EMBO/soda-nel huggingface dataset:
Structural basis for the specificity of PPM1H phosphatase for Rab GTPases (2021)
Figure 1

Figure 1: Structure of Rab-specific PPM1H phosphatase.

(A) Domain organization of PPM1H, PPM1J, and PPM1A. The annotation of regions (anchor, flap domain) is discussed in the text. The loop deletion (188-226) that was engineered to improve diffraction is indicated.

(B) Ribbon model of the enzyme with a view to the catalytic cleft that contains two Mg2+ ions (cyan spheres). The N-terminal region is magenta (33-71), the flap domain is a wheat colour, and the β-sheet motif is green. The loop deletion (188-226) connects α1/α2 on the opposite face relative to the active site. The back view of the enzyme is also shown with a 180° rotation around the axis indicated. Parts of the anchor (RPxFL motif, magenta) that interact with the globular core are shown as stick models, and discussed in the text. The β1 strand is orange to emphasize its non-canonical conformation due to the presence of the preceding anchor.

(C) Comparisons of the flap domain of PPM1H with PPM1A (left) and the tudor domain (right). Apart from a conserved loop (dotted circle) which forms an interface with the catalytic domain, the sequences and structures of flaps are diverse among the PPM family.

(D) The indicated amounts of recombinant wild-type and mutant PPM1H or PPM1J (with a His-Sumo N-terminal tag, expressed in E. coli) were incubated in vitro with 2.5 µg pThr72 phosphorylated Rab8a (left) or pThr73 phosphorylated Rab10 (right) for 20 min in the presence of 10 mM MgCl2 in 40 mM HEPES pH 7.5 buffer. Reactions were terminated by addition of SDS Sample Buffer and analyzed by Phos-tag gel electrophoresis that separates phosphorylated (slow migrating) and dephosphorylated Rabs. The gel was stained with Instant Blue Coomassie. D288A is a substrate-trapping (inactive) variant of PPM1H and was used as a control.
Structural basis for the specificity of PPM1H phosphatase for Rab GTPases (2021)
Figure 2

Figure 2: Crosslinking and docking analysis suggest that the flap domain binds to pRab8a.

(A) SDS-PAGE analysis of the PPM1H(D288A): pRab8a complex in the presence of DSBU crosslinker. Control migration of proteins are on the left. NP, non-phosphorylated Rab8a; P, phosphorylated Rab8a. The migration of control and crosslinked proteins is marked on the right.

(B) Crosslinked peptides from PPM1H and pRab8a are mapped onto the sequence. The flap domain forms extensive crosslinks with pRab8a. In addition, two flexible loops (104-142, 188-226) also have multiple crosslinks with the substrate.

(C) Ribbon model of pRab8a (left) and the switch 2 phosphopeptide (right) docked onto the active site of PPM1H. The crosslinks shown between PPM1H and pRab8a are within accepted distance constraints (32 Å) for DSBU (He et al., 2015).

(D) Kinetics of phosphate hydrolysis. 25 nM recombinant wild-type PPM1H was incubated with increasing concentrations of pThr72 phosphorylated Rab8a (GTPγS or GDP) as described in Materials and Methods. Initial velocity (V0) was calculated by dividing the concentration of released phosphatase (μM) by time (min) and plotted against substrate concentration for pThr72 phosphorylated Rab8a[GTP bound conformation] (blue) and pThr72 phosphorylated Rab8a[GDP bound conformation] (red). The experiments were repeated twice, and both data points are shown in curves. Line fittings for the left and middle panel were performed using the mean of the two values. Kinetic constants (Kcat, Vmax, Km) were obtained using GraphPad software, and their uncertainties (±) correspond to the SE of mean.

(E) Kinetic analysis of PPM1H as in (D) using 50 nM PPM1H and phosphopeptide substrates, as described in Materials and Methods. Initial velocity (V0) was calculated by dividing the concentration of phosphatase (μM) by time (min) and plotted against substrate concentration for pThr72 Rab8a phospho-peptide (blue) and Rab10 pThr73 phospho-peptide (red). Each experiment was performed twice (individual data points shown).

