Structural basis of inactivation of Ras and Rap1 small GTPases by Ras/Rap1-specific endopeptidase from the sepsis-causing pathogen Vibrio vulnificus

Abstract

Multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are secreted by Gram-negative bacteria and function as primary virulence-promoting macromolecules that deliver multiple cytopathic and cytotoxic effector domains into the host cytoplasm. Among these effectors, Ras/Rap1-specific endopeptidase (RRSP) catalyzes the sequence-specific cleavage of the Switch I region of the cellular substrates Ras and Rap1 that are crucial for host innate immune defenses during infection. To dissect the molecular basis underpinning RRSP-mediated substrate inactivation, we determined the crystal structure of an RRSP from the sepsis-causing bacterial pathogen Vibrio vulnificus (VvRRSP). Structural and biochemical analyses revealed that VvRRSP is a metal-independent TIKI family endopeptidase composed of an N-terminal membrane-localization and substrate-recruitment domain (N lobe) connected via an inter-lobe linker to the C-terminal active site-coordinating core β-sheet-containing domain (C lobe). Structure-based mutagenesis identified the 2His/2Glu catalytic residues in the core catalytic domain that are shared with other TIKI family enzymes and that are essential for Ras processing. In vitro KRas cleavage assays disclosed that deleting the N lobe in VvRRSP causes complete loss of enzymatic activity. Endogenous Ras cleavage assays combined with confocal microscopy analysis of HEK293T cells indicated that the N lobe functions both in membrane localization via the first α-helix and in substrate assimilation by altering the functional conformation of the C lobe to facilitate recruitment of cellular substrates. Collectively, these results indicate that RRSP is a critical virulence factor that robustly inactivates Ras and Rap1 and augments the pathogenicity of invading bacteria via the combined effects of its N and C lobes.

Keywords: MARTX toxin; Ras/Rap1-specific endopeptidase; Vibrio vulnificus; bacterial pathogenesis; effector; host–pathogen interaction; sepsis; toxin; virulence factor.

Figure 1.

Overall structure of the MARTX toxin effector RRSP from V. vulnificus CMCP6. A, schematic representation of MARTX effector domains of V. vulnificus CMCP6, of which VvRRSP (residues 3580–4089) is shown in green. B, four VvRRSP molecules in the asymmetric unit (chains A–D) are colored green, magenta, cyan, and yellow, respectively. C, superimposition of VvRRSP molecules in the asymmetric unit. D, size-exclusion chromatographic analysis of purified VvRRSP (residues 3596–4072) in black, with bovine serum albumin (BSA; magenta) used as a size marker. E, in vitro KRas cleavage assay of purified VvRRSP used for structure determination. KRas* indicates KRas cleaved by VvRRSP.

Figure 2.

Molecular details of VvRRSP. A, structure of VvRRSP divided into subdomains. The N1, N2, C1, and C2 domains of VvRRSP are colored blue, gray, green, and magenta, respectively. The linker region between the N and C lobes (inter-lobe linker) is colored orange. B, three-helix bundle within the N1 domain. Residues forming the hydrophobic core of the bundle are indicated. C, superimposition of the VvRRSP N1 domain (blue) onto the PMT (PDB code 2EBF) C1 domain (orange). D, structural stabilization of the N1 domain. The critical residues Phe-3636 of α3 and Phe-3670 of α4 that are involved in structural stabilization are indicated. Residues involved in forming the groove in the N2 domain are shown in light blue surface representation and are indicated. E, structural integration between the C lobe and the N2 domain. Helix α20 in the C2 domain (magenta), α12 in the C1 domain (green), and α7 in the N2 domain (light blue) are displayed in cartoon representation. Residues involved in hydrophobic and charge interactions between the domains are indicated. F, inter-lobe linker connecting the two lobes shown in stick representation. The N2 domain in the N lobe and the C1 and C2 domains in the C lobe are colored light blue, green, and magenta, respectively. The enlarged box shows residues Tyr-3723 and Leu-3724 that may be critical for controlling flexibility between the N and C lobes. G, in vitro KRas cleavage assays showing the importance of residues Tyr-3723 and Leu-3724. KRas* indicates cleaved KRas.

