Research ArticleBiochemistry

The bacterial Ras/Rap1 site-specific endopeptidase RRSP cleaves Ras through an atypical mechanism to disrupt Ras-ERK signaling

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Science Signaling  02 Oct 2018:
Vol. 11, Issue 550, eaat8335
DOI: 10.1126/scisignal.aat8335
  • Fig. 1 Subdomain architecture of RRSP from V. vulnificus strain CMCP6.

    (A) Ribbon representation of RRSP structure. The C1 subdomain includes a conserved MLD (blue). The C2 subdomain can be divided into C2A (green) and C2B (red), which are connected by a helical linker (yellow). The 23 α helices and nine β sheets are labeled. (B) Amino acid sequence of RRSP color-coded to match the structure in (A) with secondary structure elements (α helices and β sheets) labeled.

  • Fig. 2 Transitive homology reveals that RRSP is a member of the EreA/ChaN-like family of enzymes.

    (A) Comparison of the RRSP C2B domain structure (α helices in teal, β sheets in magenta, and flexible linkers in pink) with that of other whole enzymes of the EreA/ChaN-like superfamily: HopBA1 [PDB ID 5T09 (26)], ChaN [PDB ID 2G5G (27)], and Bcr135 [PDB ID 3B55 (28)]. Structural representations were made with PyMOL. (B) Alignment of the primary sequences and structures of the three clusters of amino acids comprising the putative catalytic site of RRSP with those of other known members of the EreA/ChaN-like superfamily enzymes. The conserved residues (black dots) and the nonconserved His3931 (asterisk) that were mutated to alanine in this study are noted. Multiple alignment was performed with ESPript 3.0 (72).

  • Fig. 3 RRSP residues that contribute to KRAS processing.

    (A) Ribbon representation of the RRSP catalytic site. Sticks indicate the side groups of the residues substituted with alanine in our experiments. Amine groups and carboxyl groups are colored in blue and red, respectively. Polar interactions between residues are depicted by black dashed lines with distances indicated in Å. (B) Representative SDS–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the reaction product(s) of 10 μM rKRAS and 10 μM of each indicated rRRSP mutant after a 30-min in vitro incubation (n = 3 independent experiments). (C) Quantification of rKRAS cleavage efficiency of rRRSP mutants shown in (B). Bars are color-coded by mutant. The bar graph reports means ± SD of three independent biological replicates [analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test, ****P ≤ 0.0001]. WT, wild-type.

  • Fig. 4 RRSP does not require a metal cofactor.

    (A and B) SDS-PAGE gels showing cleavage of 10 μM rKRAS that had been buffer-exchanged to remove MgCl2 by 10 μM RRSP that had been preincubated with increasing amounts of EDTA (A) or 1,10-phenanthroline (B) for 30 min. (C) Cleavage of 10 μM MgCl2-free rKRAS by 10 μM thermolysin (T) that had been preincubated with EDTA (A) or 1,10-phenanthroline (B) for 30 min. SDS-PAGE gels are representatives of three independent biological replicates. (D and E) EC50 curves for cleavage of 10 μM MgCl2-free rKRAS by increasing concentrations of untreated (Unt) rRRSP compared to rRRSP that had been pretreated with 5 mM EDTA (D) or 1,10-phenanthroline treated (E). At the highest concentration of rRRSP, KRAS/rRRSP was 1:1 as in (A) and (B). (F) EC50 curves for cleavage of 10 μM MgCl2-free rKRAS by rRRSP in the presence of 5 mM MgCl2 or ZnCl2 or by rRRSP pretreated with 1,10-phenanthroline. All curves are derived from SDS-PAGE analysis (n = 3 independent experiments) reported as the means ± SD. EC50 values calculated from nonlinear curve fit ± SE.

  • Fig. 5 RRSP-cleaved KRAS maintains its tertiary structure and nucleotide binding.

