the bacterial ras/rap1 site-specific endopeptidase rrsp ...the best-characterized downstream...

SC I ENCE S I GNAL ING | R E S EARCH ART I C L E

B IOCHEM ISTRY

1Department of Microbiology-Immunology, Feinberg School of Medicine, North-western University, Chicago, IL 60611, USA. 2Center for Structural Genomics of In-fectious Diseases, Feinberg School of Medicine, Northwestern University, Chicago,IL 60611, USA. 3Department of Chemistry, University of Illinois at Chicago, Chicago,IL 60607, USA. 4National Cancer Institute-RAS Initiative, Cancer Research Technol-ogy Program, Frederick National Laboratory for Cancer Research, Leidos BiomedicalResearch, Frederick, MD 21702, USA. 5Department of Biochemistry and MolecularGenetics, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611,USA. 6Department of Biochemistry and Molecular Genetics, University of Illinois atChicago, Chicago, IL 60607, USA.*Present address: GSK Vaccines, Rockville, MD 20850, USA.†Corresponding author. Email: [email protected]

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The bacterial Ras/Rap1 site-specific endopeptidaseRRSP cleaves Ras through an atypical mechanismto disrupt Ras-ERK signalingMarco Biancucci1*, George Minasov1,2, Avik Banerjee3, Alfa Herrera1, Patrick J. Woida1,Matthew B. Kieffer1, Lakshman Bindu4, Maria Abreu-Blanco4, Wayne F. Anderson2,5,Vadim Gaponenko6, Andrew G. Stephen4, Matthew Holderfield4, Karla J. F. Satchell1,2†

The Ras–extracellular signal–regulated kinase pathway is critical for controlling cell proliferation, and its aberrantactivation drives the growth of various cancers. Because many pathogens produce toxins that inhibit Ras activity,efforts todevelop effective Ras inhibitors to treat cancer could be informedby studies of Ras inhibition bypathogens.Vibrio vulnificus causes fatal infections in amanner that depends onmultifunctional autoprocessing repeats-in-toxin,a toxin that releases bacterial effector domains into host cells. One such domain is the Ras/Rap1-specific endo-peptidase (RRSP), which site-specifically cleaves the Switch I domain of the small GTPases Ras and Rap1. We solvedthe crystal structure of RRSP and found that its backbone shares a structural foldwith the EreA/ChaN-like superfamilyof enzymes. Unlike other proteases in this family, RRSP is not a metalloprotease. Through nuclear magneticresonance analysis and nucleotide exchange assays, we determined that the processing of KRAS by RRSP did notrelease any fragments or cause KRAS to dissociate from its bound nucleotide but instead only locally affected itsstructure. However, this structural alteration of KRAS was sufficient to disable guanine nucleotide exchangefactor–mediated nucleotide exchange and prevent KRAS from binding to RAF. Thus, RRSP is a bacterial effector thatrepresents a previously unrecognized class of protease that disconnects Ras from its signaling network whileinducing limited structural disturbance in its target.

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INTRODUCTIONBacterial toxins have evolved to hijack cellular pathways by compro-mising the innate immune response, thus promoting bacterial inva-sion, and by modifying epithelial and endothelial cells to promotesystemic spread (1). Inhibition or deregulation of signaling throughsmall guanosine triphosphatases (GTPases) is one of themost commonstrategies for avoiding host innate responses because these proteinsserve as central nexuses in many cellular signal transduction pathways.Ras is a small GTPase that directs extracellular signals throughmitogen-activated protein kinase (MAPK) signaling cascades to promote cellproliferation, differentiation, and survival by specific target gene ex-pression (2). The activity of Ras is finely controlled by guanine nu-cleotide exchange factors (GEFs), which promote formation of theguanosine 5′-triphosphate (GTP)–bound active state, and by GTPaseactivating proteins, which accelerate the intrinsicGTPase activity to pro-mote formation of the guanosine diphosphate (GDP)–bound inactivestate (3–5). Cycling between GDP and GTP binding causes conforma-tional changes of the Switch I (residues 30 to 40) and Switch II (residues60 to 76) regions of Ras (5). In theGTP-bound active conformation, Rasinteracts with downstream effectors to modulate cytoplasmic signalingnetworks (6). The best-characterized downstream effectors of Ras are

kinases of the Raf family, which mediate the activation of Ras-MAPKsignal transduction pathways (6). Given its prominent role in cell pro-liferation, Ras signaling is frequently constitutively activated in cancercells due to mutations in KRAS, NRAS, or HRAS. However, there arecurrently no drugs targeting Ras in cancer and very few Ras-specificinhibitors useful for studying this protein in cancer cell signaling.