(F) Side by side comparison of the catalytic activity against protein and peptide by in vitro malachite green time course analysis. 50 nM recombinant wild-type PPM1H was incubated with 16 μM Rab8a GTPγS pThr72 phosphorylated protein (blue) or Rab8a pThr72 phosphorylated peptide (red) for indicated times and analysed as described in Materials and Methods. The experiments were repeated 3 times. The error bars represent SE of mean of the technical replicates.
Structural basis for the specificity of PPM1H phosphatase for Rab GTPases (2021)
Figure 3

Figure 3: PPM1H is a dimer.

(A) Model of the dimeric form of MnPPM1HWT-LD. Each monomer of PPM1H has an A or B prefix. Flap domains mediate the dimer and are shown in dark blue/wheat colors. The view is down a pseudo two-fold formed by α3 in the flap domain. Dotted lines denote the flexible loop (183-235) that connects α1/α2 in the catalytic domain. The dimeric organization of PPM1H positions this loop next to substrates, shown as docked models of pRab8a in green ribbons (transparent). The phosphoryated switch 2 helix of pRab8a is red. Yellow spheres indicate site of a double mutation in the flap domain (G357E+A359E) that disrupts a contact with α2 of the catalytic domain.

(B) SEC-MALS analyses of PPM1HWT-LD and PPM1H2Glu-LD, showing that the WT enzyme is a dimer (104.5±2 kDa) while the double mutant is a monomer (53.6±1 kDa). The calculated molecular weight of His6-tagged PPM1HWT-LD used for the experiment is approximately 52.1 kDa. The errors were calculated from the SE of technical replicates from the data represented as a line across the central peaks in the gel filtration column. Light scattering from 15/18 possible angles (DAWN-EOS, Wyatt Corp) and the refractive index change relative to buffer (Optilab TrEX) were collected every second along these time points. Subsequently, data were processed using Astra 7.1 software (Wyatt Corp) to generate a weight-averaged molecular mass (y-axis) plotted against time (x-axis). There were approximately 130 technical data points for PPM1HWT-LD and 60 technical data points for PPM1H2Glu-LD.

(C) His6-SUMO-tagged full-length variants of PPM1H were used for catalytic assays (left panel). 3 μg of recombinant wild-type or indicated mutant PPM1H proteins were resolved on 4-12% Bis-Tris gradient gel and stained with Instant Blue Coomassie. Right, mass photometry histogram for 40nM His6-SUMO-PPM1H WT (blue) and His6-SUMO-PPM1H 2Glu (brown), where WT is 130kDa (±15.8 kDa, with 1004 single molecules counted) and 2Glu is 83kDa (±13.9 kDa, with 4117 single molecules counted). The calculated molecular weight of the fusion protein is approximately 68.5 kDa.

(D) In vitro malachite green assay time course of recombinant His6-SUMO-tagged PPM1H wild-type (blue), 2Glu (red), or D288A (grey) against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS, left), 32 μM pThr72 phosphorylated Rab8a peptide (middle), or 32 μM pThr73 phosphorylated Rab10 peptide (right). The experiments were repeated 4 times. The error bars represent SE of mean of the technical replicates.
Structural basis for the specificity of PPM1H phosphatase for Rab GTPases (2021)
Figure 4

Figure 4: The flap domain of PPM1H is a determinant of Rab specificity.

(A) Domain organization of chimeric variants for in vitro and cellular assays.

(B) 3 μg of recombinant proteins were resolved on 4-12% Bis-Tris gradient gel and stained with Instant Blue Coomassie.

(C) In vitro malachite green assay time course to determine enzyme activity of recombinant PPM proteins (25 nM for the protein substrate, 50 nM for the peptide substrate) against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS). The experiments were repeated 3 times. The error bars represent SE of mean of the technical replicates.

(D) In vitro malachite green assay against 32 μM pThr72-Rab8a peptide (left) and pThr73-Rab10 peptide mimic (right). The experiments were repeated 3 times. The error bars represent standard error (SE) of mean of the technical replicates.