    (A) Ribbon representation of the structure of GDP-bound KRAS [PDB ID 4OBE (73)]. Regions structurally affected by RRSP cleavage are colored in red, and the cleavage site is indicated by the black arrow. (B) Representative nucleotide exchange curves for rKRAS and RRSP-cleaved rKRAS (rKRAS*) in the presence of EDTA and either mant-GDP (mGDP) or mant-GppNHp (mGppNHp) (n = 3 independent experiments). (C) Bar graph of the observed nucleotide exchange rates (kobs) of rKRAS and rKRAS* in the presence of mGDP or mGppNHp. Data were fit in GraphPad Prism 6.0 to a one-phase exponential association curve to determine kobs values. Data in (C) are means ± SD from three independent biological replicates (ANOVA followed by Dunnett’s multiple comparisons test; ns, nonsignificant).

  • Fig. 6 Real-time assay of rKRAS processing by RRSP.

    (A) Fluorescence of unprocessed rKRAS bound to mant-GDP– and RRSP-processed rKRAS (rKRAS*) that was loaded with mant-GDP before incubation with RRSP. Fluorescence was quantified as intensity unit. (B) Representative curves of real-time cleavage assays of mant-GDP–bound rKRAS incubated with wild-type RRSP at the indicated ratios (n = 3 independent experiments). (C) Bar graphs of the observed rKRAS cleavage rates (kobs) of wild-type rRRSP. (D) Representative curves of real-time cleavage assays of mant-GDP–bound rKRAS incubated with wild-type and the indicated mutant forms of RRSP at 1:10 molar ratio (rRRSPs/rKRAS) (n = 3 independent experiments). (E) Bar graphs of the observed rKRAS cleavage rates (kobs) of wild-type and mutant forms of rRRSP. Lines/bars are color-coded by mutant as in Fig. 3. Data were fit in GraphPad Prism 6.0 to a one-phase exponential association curve to determine kobs values. Data in (A), (C), and (E) are means ± SD from three independent biological replicates (ANOVA followed by Dunnett’s multiple comparisons test, ****P ≤ 0.0001). ND, not detected.

  • Fig. 7 RRSP processing prevents the interaction of KRAS with GEFs and RAF.

    (A) Ribbon and surface representation of GDP-bound KRAS [PDB ID 4OBE (73)]. Regions that are structurally affected by RRSP cleavage are shaded in red, and the cleavage site is indicated by the black arrow. RAF- and SOS-binding interfaces are indicated with dotted lines. (B and C) Representative curves of intrinsic and SOS-catalyzed nucleotide exchange for rKRAS (B) and rKRAS* (C) (n = 3 independent experiments). (D) Bar graph of the intrinsic and SOS-catalyzed nucleotide exchange rates (kobs) of rKRAS (filled bars) and rKRAS* (open bars). Each bar represents the mean ± SD of three independent biological replicates (ANOVA followed by Dunnett’s multiple comparisons test, *P ≤ 0.05 and ****P ≤ 0.0001). Data were fit in GraphPad Prism 6.0 to a one-phase exponential dissociation curve to determine the kobs. (E) Proximity-based AlphaLISA assay between GST-RAF-RBD and avi-KRAS after preincubation of avi-KRAS with increasing concentrations of each rRRSP mutant. Emission data were normalized to control without addition of RRSP and plotted as 1/x to show percent (%) binding inhibition by rRRSP and mutant variants. Lines are color-coded by mutant as in Fig. 3. Each curve represents the mean ± SD of three independent biological replicates. (F) Binding responses and dissociation constant (Kd) from Biacore SPR analysis of the interaction between immobilized RAF-RBD and injected GppNHp-bound rKRAS or rKRAS*. Representative plot of time (s) versus response unit (RU, defined as binding of 1 pg/mm2) from a single injection is shown (n = 3 independent experiments).

  • Fig. 8 Glu3930 and His4030 are essential for RRSP cytotoxicity.

    (A and B) Representative immunoblots of lysates from HeLa cells incubated with LFNRRSP, the LFNRRSP E3930A and H4030A mutants, and anthrax toxin PA as indicated and probed with antibodies that recognize all RAS proteins (Pan-RAS) (A) or total ERK1/2 and pERK1/2 (B) (n = 3 independent experiments). (C) Percentage of RAS that was cleaved in HeLa cells by wild-type and mutant LFNRRSP as determined by densitometry analysis. (D) The ratio of pERK1/2 to total ERK1/2 in lysates from HeLa cells after treatment with wild-type and mutant LFNRRSP. (E) Schematic representation of MARTX toxin effector domain organization in V. vulnificus CMCP6 strains. (F) Representative immunoblots showing RAS in lysates from T84 cells that has been incubated with V. vulnificus producing the indicated MARTX toxins (n = 3). (G) Percentage of Ras that was cleaved in (E) as determined by densitometry analysis. All graphs (C, D, and G) show means ± SD from three independent biological replicates (ANOVA followed by Dunnett’s multiple comparisons test, *P ≤ 0.05, **P ≤ 0.01, and ****P ≤ 0.0001).