Novel strategies to inhibit Ras could be informed by understandingthe natural event of Ras inhibition by bacteria. Several bacteria producesecreted protein toxins that target Ras directly. Multifunctional auto-processing repeats-in-toxin (MARTX) toxins are 3500– to 5300–aminoacid protein toxins secreted by Gram-negative bacteria throughmodified type I secretion systems (7). These toxins translocate multipleeffector domains across the eukaryotic plasma membrane (8), wherethey are released from the holotoxin into the cytosol by inositolhexakisphosphate–induced autoprocessing (9). The motile,Gram-negative opportunistic human pathogen Vibrio vulnificus causesrapidly progressing, life-threatening sepsis after contaminated sea-food is ingested or open wounds are exposed to seawater in a mannerthat depends on the production of a MARTX toxin (7, 10, 11). Up toseven different variants of the toxin carrying distinct repertoires of ef-fector domains have been identified in differentV. vulnificus isolates (7).Strains producing theMARTXVv toxin variant that contains the effectordomain known as Ras/Rap1-specific endopeptidase (RRSP; also knownas DUF5, Photorhabdus asymbiotica rtxA homology domain, andPasteurella multocida toxin (PMT) C1-C2 homology domain) showabout 50-fold increased virulence in mice compared to an isogenicstrain that produces a toxin without RRSP (12). This potent cyto-toxicity of RRSP is due to its direct inhibition of MAPK signalingby proteolytically processing the small GTPases Ras and Rap1. Thisprocessing of Ras prevents the subsequent phosphorylation and activationof extracellular signal–regulated kinases (ERKs) (13). RRSP specificallyrecognizes the Switch I domain of Ras andRap1 and cleaves it between

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Tyr32 and Asp33 (13, 14). This activity is also found in toxins of otherbacteria, including Photorhabdus spp. insect pathogens andAeromonasspp. fish pathogens (13, 14).

Despite the extensive characterization of substrate specificity, thecatalyticmechanism bywhich RRSP cleaves Ras remains unknown be-cause RRSP shows no primary sequence similarity to known proteasefamilies. Furthermore, how clipping the Switch I domain leads to in-activation of Ras remains unknown. Here, we solved the crystal struc-ture of the MARTXVv toxin RRSP effector domain and found it to besimilar to that of members of the EreA/ChaN-like superfamily of hy-drolytic enzymes (15). Structural alignment identified conserved cata-lytic residues that were essential for RRSP enzymatic activity and forcytotoxicity. However, RRSP, unlike other known proteases in thisfamily, cleaved its substrate in the absence of metal cofactors. We fur-ther show the structural and functional consequences of KRAS cleav-age by demonstrating that RRSP processing induced amainly localizedperturbation within and adjacent to the Switch I region without com-promising either the overall structural integrity or the nucleotide-binding site of KRAS.However, cleavedKRAS could not be stimulatedfor nucleotide exchange by a GEF and was unable to bind RAF1. Thus,the RRSP toxin effector domain is a unique class of protease that ishighly potent for pathogenesis because it completely disconnects theRas-ERK signaling network, an activity that may prove beneficial tostudies of cancer cell signaling.

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RESULTSCrystal structure reveals subdomain architecture of RRSPfrom the V. vulnificus MARTX toxinTo understand the catalytic mechanism of RRSP, we determined thecrystal structure of RRSP from V. vulnificus strain isolate CMCP6 [Na-tional Center for Biotechnology Information locus NC_004460 (16);residues 3594 to 4087] at 3.45 Å resolution. The structure was depositedinto the Protein Data Bank (PDB) with PDB ID 5W6L (Table 1). Therewere two molecules in the asymmetric unit, and the structures of thetwo chains were nearly identical [root mean square deviation (RMSD)of 0.488 Å], with chain A selected for further analysis.

The structure of RRSP is composed of two subdomains: C1 (residues3596 to 3742) and C2 (residues 3743 to 4087) (Fig. 1A). The C1 sub-domain includes seven a helices (a1 to a7), whereas the C2 subdomainis formed by 16 a helices (a8 to a23) and nine b-strands (b1 to b9)(Fig. 1B). The C2 subdomain can be divided intoC2A (residues 3743 to3830, containing a8 to a11 and b1 to b3) and C2B (residues 3856 to4087, containing a13 to a23 and b4 to b9), both with a/b folds andconnected by the long helix a12.

The extreme N terminus of RRSP (a1 to a4) is a characterizedplasma membrane localization domain (MLD) found in many bacte-rial toxins (17, 18). It is essential for targeting RRSP to the negativelycharged plasma membrane through positively charged lysine and ar-ginine residues and a phenylalanine forming a classic basic hydropho-bic motif (17, 18). Nuclear magnetic resonance (NMR) analysis hadpreviously showed that the RRSP N terminus is a four-helix bundlein solution with residues essential for membrane targeting found inloops 1 and 3 (19). The RRSP structure here likewise reveals a four-helix bundle within a larger seven-helix C1 subdomain with a5 to a7interfacingwith the C2 subdomain (Fig. 1A). The resolved structure ofthe C1 MLD subdomain can be superimposed with similar targetingdomains from PMT, Vibrio cholerae Rho inactivation domain, andclostridial glucosylating toxins (fig. S1).