(E, F) (E) HEK293 cells overexpressing indicated constructs were treated with ± 200 nM MLi-2 for 90 min and then lysed. 10 μg whole cell lysate was subjected to immunoblot analysis with the indicated antibodies at 1 μg/ml final concentration and membranes were analysed using the OdysseyClx Western Blot imaging system. Left, each lane represents cell extract obtained from a different dish of cells (two biological replicates per condition without MLi-2 treatment, one biological replicate per condition with MLi-2 treatment). Right, the ratio of phospho-Rab8a/total Rab8a was quantified using Image Studio software and data presented relative to the phosphorylation ratio observed in PPM1H wild-type expressing cells. (F) As in (E) assessing phospho-Rab10 levels. For quantitation the two replicates from the presented blots were used. The data points are shown on the graphs with the bar levels representing the mean value of the biological replicates.
Structural basis for the specificity of PPM1H phosphatase for Rab GTPases (2021)
Figure 5

Figure 5: Mutagenesis and functional assays identify determinants of catalysis.

(A) Sites for targeted mutagenesis on the flap domain and active site Lys88 residue. The central ribbon model provides context for the conserved loop (left) and epitopes distant from the active site in the flap domain (right). Residues subjected to mutagenesis are shown as stick models, with dotted envelopes in the central panel for emphasis. Docked pRab8a (yellow ribbons, right) highlights its proximity to mutagenized residues in the flap domain. The sequences of the conserved loop motif are shown below the left panel for several PPMs with known 3-D structures (PPM1H, PPM1K, PPM1B, PPM1B).

(B) In vitro malachite green assay time course of recombinant PPM1H mutants against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS, left), 32 μM pThr72 phosphorylated Rab8a peptide (middle), or 32 μM pThr73 phosphorylated Rab10 peptide (right). The experiments were repeated 4 times. The error bars represent SE of mean of the technical replicates.

(C) 3 μg of recombinant wild-type or indicated mutant PPM1H proteins were resolved on 4-12% Bis-Tris gradient gel and stained with Instant Blue Coomassie.

(D) HEK293 cells overexpressing indicated constructs were treated and analysed as described in Figure 4E.

(E) Activity of 25 nM PPM1H flap domain mutants against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS) using the malachite green assay time course. The experiments were repeated 3 times. The error bars represent SE of mean of the technical replicates. Quality of purified proteins (right panel) is shown using the protocol in (C).

(F) HEK293 cells overexpressing indicated constructs were treated and analysed as described in (D), with pRab10 analysed in the upper panel, and pRab8a in the lower panel.
Structural basis for the specificity of PPM1H phosphatase for Rab GTPases (2021)
Figure 6

Figure 6: Anchor of PPM1H is a folding motif.

(A) Interactions between the anchor of PPM1H against the electrostatic surface of the core catalytic domain. The region is placed in context with the dotted box on a ribbon model of PPM1H (right).

(B) Incremental deletions of the N-terminus from residues 1-37 to 1-44, one residue at a time. Upon deletion of R43 (44-end), a reduced level of soluble PPM1H expression has no significant catalytic activity.

(C) Sequence alignment of the N-terminal regions of the evolutionarily related PPM1H/J/M enzymes. The degree of conservation is above the alignment, and residue numbers correspond to PPM1H. A conserved anchor motif (RPxFL) is annotated below the sequences.
Streptococcus pneumoniae highjacks host autophagy by deploying CbpC as a decoy for Atg14 depletion (2020)
Figure 1

Figure 1 - The pneumococcal CbpC protein can activate autophagy, but can also facilitate intracellular pneumococcal survival.

A Pneumococcal CBPs used in (B).

B Confocal images of HeLa cells transiently expressing GFP-CBPs or GFP. The dotted lines show each cell shape. Scale bars, 10 µm.

C Confocal images of HeLa cells transiently expressing GFP-CbpCT4 and mCherry-LC3 (upper). The fluorescence intensities of GFP-CbpCT4 (green) and mCherry-LC3 (red) along the arrow are shown in the graph at the bottom.

