Inhibition of bacterial ubiquitin ligases by SidJ-calmodulin catalysed glutamylation

Abstract

The family of bacterial SidE enzymes catalyses phosphoribosyl-linked serine ubiquitination and promotes infectivity of Legionella pneumophila, a pathogenic bacteria that causes Legionnaires' disease^1-3. SidE enzymes share the genetic locus with the Legionella effector SidJ that spatiotemporally opposes the toxicity of these enzymes in yeast and mammalian cells, through a mechanism that is currently unknown^4-6. Deletion of SidJ leads to a substantial defect in the growth of Legionella in both its natural hosts (amoebae) and in mouse macrophages^4,5. Here we demonstrate that SidJ is a glutamylase that modifies the catalytic glutamate in the mono-ADP ribosyl transferase domain of the SdeA, thus blocking the ubiquitin ligase activity of SdeA. The glutamylation activity of SidJ requires interaction with the eukaryotic-specific co-factor calmodulin, and can be regulated by intracellular changes in Ca²⁺ concentrations. The cryo-electron microscopy structure of SidJ in complex with human apo-calmodulin revealed the architecture of this heterodimeric glutamylase. We show that, in cells infected with L. pneumophila, SidJ mediates the glutamylation of SidE enzymes on the surface of vacuoles that contain Legionella. We used quantitative proteomics to uncover multiple host proteins as putative targets of SidJ-mediated glutamylation. Our study reveals the mechanism by which SidE ligases are inhibited by a SidJ-calmodulin glutamylase, and opens avenues for exploring an understudied protein modification (glutamylation) in eukaryotes.

Extended data Fig. 1. SidJ does not possess intrinsic deubiquitinase activity

a) sdeC-sdeA genetic locus in Legionella genome. b) left, GFP-SidJ ectopically expressed and purified from HEK293T cells was incubated with canonical HA-ubiquitin chains purified from mammalian cells, canonical DUB USP2 was used as a postive control. right, GFP or GFP-SidJ was incubated with purified SdeA-ubiquitinated Rab33b. Experiment was repeated twice independently with similar results. c) Full length SidJ was incubated with various substrates modified with canonical ubiquitination or PR ubiquitination to probe the cleavage activity. USP2 was used as positive controls for cleaving canonical Ub chains and PR-Ub substrates respectively. Experiment was repeated twice independently with similar results. d) Legionella purified SidJ was analyzed by mass spectrometry and protein quantification was performed using the MaxQuant iBAQ algorithm. Experiment was repeated twice independently with similar results.

Extended data Fig. 2. SidJ binds to Calmodulin

a) GFP and GFP-SidJ were ectopically expressed in HEK293T cells and immunoprecipitated. The samples were analyzed by SDS-PAGE followed by coomassie staining. Experiment was repeated twice independently with similar results. b) Purified SdeA from HEK293T cells expressing SdeA alone or in combination with SidJ or SidJ (1-819) were used in ε-NAD⁺ hydrolysis assays. Experiment was repeated twice independently with similar results. c) A549 cells infected with WT Legionella pneumophilia for different time periods was loaded with Fura2AM for 30 min at 37°C followed by ratiometric measurement of intracellular Ca²⁺ using a plate reader. Infection with bacteria did not change total Ca²⁺ levels in the cell. n=3 biologically independent experiments, Data points indicate mean and error bars represent standard deviation d) A549 cells expressing ER-GFP and ER-cepia were infected with Legionella pneumophilia (Ds-Red lp02) followed by time-lapse imaging. Fluorescence intensity of ER-cepia at each region is proportional to local Ca²⁺ levels. ER-GFP fluorescence is independent of Ca²⁺ concentration. The ER has a heterogenous and dynamic distribution of Ca²⁺. The bacteria make transient contacts with the ER and may be influenced by local Ca²⁺ fluxes in the cell. ER-cepia is marked in red, ER-GFP in green and bacteria are marked by white dotted lines. Time-lapse images were taken at 1s intervals for 2min. Images shown in the montage are at 10s intervals. Experiment was repeated thrice independently with similar results. e) Crystal structure of SdeA (PDB: 5YIM) is shown in cartoon representation highlighting the missing peptide of SdeA in SidJ-treated samples (shown in colors red and blue), solvent exposed part of this peptide containing the catalytic glutamate (E860, shown in green) is marked.

