates the bands corresponding to PLD. d–f, Kinetic analysis of PLD activity. PLDWT and a subset of superPLD mutants were diluted accordingly to adjust to equal amounts based on SDS-PAGE (d) and incubated with indicated concentration of DOPC. The purification of each PLD mutant was performed in at least three independent experiments (except for 1-4 and 1-12, which were purified in two independent experiments) with similar results, and the representative data are shown in panels a–d. Activity assays were performed using the Amplex Red Phospholipase D Assay Kit. The rates of reaction are plotted against the substrate concentration (e), and the data were fit to the Michaelis–Menten equation to obtain the kinetic parameters (f). Horizontal lines indicate average, and vertical error bars indicate standard deviation (n = 4 independent replicates). g, Thermal stability of PLDWT and a subset of superPLD mutants spanning a wide range of activities (see Fig. 2d), indicating no clear correlation between thermal stability and PLD activity. Melting temperature (Tm) was determined in a real-time thermocycler using SYPRO Orange dye. Horizontal lines represent the mean and error bars represent the standard deviation (n = 2, 4, 6, 2, 7, 8, and 4, respectively, and n refers to the number of independent replicates). h, Chemical stability of PLDWT and superPLDhigh (2-48) purified from the Gami-2 strain, indicating increased chemical stability for superPLD. Each enzyme was incubated with indicated concentration of urea in PBS for 12 h at 37 °C, followed by an activity assay using the Amplex Red Phospholipase D Assay Kit. Relative rates of reaction compared to control samples (enzymes incubated in PBS only) are plotted. Horizontal lines indicate average, and vertical error bars indicate standard deviation (n = 4 independent replicates).

Source data

Extended Data Fig. 5 Application of superPLD to manipulate PA signalling.

a–b, Quantification of nuclear YAP levels to evaluate Hippo signalling activity. HEK 293T cells expressing plasma membrane-targeted optoPLD (PLDdead, PLDWT or superPLDmed) were immunostained for YAP (a; scale bars: 10 µm), and percentage of the total YAP signal that is colocalized with the DAPI (nucleus) signal is plotted for each transfected cell (b). Horizontal lines indicate average, and vertical error bars indicate standard deviation (n = 92, 122, and 119, respectively, and n refers to the number of cells examined over three independent experiments). Statistical significance was determined by one-way ANOVA followed by Sidak’s multiple comparisons test. ***, p < 0.001. The p values for the indicated pairwise comparisons are <0.0001, <0.0001, and 0.14, respectively. c, Representative Western blots used to quantify p-AMPK levels (see Fig. 5d). Cells expressing plasma membrane-targeted optoPLD were treated with a CaMKK inhibitor (STO-609) for 6 h to block CaMKK-mediated AMPK activation, followed by a 30 min incubation with 488 nm light. d, Representative Western blots used to quantify p-S6K levels (see Fig. 5e). Cells expressing plasma membrane-targeted optoPLD were treated with an AMPK inhibitor (dorsomorphin) for 1 h, followed by a 30 min incubation with 488 nm light. Experiments were repeated at least three times with similar results for c and d.

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Extended Data Fig. 6 Mutations identified in various superPLD clones.

a, PLD mutant clones are shown in order of PLD activity in cells determined by IMPACT (increasing from left to right), and black dots indicate the presence of a particular point mutation in that PLD mutant. Mutants with identical sets of mutations are not shown in the plot. The mutated residues are coloured based on conservation across PLDs from different species, determined by ConSurf;1,2,3 red: high conservation, white: medium, blue: low. b, Activity assay of PLD mutants containing different combinations of four commonly occurring mutations that were generated in the PLDWT background (A258T, G381V, G429D and T450A). Cells expressing plasma membrane-targeted optoPLD with the indicated set of mutations were labelled with IMPACT using 1 mM azidopropanol and 1 µM BCN-BODIPY. IMPACT fluorescence intensity normalized to optoPLD expression was determined by flow cytometry, and the relative values for each mutant compared to the PLDWT are plotted as relative IMPACT labelling (n = 2 independent transfection replicates). The effect of each mutation occurred mostly in a combinatorial manner (that is, most mutations exhibited multiplicative effects in either increasing (G381V, T450A) or slightly decreasing (A258T) the activity, though G429D slightly increased activity alone but had negligible effects when combined with other mutations).

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Extended Data Fig. 7 Analysis of the effects of acquired mutations on PLD activity.

a, Relative activity of PLDs with frequently occurring mutations that were individually installed into either the PLDWT (black) or G381V (magenta) background. Cells were labelled and analysed by flow cytometry as described in Extended Data Fig. 6. Horizontal lines indicate average (n = 2 independent transfection replicates) of relative mean intensities of IMPACT fluorescence. Dashed grey lines indicate the activity of PLDWT, which is normalized to 1. b–c, Systematic comparison to evaluate the effect of individual mutations in the context of actual superPLD mutants isolated from the screen. The graphs show related mutants grouped separately. Each group contains a “template mutant” consisting of a specific set of mutations (which came from directed evolution experiments), which is shown left-most within each group, as well as other mutants that contain all the mutations in the template mutant plus one additional mutation that is indicated. d, Three different types of effects caused by individual mutations, determined based on point mutation analysis and systematic comparison analysis of mutants.