D Lysates from 293T cells transiently expressing GFP-Cbps or GFP were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against LC3, GFP, or actin.

E Lysates from 293T cells transiently expressing GFP-CbpCT4, GFP-LytR, or GFP in the presence or absence of chloroquine were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against LC3, GFP, or actin.

F MEFs were infected with S. pneumoniae R6 WT or ΔcbpF for the indicated periods, and the intracellular survival of bacteria expressed as the number of CFUs.

G p62-KO MEF cells infected with S. pneumoniae R6 ΔcbpF/pCbpFR6-FLAG for 2 h were fixed and stained with DAPI and an anti-FLAG antibody, and representative epifluorescence images are shown. Scale bars, 10 µm.

Data information: In (F), data represent mean ± SEM of 3 biological replicates. Student's t-test was used to calculate statistical significance. *P < 0.05, **P < 0.01
Streptococcus pneumoniae highjacks host autophagy by deploying CbpC as a decoy for Atg14 depletion (2020)
Figure 2

Figure 2 - Identification of Atg14 as a CbpC-interacting protein.

A Quantification of the band intensities following streptavidin-based pulldown assays using the in vitro-translated biotinylated CbpCT4 and FLAG-tagged autophagy-related proteins shown in Fig EV2A.

B Quantification of the band intensities following GST-pulldown assays with 293T cell lysates expressing the autophagy-related proteins and recombinant GST-CbpFR6 shown in Fig EV2D.

C Lysates from 293T cells transiently expressing 3Myc-Atg14 or Atg5-3Myc, and GFP-CbpCT4 or GFP were immunoprecipitated with GFP-Trap, and the bound proteins were analyzed by immunoblotting.

D Lysates from 293T cells transiently expressing GFP-CbpCT4 or GFP, and 6Myc-Atg14 were immunoprecipitated with an anti-Myc antibody and Protein G PLUS-Agarose. The bound proteins were analyzed by immunoblotting.

E Recombinant GST-CbpFR6 or GST, and 3Myc-Atg14 were used for GST-pulldown assays. The bound proteins were analyzed by immunoblotting using an anti-Myc antibody.

F MEFs infected with R6 ΔcbpF expressing CbpFR6-FLAG for 2 h were subjected to IP and assayed using anti-HA agarose beads. The bound proteins were analyzed by immunoblotting using an anti-FLAG antibody. Schematic representations indicating assay designs in C-F are shown below the blots.
Streptococcus pneumoniae highjacks host autophagy by deploying CbpC as a decoy for Atg14 depletion (2020)
Figure 3

Figure 3 - CbpC could bind to p62 and act as a decoy for autophagic degradation of Atg14 to suppress autophagic degradation.

A Lysates from MEFs stably expressing HA-Atg14 infected with S. pneumoniae TIGR4 WT or ΔcbpC for 1, 2, or 3 h in the presence or absence of cycloheximide were subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies.

B MEFs/HA-Atg14 cells were infected with S. pneumoniae TIGR4 WT or ΔcbpC for 1, 2, or 3 h in the presence or absence of Bafilomycin A1 (BafA1). The lysates were subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies.

C-F (C, E) A549 cells were infected with S. pneumoniae R6 WT or ΔcbpC for 2 h and fixed and stained with DAPI and an anti-Atg14 or -GM130 antibody. Representative epifluorescence images are shown. Scale bars, 10 µm. (D, F) The percentages of perinuclear-localizing Atg14 containing cells in (C) or intact Golgi apparatus-containing cells in (E) were quantified.

G S. pneumoniae (Sp) invasion-permissive A549 cells (marked with GFP) and Sp invasion-non-permissive (pIgR knocked down) A549 cells were co-cultured at a 1:3 ratio. Atg14-disappearance experiments were conducted, and representative epifluorescence images are shown. Scale bars, 10 µm. The lines show each cell shape and the arrows show invasion-permissive A549 cells marked with GFP.

