Research ArticleMICROBIOLOGY

Inverse control of Rab proteins by Yersinia ADP-ribosyltransferase and glycosyltransferase related to clostridial glucosylating toxins

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Science Advances  11 Mar 2020:
Vol. 6, no. 11, eaaz2094
DOI: 10.1126/sciadv.aaz2094
  • Fig. 1 Domain architecture of YART and YGT and analysis of the translocation and protease domains.

    (A) Domain comparison of YART, TcdB, and YGT. The N-terminal domain of YART is an ADP-ribosyltransferase (ART), whereas the N terminus of YGT and TcdB harbors glycosyltransferases (GTD). Like TcdB, YART and YGT have an autocatalytic cysteine protease domain (CPD) and a translocation domain (TD). The C terminus of the TD of TcdB contains a receptor-binding region (RBR). Only TcdB has a CROP domain. Sequence identities of regions indicated by amino acid numbers are in percentage. Arrowheads mark split products of YART and YGT. Cys500 and Leu917 of YART and Cys668 and Leu1092 of YGT indicate the essential cysteine of CPDs and the critical leucine of TDs. (B and C) Membrane activity in lipid bilayer by YART, YGT, and their mutants YART L917K and YGT L1092K. Pore formation was induced by acidification to pH 4.5. The mutants did not induce increase in electric conductance, even after prolonged incubation (10 min). (D and F) In vitro processing of YART1–632 and YGT1–1998 at the indicated concentrations of InsP6, resulting in fragments YARTC-term and YGT1–781. (E and G) Inhibition of autocatalytic cleavage in mutants YGT1–1998 L781A, YGT1–1998 C668A, and YART1–632 C500A in the presence of InsP6. Western blots with anti-His antibody are shown. WT, wild type.

  • Fig. 2 ADP-ribosylation by YART and glycosylation by YGT.

    (A) Sequence comparison of ADP-ribosyltransferases [YART, pertussis toxin (PT; P04977), cholera toxin (CT; P01555), Clostridium botulinum C3 toxin (C3; P15879), and C. difficile ADP-ribosyltransferase (CDTa; Q9KH42)]. Conserved amino acids are marked in red; R-S-E amino acid motifs are indicated by asterisk. (B) Spot test of S. cerevisiae growth. Yeast cells, expressing YARTA or mutants under control of a galactose-dependent promoter, were spotted on glucose (GLC) and galactose (GAL) plates in fivefold dilutions. OD, optical density. (C) ADP-ribosylation of Rab proteins. Rab5A (▲), Rab31 (■), and Rab17 (●) (each 5 μg) were incubated at the indicated concentrations of YARTA with 1 μM [32P]NAD+ for 15 min, followed by SDS–polyacrylamide gel electrophoresis (PAGE) and phosphorimaging (data are means ± SD; n = 3). (D) Substrates of YART. Indicated guanosine triphosphatases (GTPases) (Rab31, 0.2 μg; other, 2 μg) were incubated with YARTA (3 μM) and [32P]NAD+ as in (C). Coomassie staining and autoradiogram are shown. (E) Sequence alignment of the DXD region of glucosyltransferase toxins [YGT, C. perfringens TpeL (A2PYQ6), C. novyi α-toxin (Q46149), and C. difficile TcdB (P18177) and TcdA (P16154)]. (F) Growth spot test of YGTG or mutants in yeast cells as in (B). (G) Glycosylation of Rab5A (▲) and Rab31 (■) (each 5 μg) with YGTG and UDP-[14C]GlcNAc (10 μM) for 15 min. Analysis of labeled proteins as in (C). (H) Substrates of YGT. Indicated GTPases (each 2 μg) were incubated with YGTG (0.5 μM) and UDP-[14C]GlcNAc as in (G).

  • Fig. 3 Structure of YGTG.

    (A) The YGTG structure displays a typical GT-A glycosyltransferase fold (yellow) comprising a seven-stranded β sheet surrounded by α helices and a C-terminal five α-helical bundle (violet). UDP (black sticks) and Mn2+ (purple sphere) are bound centrally. Helix 17 and the loop that encloses the ligand binding site are highlighted in blue. (B) Structural superposition of YGTG [colored as in (A)] with TcdB from C. difficile [Protein Data Bank (PDB) ID: 2BVL, green] showing the GT-A glycosyltransferase fold. While the N-terminal four-helix bundle of TcdB is absent in YGTG, four helices at the C terminus of YGTG are not conserved in clostridial toxins. (C to F) Closeup views of the active site showing the binding mode of UDP (black) with Mn2+ and K+ ions represented as spheres (purple and brown, respectively) and YGTG represented as a cartoon with relevant residues in sticks and colored as in (A). (C) The uridine base moiety is coordinated by a π-stacking interaction with Trp23 and by several direct and water-mediated hydrogen bonds (dashed lines). (D) The ribose moiety is coordinated by a series of hydrogen bonds with its hydroxyl groups. (E) The diphosphate from UDP is stabilized via interactions with the Mn2+ ion. Mn2+ is coordinated with aspartate side chains of the DXD motif, Glu427, and water molecules. Note the presence of a K+ ion in close vicinity. (F) The C-terminal loop of the GT-A fold encloses the nucleotide-binding pocket comprising direct interactions between the diphosphate moiety and Ser430, Ser431, and Trp432.

  • Fig. 4 YART- and YGT-induced modifications of Rab5.