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Extended Data Fig. 8 Mapping correlations between structural shifts and mutated sites in superPLDs.

a–b, Alignments of PLDWT with either superPLDhigh (2-48) (a) or superPLD clone 2-23 (b), with the differences between the two structures coloured by root mean square deviation (RMSD) using the ColorByRMSD script4. The distances between aligned Cα atom pairs are stored as B-factors of these residues, which are coloured by a colour spectrum, with cyan specifying the minimum pairwise RMSD and magenta indicating the maximum. Unaligned residues are coloured grey. Sites of mutations in each superPLD are shown in yellow, and mutated residues are shown as yellow sticks except for G381V, which exists on the missing flexible loop that is shown as a yellow dashed line. Loops 1–4 at the entrance to the active site are highlighted in red dashed boxes. c, Alignment of PLD from Streptomyces sp. PMF and PLD from Streptomyces antibioticus, with the differences between the two structures coloured by RMSD, demonstrating a high level of structural homology. Loops 1 and 4 are highlighted in red dashed boxes. d, Zoomed-in image of loops 1 and 4. PLD from Streptomyces sp. PMF, PLD from Streptomyces antibioticus, and superPLDhigh (2-48) are shown in green, cyan, and magenta, respectively. Note that the positions of residues Y186, W190 and Y383 are identical in the two wildtype PLDs and found to be largely shifted in superPLD (except for Y383, which is unresolved but likely substantially displaced as discussed in the main text).

Extended Data Fig. 9 Analysis of the active site structure of superPLD.

a–c, Structures of the active sites of superPLDhigh (2-48) (a), superPLDhigh overlaid with PLDWT (PDB ID: 1V0Y; green) (b), and superPLDhigh overlaid with PLD from Streptomyces antibioticus engineered to produce phosphatidylinositol5 (PDB ID: 7JRV; light blue) (c). Loops 1 and 4 in each structure are shown in dark purple (superPLD 2-48) and dark blue (engineered S. antibioticus PLD). A phosphate ligand is modelled in the electron density found in the active site of superPLDhigh. Polder omit map for the region around the modelled phosphate molecule is shown as a mesh, contoured at 0.52 σ using a carve distance of 3 Å6. d, LC–MS analysis of a lipid extract from purified superPLD, demonstrating the existence of multiple PA species that co-purified with superPLDhigh (2-48). e, Comparison of the effects of site-directed mutation on PLDWT (green) vs. superPLDhigh (2-48; magenta). HEK 293T cells expressing plasma membrane-targeted optoPLD versions of PLDWT or superPLDhigh (2-48) with the indicated point mutation were labelled with 10 mM (for PLDWT) or 100 μM (for superPLDhigh) azidopropanol, followed by click chemistry tagging with 1 µM BCN-BODIPY. IMPACT fluorescence intensity normalized to optoPLD expression was determined by flow cytometry, and the relative values for each mutant compared to the appropriate parental PLD (that is, PLDWT or superPLDhigh (2-48)) are plotted as relative IMPACT labelling. Plots are replicates (n = 5 for Y459F, n = 3 for N184Q, N184A, Q335N, H440Y and H440A, n = 2 for the others, and n refers to the number of independent transfection replicates) from flow cytometry analysis, and the line indicates the average.

Extended Data Fig. 10 Disulfide bonds in superPLD and PLDWT structures.

a–e, Crystal structures of superPLDhigh (2-48) (a, c, e) and PLDWT (PDB ID: 1V0Y) (b, d) with cysteine residues shown in red. Disulfide bonds in PLDWT are highlighted with red dashed boxes. In superPLD structures, C295 was not resolved, and C341 residue was flipped, indicating that the C295–C341 disulfide bond is reduced. Sites of mutations in superPLDhigh are shown in yellow, with mutated residues shown as yellow sticks (except for G381V, which is in an unresolved flexible loop and is shown as a yellow dashed line). f–g, Identification of a mutation in superPLD that is responsible for enhanced tolerance to disabled disulfide bond formation. Mutations in superPLDhigh (2-48) that occur at three positions near C415 (G328S, G381V, and G406S) were, within the superPLDhigh (2-48) background, reverted to the residue that occurs in PLDWT, and the relative effects of the C415S mutation on PLD activity in these constructs (f) were compared to the effect of the C415S mutation in PLDWT (g). IMPACT fluorescence intensity normalized to optoPLD expression was determined by flow cytometry. Horizontal lines indicate average (n = 3 independent transfection replicates) of relative mean intensities of IMPACT fluorescence of cells expressing the indicated mutant PLD compared to the parental PLD (for example, PLDWT or superPLDhigh (2-48)), as measured by flow cytometry.

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Supplementary information

Supplementary Information

Supplementary Tables 1–4 and references.

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Source code used for data analysis presented in this paper.

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Tei, R., Bagde, S.R., Fromme, J.C. et al. Activity-based directed evolution of a membrane editor in mammalian cells. Nat. Chem. 15, 1030–1039 (2023). https://doi.org/10.1038/s41557-023-01214-0

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