H The percentages of perinuclear-localizing Atg14 containing cells in (G) were quantified.

I Lysates from 293T cells transiently expressing GFP-CbpC or GFP, and HA-Atg14 were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against HA or GFP.

J Lysates from 293T cells transiently expressing GFP-CbpCT4 or GFP were immunoprecipitated using GST-GFP-Nanobody. Additionally, beads were mixed with lysates from 293T cells transiently expressing p62-3Myc and HA-Atg14, and bound proteins were analyzed by immunoblotting.

K A549 cells treated with the indicated siRNAs were infected with S. pneumoniae R6 WT or ΔcbpF for the indicated durations. The cells were fixed and stained with DAPI and an anti-Atg14 antibody, and percentages of perinuclear-localizing Atg14 containing cells were quantified.

L Lysates from MEFs stably expressing GFP-CbpCT4 or GFP in the presence or absence of rapamycin or chloroquine were subjected to SDS-PAGE and immunoblotted using antibodies against LC3 or p62.

M Quantification of NanoBRET signals in 293A cells transiently expressing HaloTag-Stx17 and Nanoluc-Atg14 in the presence or absence of GFP-CbpC or GFP.

Data information: In (D, F, H, K, M), data represent mean ± SEM of 3 biological replicates. Student's t-test was used to calculate statistical significance. *P < 0.01, N.S., not significant.
Streptococcus pneumoniae highjacks host autophagy by deploying CbpC as a decoy for Atg14 depletion (2020)
Figure 4

Figure 4 - The loop structure in the CbpC dp3 domain enabled interaction with the CCD of Atg14.

A Diagram of the Atg14 derivatives used in (B).

B GST-pulldown assays performed using GST-CbpFR6 or GST and lysates from 293T cells expressing GFP-Atg14 derivatives or GFP. The bound proteins were analyzed by immunoblotting using an anti-GFP antibody.

C Schematic representation of the CbpFR6 derivatives used in (D) and (E). The domains are color-coded according to the crystal structure report for CbpFR6 (lower left) [10].

D, E GST-pulldown assays using GST-CbpFR6 derivatives and lysates from 293T cells expressing GFP-Atg14 CCD or GFP. The bound proteins were analyzed by immunoblotting using an anti-GFP antibody. Each GST-CbpFR6 derivative-bound bead was confirmed by Coomassie brilliant blue (CBB) staining.

F Structure of CbpFR6 (PDB ID: 2V04). The overall structure (left) and dp3 domain (right) of homology-modeled structures are shown. The side chains of Y83 and E95 and bound choline molecules are shown as green and yellow sticks, respectively.

G The amino acid sequences of the CbpCT4/CbpFR6 mutants used in (H), (I), and (J).

H GST-pulldown assays performed using GST-CbpFR6 mutants or GST and lysates from 293T cells expressing GFP-Atg14 CCD or GFP. The bound proteins were analyzed by immunoblotting using an anti-GFP antibody. Each GST-CbpFR6 mutant-bound bead was confirmed by CBB staining.

I Lysates of 293T cells transiently expressing GFP-CbpCT4 mutants or GFP were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against LC3, GFP, or actin.

J Confocal microscopy images of HeLa cells transiently expressing the indicated GFP-CbpCT4 mutants. Scale bars, 10 µm.
Streptococcus pneumoniae highjacks host autophagy by deploying CbpC as a decoy for Atg14 depletion (2020)
Figure 5

Figure 5 - Domain analysis of the CbpC-p62 interaction.

A Schematic representation of the CbpCT4 variants used in (B), (E), (H) and (I).

B Lysates from 293T cells transiently expressing p62-3Myc and GFP-CbpCT4 variants were immunoprecipitated using a GST-GFP-Nanobody fusion protein. Bound proteins were analyzed by immunoblotting.

C Diagram of the ∆DD mutation in dp5 loop domain.