Figure 3.

C2 catalytic domain. A–E, structural comparison of VvRRSP with the Ere family protein Bcr135 (PDB code 3B55). A, superimposition of VvRRSP onto Bcr135. A, B, and D, structures of the VvRRSP C2 domain and Bcr135 are colored yellow and orange, respectively. Functional residues of VvRRSP and Bcr135 are colored magenta and cyan, respectively. C and E, topologies of the parallel β-sheet core and neighboring helices of the VvRRSP C2 domain and Bcr135 colored yellow and orange, respectively. Functional residues are shown as elliptical shapes in magenta for RRSP and cyan for Bcr135. F, effects of VvRRSP catalytic residues on KRas cleavage in vitro. KRas* indicates cleaved KRas.

Figure 4.

Essential residues of the C2 catalytic domain for hydrolysis of endogenous Ras. A, analysis of VvRRSP catalytic residues in endogenous Ras cleavage. HEK293T cells transfected with plasmids expressing the indicated Strep-tagged proteins were lysed and immunoblotted with anti-Strep or anti-Ras (Pan-Ras) antibodies. Actin was used as a loading control. EV, empty vector. Data are representative of at least three independent experiments with similar results, and values are expressed as mean ± S.D. B, morphological differences of HEK293T cells transfected with GFP fusion plasmids expressing the indicated RRSP proteins. White arrows indicate severely shrunken cells. C, confocal microscopy analysis of WT and mutant RRSPs during cleavage of endogenous Ras. HEK293T cells were transfected with the indicated RRSP plasmids expressing GFP fusion proteins. After 16 h, cells were stained with anti-Pan-Ras antibody (red), and nuclei were stained with Hoechst (blue). Scale bars = 10 μm. D, analysis of VvRRSP residues involved in substrate recognition during KRas cleavage in vitro. E, superimposition of the RRSP_{E3930L/H4030L} mutant structure (light orange) onto the WT RRSP structure (white). Functional residues are shown in stick representation. Residues located in the disordered region in the WT RRSP structure are indicated in stick representation (green) in the structure of mutant RRSP. F, pulldown assays of RRSP proteins using GST-fused KRas.

Figure 5.

VvRRSP is a metal-independent Ras-specific endopeptidase. A, in vitro KRas cleavage assays showing metal-independent RRSP activity. KRas* indicates cleaved KRas. B, effects of protease inhibitor cocktails including phenylmethanesulfonyl fluoride, pepstatin, leupeptin, benzamidine, and bestatin on the KRas cleavage activity of RRSP. Trypsin was used as a control. C, in vitro KRas cleavage assay showing that benzamidine inhibits the activity of RRSP. Ethanol was used as a control. D, effect of benzamidine (4 mm) on the KRas cleavage activity of RRSP. Data are representative of two independent experiments with similar results. E, superimposition of the structure of KRas complexed with GTP (PDB 5W22, white) onto that of KRas complexed with GTP and benzamidine (PDB 4DSO, magenta). Switch I and II regions in the structure of KRas complexed with GTP and benzamidine are colored yellow and green, respectively. Benzamidine and GTP are shown in stick representation.

Figure 6.

Roles of the N lobe in VvRRSP. A, schematic representation of VvRRSP consisting of the four domains N1 (blue), N2 (light blue), C1 (green), and C2 (magenta). B, in vitro KRas cleavage assay of RRSP domains. KRas* indicates cleaved KRas. C, effects of the N domain on endogenous Ras cleavage. HEK293T cells transfected with plasmids expressing the indicated Strep-tagged proteins were lysed and immunoblotted with anti-Strep or anti-Ras (Pan-Ras) antibodies. Arrows indicate RRSP expression bands. Actin was used as a loading control. EV, empty vector. Data are representative of at least three independent experiments with similar results, and values are expressed as mean ± S.D. D, roles of the N domain in localization and enzymatic activity of RRSP in cells. HEK293T cells were transfected with the indicated RRSP constructs expressing GFP fusions. After 16 h, cells were stained with anti-Pan-Ras antibody (red), and nuclei were stained with Hoechst (blue). Scale bars = 10 μm.