  • Table 1 X-ray data collection and refinement statistics of V. vulnificus RRSP (PDB ID 5W6L).
    CrystalRRSP
    Data collection
      X-ray wavelength0.97872
      Space groupI23
      a, b, c (Å)247.1, 247.1, 247,1
      α, β, γ (°)90.00, 90.00, 90.00
      Resolution (Å)30 to 3.45 (3.51 to 3.45)*
      No. of unique reflections33,078 (1638)
      Data redundancy6.8 (5.9)
      Data completeness (%)100.0 (99.9)
      Rsym (%)11.3 (86.6)
      I/sig18.4 (2.0)
    Refinement
      Resolution (Å)29.97 to 3.45
      No. of unique reflections31,061
      No. of reflections (Rfree)1562
      Rwork/Rfree (%)22.79:24.91
    Number of atoms
      Protein7662
      Ligand/ion17
      Solvent15
    RMS deviations
      Bond length (Å)0.009
      Bond angle (°)1.235
    Average B factors (Å2)
      All protein atom125
      Ligand/solvent134
    Ramachandran plot
      Outliers (%)0.0
      Allowed (%)6.0
      Favored (%)94.0
      Rotamer outliers (%)1.0
      No. of C-β deviations0
      All-atom clashscore2.0

    *Values in parenthesis are for the highest resolution shell.

    Rsym = Σ|I−(I)|/ ΣI, where I is the observed intensity of a reflection and (I) is the average intensity of all the symmetry related reflections.

    ‡For Rfree calculation, 10% of reflections were randomly excluded from the refinement.

    Supplementary Materials

    • www.sciencesignaling.org/cgi/content/full/11/550/eaat8335/DC1

      Fig. S1. Similarity of membrane-targeting domains from RRSP and other bacterial toxins.

      Fig. S2. Dali server search results using RRSP_chainA as the query.

      Fig. S3. PMT does not cleave rKRAS.

      Fig. S4. Space-filling model showing cleft with putative active site residues.

      Fig. S5. EC50 for processing of KRAS + Mg2+.

      Fig. S6. Divalent cations do not stimulate the activity of chelated rRRSP.

      Fig. S7. rKRAS* shows localized structural perturbation.

      Fig. S8. DSF analysis of rRRSP mutants.

      Fig. S9. SPR of KRAS binding to RRSP.

      Fig. S10. NMR of KRAS in the presence of rRRSP H4030A.

      Fig. S11. Schematic representation of the LFNA+PA system.

      Fig. S12. rtxA1 genes with rrsp genetic modifications induce cell rounding but not RRSP processing in HeLa cells.

      Table S1. Oligonucleotides used in the study.

      Table S2. Sequence of gBlocks used in the study.

      References (7480)

    • This PDF file includes:

      • Fig. S1. Similarity of membrane-targeting domains from RRSP and other bacterial toxins.
      • Fig. S2. Dali server search results using RRSP_chainA as the query.
      • Fig. S3. PMT does not cleave rKRAS.
      • Fig. S4. Space-filling model showing cleft with putative active site residues.
      • Fig. S5. EC50 for processing of KRAS + Mg2+.
      • Fig. S6. Divalent cations do not stimulate the activity of chelated rRRSP.
      • Fig. S7. rKRAS* shows localized structural perturbation.
      • Fig. S8. DSF analysis of rRRSP mutants.
      • Fig. S9. SPR of KRAS binding to RRSP.
      • Fig. S10. NMR of KRAS in the presence of rRRSP H4030A.
      • Fig. S11. Schematic representation of the LFNA+PA system.
      • Fig. S12. rtxA1 genes with rrsp genetic modifications induce cell rounding but not RRSP processing in HeLa cells.
      • Table S1. Oligonucleotides used in the study.
      • Table S2. Sequence of gBlocks used in the study.
      • References (7480)

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