Extended data Fig. 3. SdeA E860 glutamylation mass spectra

a) Annotated mass spectra for di-glutamylation of SdeA E860 from immunoprecipitated GFP-SdeA co-expressed with SidJ samples b) In vitro glutamylation of SdeA was carried in various conditions as indicated followed by ε-NAD⁺ hydrolysis assays to measure the Ub ADP-ribosylation activity of SdeA. Experiment was repeated thrice independently with similar results.

Extended data Fig. 4. In vitro glutamylation of SdeA

a) Samples of in vitro glutamylation reactions containing SdeA and SidJ with or without ATP were TMT-labeled and analyzed by quantitative mass spectrometry. Mono- and di-glutamylation of the catalytic E860 of SdeA was enriched in samples containing ATP. In-vitro glutamylation was performed in n=3 biologically independent experiments. Samples were labeled with TMT 6 plex reagent and analyzed in one LC-MS run. Significant differences between samples were detected by a two-sided Student's t-test. b) Annotated mass spectra for mono-glutamylation of SdeA E860 from SidJ glutamylase in vitro reactions. c) Annotated mass spectra for di-glutamylation of SdeA E860 from SidJ glutamylase in vitro reactions.

Extended data Fig. 5. Analysis of isobaric glutamylated peptide species

a) Extracted Ion Chromatogram (XIC) of +4 charged -QVGRHGEGTESEFSVYLPEDVALVPVK- + 1 Glutamate, showing no other co-existing mono-glutamylated versions of the catalytic peptide besides E860 glutamylation b) XIC of the catalytic peptide plus two glutamates (charge +4) is separated into three different peaks that could be assigned to Di-Glutamylation of E860, as well as two mono-glutamylations on the peptide on E860 plus E857 as well as E862. Annotated spectra are shown below. c) XIC of the catalytic peptide plus three glutamates (charge +4) is separated into three different peaks that could be assigned to di-glutamylation of E860 plus mono-glutamylation of E857 and parallel mono-glutamylation of E857, E860 and E862, a third peak could not be clearly assigned. Annotated spectra of the annotated species are shown below. For panels a-c, In-vitro glutamylation and label-free LC-MS analysis were performed in 3 biologically independent experiments with similar results. Corresponding quantitative information is shown in Figure 3d.

Extended data Fig. 6. Cryo-EM data processing and 3D reconstruction

a) Size-exclusion profile of SidJ/CaM complex, elution fractions were analysed by SDS PAGE. Marked fractions were used for cryo-EM sample preparation. This experiment was repeated thrice independently with similar results. b) A representative electron micrograph for the cryo-EM dataset collected. c) Reference-free representative 2D class averages of the SidJ/CaM complex. Secondary structure features are already visible in projection images. Used number of particles to obtain a 2D class average is mentioned accordingly in each subpanel. d) Gold-standard Fourier Shell Correlation plot between two independently refined half-maps, FSC_0.143=4.15 Å resolution. FSC between phase-randomized half-maps show, as expected, a rapid drop of correlation beyond randomization point. e) Crystal structure of SidJ/CaM (PDB:6OQQ) is fitted into the cryo-EM 3D reconstruction f) A part of panel d is magnified to highlight the difference between the crystal structure and the cryo-EM map.

Extended data Fig. 7. Cryo-EM single particle analysis pipeline

Data processing strategy for cryo-EM on the SidJ-CaM complex. Particle picking on 2423 micrographs using WARP resulted in the identification of 1.5 million particles. The particle coordinates were imported into Relion and particles were extracted with a 2-fold binning factor. After 2D classification and an initial 3D classification a 3D class with clear secondary structure features and 370 k particles was identified. The particles of this class were re-extracted with full pixel size and 3D refined resulting in a 4.52 Å model. Two additional rounds of 3D classification and 3D refinement improved the resolution of the model to 4.15 Å. Final particle polishing and CTF refinement of the remaining particles did not result in a nominal improvement of the resolution.