    (A) Localization of the sites of Rab5A that are modified by YGT-induced GlcNAcylation and YART-induced ADP-ribosylation. Modification sites are from MS analyses and introduced into the Rab5A structure (PDB ID: 1N6H). Switch-1 (SwI) and switch-2 (SwII) regions are indicated in blue and dark green, respectively. Light green spheres represent the Mg2+ ion, while guanosine triphosphate (GTP) (or a GTP analog) is indicated by red arrow. While YGT modifies Rab5 in Thr52 (black arrow) by GlcNAcylation, YART ADP-ribosylates Gln79 (black arrow). (B) ADP-ribosylation or GlcNAcylation of mutant Rab5 proteins. Wild-type and mutant Q79L or T52A Rab5 proteins (each 2 μg) were incubated with 1 μM YARTA or YGTG, [32P]NAD+, or UDP-[14C]GlcNAc for 1 hour at 21°C. The modifications of proteins were analyzed by SDS-PAGE and phosphorimaging. The amount of modified Rab proteins was normalized to wild-type Rab protein. Means (±SD) of three independent experiments and one representative autoradiogram and Coomassie gel are shown.

  • Fig. 5 Functional consequences of Rab5-induced modifications by YART and YGT.

    (A) YART effects on GTP hydrolysis by Rab5. Rab5A was ADP-ribosylated by active YARTA or inactive YARTA E160A and loaded with [γ-32P]GTP. Left: RabGAP5-stimulated GTP hydrolysis (time course) by Rab5A pretreated with YARTA (○), with YARTA E160A (□) or untreated control (△). Right: GTP hydrolysis after 30 min with or without RabGAP5 (untreated control, white column; YARTA pretreatment, black column; YARTA E160A pretreatment, gray column). Data are means ± SD (n = 3). (B) Pulldown of Rab5A by the R5BDs of EEA1 and Rabenosyn-5. Rab5A (Flag-tagged) was coexpressed overnight in HeLa cell with active YARTA and YGTG or with the inactive mutants YARTA E160A and YGTG AXA. Cell lysates were incubated with GST-R5BD–coupled beads (30 min). Thereafter, beads were precipitated and washed, and interacting proteins were analyzed by SDS-PAGE and Western blot. Immunoblots with anti-Flag antibody before (input) and after pulldown are shown. Left: One representative experiment. Right: Results of three independent experiments with EEA1 (black column) or with Rabenosyn-5 (gray column) (data are means ± SD). The amount of precipitated Rab5 was normalized to the pulldown of Rab5 after coexpression with YARTA and YGTG AXA.

  • Fig. 6 Effect from YART and YGT on the Rab5A distribution in HeLa cells.

    HeLa cells were transiently transfected overnight with mCherry-C1-Rab5A (top; red) together with the indicated pEGFP-C1-toxin constructs (green). Afterward, cells were washed and incubated at 21°C for an additional 3 hours. Insets show in detail Rab5A-vesicle structure. Pictures are representative of three independent experiments. Scale bar, 10 μm. (A) Effects of YARTA or YARTA E160A on the distribution of overexpressed Rab5A. (B) Effects of active YARTA and YGTG and inactive YARTA E160A and YGTG AXA on the distribution of overexpressed mCherry-C1-Rab5A. (C) Quantification by MetaMorph imaging software of the size of mCherry-C1-Rab5A vesicles in the presence of the active (YARTA) or inactive ADP-ribosyltransferase domain (YARTA E160A). Cells of view are ≥20, and n = 3. Unpaired two-sample t test was used (***P < 0.001). (D) Quantification of (B) as described in (C). n.m., not measurable. (E) For analysis of the expression of pEGFP-C1-toxin constructs, HeLa cells were lysed after transfection and expression. Proteins were analyzed by SDS-PAGE, Western blotting, and immunostaining with anti-FLAG tag antibody. G indicates GFP-YGTG and GFP-YGTG AXA; A indicates GFP-YARTA and GFP-YARTA E160A.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/11/eaaz2094/DC1

    Fig. S1. Model of the domain architecture of YART and YGT, in comparison to C. difficile TcdB and C. perfringens TpeL.

    Fig. S2. Processing, expression, and enzyme activity of YART and YGT.

    Fig. S3. Topological representation of YGTG and potassium ion coordination in the ligand-bound YGTG structure.

    Fig. S4. C-terminal loop of the glycosyltransferase domain encloses the nucleotide-binding site in the UDP-bound state and leaves it open in the ligand-free state.

    Fig. S5. ETD MS/MS spectra.

    Fig. S6. YART- and YGT-induced modifications of Rab31.

    Table S1. Data collection and refinement statistics.

    Table S2. Proteins structurally homologous to YGTG as identified by DALI and showing a Z score higher than 10.

    Table S3. Genomic DNA, primer, gblocks, plasmids, and vectors.

    Table S4. Buffer and conditions of protein purification.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Model of the domain architecture of YART and YGT, in comparison to C. difficile TcdB and C. perfringens TpeL.
    • Fig. S2. Processing, expression, and enzyme activity of YART and YGT.
    • Fig. S3. Topological representation of YGTG and potassium ion coordination in the ligand-bound YGTG structure.
    • Fig. S4. C-terminal loop of the glycosyltransferase domain encloses the nucleotide-binding site in the UDP-bound state and leaves it open in the ligand-free state.
    • Fig. S5. ETD MS/MS spectra.
    • Fig. S6. YART- and YGT-induced modifications of Rab31.
    • Table S1. Data collection and refinement statistics.
    • Table S2. Proteins structurally homologous to YGTG as identified by DALI and showing a Z score higher than 10.
    • Table S3. Genomic DNA, primer, gblocks, plasmids, and vectors.
    • Table S4. Buffer and conditions of protein purification.

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