D Structures of the dp5 and dp6 domains in CbpFR6. The side chains of D167-D168 are shown. E Lysates from 293T cells transiently expressing p62-3Myc and GFP-CbpCT4 variants were immunoprecipitated using the GST-GFP-Nanobody protein. Bound proteins were analyzed by immunoblotting.

F Diagram of the p62 variants used in (G).

G Lysates of 293T cells transiently expressing GFP-CbpCT4 and p62-3Myc variants were immunoprecipitated with the GST-GFP-Nanobody protein. Bound proteins were analyzed by immunoblotting using the indicated antibodies.

H A549 cells infected with the indicated S. pneumoniae strains for 2 h were fixed and stained with DAPI and an anti-Atg14 antibody, and the percentages of perinuclear-localizing Atg14 containing cells were quantified.

I Representative epifluorescence images in (H) are shown. Scale bars, 10 µm.

Data information: In (H), data represent mean ± SEM of 3 biological replicates. Student's t-test was used to calculate statistical significance. *P < 0.01.
Streptococcus pneumoniae highjacks host autophagy by deploying CbpC as a decoy for Atg14 depletion (2020)
Figure 6

Figure 6 - Comparison of the Atg14-binding capacities and autophagy-induction activities of CbpC family proteins.

A Comparison of the amino acid sequences of the dp3 domains from CbpCT4 orthologs of other Streptococcus strains. The identical amino acid residues in all sequences in (A) and (B) are indicated by asterisk.

B Comparison of the amino acid sequences of the dp3 domains in CbpCT4 orthologs CbpJ, CbpFT4, CbpFR6, and PcpC.

C Full-length structures of CbpCT4 (PDB ID: 2V04), CbpJ (PDB ID: 6JYX), and PcpC (homology model).

D GST-pulldown assays using the indicated GST-fusion proteins or GST and lysates from 293T cells expressing GFP-Atg14 CCD or GFP. Bound proteins were analyzed by immunoblotting using an anti-GFP antibody.

E Lysates from 293T cells expressing GFP-fusion proteins or GFP were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against LC3, GFP, or actin.

F Confocal images of HeLa cells transiently expressing GFP-fusion proteins. Scale bars, 10 µm.

G Diagram of CbpCT4/CbpFR6, CbpJ, and PcpC dp3 domain swaps with CbpJ or CbpCT4/CbpFR6.

H, I GST-pulldown assays using the indicated GST-fused proteins or GST and lysates from 293T expressing GFP-Atg14 CCD or GFP. Bound proteins were analyzed by immunoblotting using an anti-GFP antibody.

EXPANED VIEW FIGURE LEGENDS
RNA-binding protein RBM47 stabilizes IFNAR1 mRNA to potentiate host antiviral
activity (2021)
Figure 1

Figure 1. RBM47 is an interferon-stimulated gene.

A Expression of RBM47 was induced by DENV infection in 293T, HFF, HUVEC, and THP-1 cells. Cells were infected with DENV-2 at an MOI of 1 for 12 or 24 h. The expression of RBM47 mRNA (12 and 24 h post infection, hpi.) and protein (24 hpi.) were compared between virus-infected and non-infected cells (PBS). The PCR results are represented as the means ± SD of n = 3 biological replicates.

B, C Both mRNA and protein levels of RBM47 were elevated in SeV-infected (B) or IFN-α-stimulated (C) 293T, HFF, HUVEC, and THP-1 cells. Cells were infected with SeV (MOI = 1) or stimulated with IFN-α for 6 or 12 h, and the expression of RBM47 mRNA (at 6 and 12 h post treatment) and protein (at 6 and 12 h post treatment) were tested. The PCR results are represented as the means ± SD of n = 3 biological replicates.

D Three putative STAT1 binding motifs in the RBM47 gene promoter region as predicted by using JASPAR (http://jaspar.genereg.net/).

E The pGL3-RBM47 promoter plasmid (pGL3-47) or the empty vector was transfected into 293T cells, and RBM47 promoter activities were tested upon SeV infection or IFN-α stimulation.Data are represented as the means ± SD of n = 3 biological replicates.

Data information: Data in A-C and E are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).