Extended data Fig. 8. Cryo-EM model refinement

a) FSC between model and the map and cross-validation of the model fitting. FSC_0.5 = 4.3 Å for the model vs map (sum). Half-map cross validation procedure does not show significant overfitting in the refined model. b) Local resolution analysis (Relion) shows variation in the map resolution ranging from 3.9 to 5.1 Å. c) Overview of the model fitting into the map in the same orientation as in b. d) An example of the cryo-EM map quality with the atomic model fitted in, showing clear density for side chains.

Extended data Fig. 9. Ligand binding sites of SidJ/CaM complex

a) Crystal structure of SidJ/Yeast CaM (PDB:6OQQ) is shown marking the two proposed catalytic sites and the bound ligands. b) EM map showing cryo-EM density in the migrated pocket fitted with AMPPNP. c) EM map showing unassigned cryo-EM density in the canonical pocket.

Extended data Fig. 10. SidJ-dependent glutamylation during Legionella infection

a) Raw264.7 macrophages were infected with WT, ΔSidJ or ΔSidE Legionella for 3h. Lysates were used for immunoprecipitation with polyglutamylation antibody followed by immunoblotting with SdeA. n.i indicates samples that were not infected with bacteria. This experiment was repeated twice independently with similar results. b) A549 cells were infected with different strains of Legionella pneumophila for 3h. Cells were fixed and immunostained with antibodies against calnexin and poly-glutamylation (GT335). DAPI staining marks the nucleus and cytosolic bacteria. Yellow arrows indicate bacteria in infected cells. Region of interest (ROI) is defined as Calnexin stained LCV. 80x100 um² ROIs were chosen in the perinuclear region of cells followed by quantification of Mander’s coefficient (m) using Coloc2 plugin in FIJI. m represents the fraction of calnexin-positive LCVs that are also positive for poly-glutamylation. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. Number of ROIs (n) = 80 from 30 cells was used for quantification. *** indicates p<0.001 by 2-tailed type 3, Students t-test. p-value (WT vs ΔsidJ) =6.18x 10^-29, p value (WT vs ΔsidE) = 1.09x 10^-5. This experiment was repeated twice independently with similar results. c) Glutamylated proteins were isolated from WT, ΔsidE and ΔsidJ Legionella infection experiments using GT335 antibody and quantified using mass spectrometry. Correlation between WT vs ΔsidJ and ΔsidE vs. ΔsidJ quantifications are plotted (inset) showing the most correlated proteins in the abovementioned two quantifications. Legionella infection and label-free LC-MS analysis was performed with n=3 biologically independent experiments. Significant differences between samples were detected by a permutation based FDR 0.05 corrected two-sided Student's t-test. Proteins were labelled as significant, if they were above the FDR 0.05 threshold in at least one comparison (ΔsidE and WT Legionella compared to ΔsidJ infected cells). Proteins with a Log2 ratio above 2 (mean) in WT samples were labelled as highly enriched compared to ΔsidJ infected cells in samples from WT and well as ΔsidE infected cells.

Figure 1. SidJ inhibits the Ub-ADP-ribosylation activity of SdeA.

a) SidJ and SdeA constructs were expressed as indicated in HEK293T cells and ubiquitin modification was probed using the Ub antibodies abcam-Ub and CS-Ub as described previously. b) Yeast strain W303 was transformed using the indicated combination of constructs. Serial dilutions of transformed yeast were spotted on dextrose (repressing) or galactose (inducing) containing plates. SdeA EE/AA indicates mutation of E860, E862 to alanine. c) Purified SdeA from HEK293T cells expressing SdeA alone or in combination with SidJ was used in ε-NAD⁺ hydrolysis assays. Increase in the fluorescence indicates Ub-ADP ribosylation. d) SdeA purified from E.coli was incubated with HEK293T cell lysate containing SidJ or depleted of SidJ. SdeA was subsequently purified using glutathione agarose beads and used in ε-NAD⁺ hydrolysis assays. Experiments in panels a-d were repeated thrice independently with similar results. For gel source data, see Supplementary Fig. 1.

Figure 2. Calmodulin is a host-specific factor activating SidJ

a) GFP-SidJ was expressed in HEK293T cells. After immunoprecipitation of SidJ, the sample was analyzed using mass spectrometry and enriched proteins were quantified using the MaxQuant label-free algorithm. n= 3 biologically independent experiments. Statistically significant differences between samples were detected by a permutation based False Discovery Rate (FDR) 0.05 corrected one-sided Student's t-test b) Various SidJ constructs were used to pull-down Calmodulin to test the effect of IQ motif mutations and deletions. c) SdeA was expressed in HEK293T cells alone and in combination with WT SidJ or SidJ lacking the IQ motif region. Ubiquitin modification was followed using Ub-abcam and Ub-CS antibodies. d) Isothermal titration calorimetry was performed to measure the affinity between SidJ and apo or Ca²⁺-bound CaM. e) Interaction between GFP-SidJ and Calmodulin was analyzed using co-IP in low (treatment with BAPTA) or high (treatment with thapsigargin, Tg) cytosolic Ca²⁺ levels. Experiments in panels b-e were repeated thrice independently with similar results. For gel source data, see Supplementary Fig. 1.

Figure 3. SidJ is a calmodulin-dependent glutamylase

a) Co-expression of SdeA with SidJ and of SdeA alone was performed in n=3 biologically independent experiments. Samples were labeled with TMT 6 plex reagent and analyzed in one LC-MS run. Significant differences between samples were detected by a two-sided Student's t-test. Quantitative analysis of SdeA peptides under these conditions is represented in a volcano plot. b) Annotated mass spectra for glutamylation of SdeA E860 c) For quantitative information about the modification, we purified GFP-SdeA expressed alone or co-expressed with SidJ and digested it with LysC. TMT quantification revealed mass close to quantitative conversion of the peptide spanning the catalytic loop to its glutamylated form. n=3 biologically independent experiments. Significant differences between samples were detected by a two-sided Student's t-test. d) Intensities of differentially modified versions of the QVGRHGEGTESEFSVYLPEDVALVPVK peptide (catalytic center of mART domain) are plotted as fraction of total SdeA intensity over the time of an in vitro reaction, proving E860 mono-glutamylation to be the primary reaction of SidJ. In vitro glutamylation and label-free LC-MS analysis was performed in n=3 biologically independent experiments. Data points are mean centered; error bars indicate standard deviation.

Figure 4. Glutamylation of SidEs and host proteins during Legionella infection

a) Comparison of the crystal structure of SidJ/Yeast CaM (PDB:6OQQ) with the Cryo-EM structure showing r.m.s.d values of various regions marked accordingly. b) A549 cells were infected with different strains of Legionella pneumophila for 3h. Cells were fixed and immunostained with antibodies against calnexin (Caln) and polyglutamylation (Glu). DAPI staining marks the nucleus and cytosolic bacteria (Leg). Number of LCVs (marked by calnexin) that are positive for poly-glutamylation are counted in FIJI, % of polyglutamylated LCVs is plotted for cells infected with different strains of Legionella. Data represents 100 LCVs taken from 30 cells over n=3 biologically independent experiments. Error bars indicate standard deviation. *** indicates p<0.001 using 2 tailed, type-3 Student's t-test. p value= 8.45x 10^-15 (WT vs ΔsidJ); p value =5.14X10^-11 (WT vs ΔsidE). c) Glutamylated proteins were isolated from WT and ΔsidJ Legionella infection experiments using GT335 antibody and quantified using mass spectrometry. Data is represented in volcano plot (inset) showing the most enriched proteins in WT. n=3 biologically independent experiments. Significant differences between samples were detected by a permutation based FDR 0.05 corrected two-sided Student's t-test. Proteins with Log2 ratio above 2 (mean) were labelled as highly enriched in WT compared to ΔsidJ infected cells. For gel source data, see Supplementary Fig. 1.