The C2 subdomain is sufficient to confer cytotoxicity in mamma-lian and yeast cells (20). In a Dali server search (21) using the entireRRSP structure (fig. S2), the highest scoring hit (z score = 31.7)was the

Table 1. X-ray data collection and refinement statistics of V. vulnificusRRSP (PDB ID 5W6L).

Crystal
RRSP
Data collection

X-ray wavelength
0.97872
Space group
I23
a, b, c (Å)
247.1, 247.1, 247,1
a, b, g (°)
90.00, 90.00, 90.00
Resolution (Å)
30 to 3.45 (3.51 to 3.45)*
No. of unique reflections
33,078 (1638)
Data redundancy
6.8 (5.9)
Data completeness (%)
100.0 (99.9)
Rsym (%)†
11.3 (86.6)
I/sig
18.4 (2.0)
Refinement

Resolution (Å)
29.97 to 3.45
No. of unique reflections
31,061
No. of reflections (Rfree)‡
1562
Rwork/Rfree (%)
22.79:24.91
Number of atoms

Protein
7662
Ligand/ion
17
Solvent
15
RMS deviations

Bond length (Å)
0.009
Bond angle (°)
1.235
Average B factors (Å2)

All protein atom
125
Ligand/solvent
134
Ramachandran plot

Outliers (%)
0.0
Allowed (%)
6.0
Favored (%)
94.0
Rotamer outliers (%)
1.0
No. of C-b deviations
0
All-atom clashscore
2.0
*Values in parenthesis are for the highest resolution shell. †Rsym = S|I−(I)|/SI, where I is the observed intensity of a reflection and (I) is the averageintensity of all the symmetry related reflections. ‡For Rfree calculation,10% of reflections were randomly excluded from the refinement.

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structure of the C terminus of PMT (PDB ID2EBF) (15). In addition tothe C1MLD of RRSP showing high similarity to the C1MLD of PMTas noted above, the RRSP-C2 and PMT-C2 domains also showed ahigh degree of similarity (RMSD of 3.3 Å), although the amino acidsequence identity is only 25% (fig. S3A). This similarity could suggestconservation of function. The PMTC-terminal region consists of threewell-defined domains: C1, C2, and C3 (15). The PMT catalytic domainC3 induces the constitutive activation ofGa proteins of theGq/11, G12/13,and Gi families by deamidating a crucial glutamine in the Switch IIdomain and blocking GTP hydrolysis activity (22, 23). RRSP does notcontain a domain corresponding to the PMT-C3 domain. Furthermore,PMT causes the transactivation of the MAPK cascade by targetingGq/11 through the p63RhoGEF-RhoApathway, a process that dependson functional Ras (24), making it unlikely that PMT-C2 would cleave


Ras. Purified recombinant PMT contain-ing only the C1 and C2 domains (rPMT-C1C2) mixed at equimolar concentrationwith recombinant humanKRAS (rKRAS)for 30 min at 37°C did not show Ras-processing activity, whereas recombinantRRSP (rRRSP) completely cleaved rKRASin a parallel reaction (fig. S3B). Theseresults demonstrate that, although thePMT-C1C2 domains have a similar foldto RRSP, PMT is not able to cleave Ras.These biochemical data reinforce previouscell biological data that PMT-C1C2 is notcytotoxic and does not affect mitogenicsignaling (20, 25).

The Dali search also revealed homol-ogy of the RRSP-C2B subdomain to thebacterial type III secretion system (T3SS)effector proteinHopBA1 of Pseudomonassyringae [PDB ID 5T09 (26); z score =11.5], the heme-binding ChaN proteinfrom Campylobacter jejuni [PDB ID2G5G (27); z score = 11.0], and two pro-teins of unknown function from Bacilluscereus, Bcr135 and Bcr136 [PDB IDs3B55 (28) and 2QGM (29); z scores = 9.2and 9.1, respectively] (Fig. 2A and fig. S2).All of these proteins have no knownfunction. Thus, alignment of RRSP toother proteins of known structure doesnot inform about the catalytic mecha-nism by which RRSP can cleave Ras oris cytotoxic to cells.

Transitive homology analysisshows unrecognizedcatalytic residues in RRSPTransitivity suggests that the function ofa protein can be deduced if two proteinswith little or no similarity can be clus-tered with a third protein to which bothshare similarity (30). The catalytic sitesfor the erythromycin esterases EreA andEreB (31) and the Tiki metalloproteases(32) were previouslymodeled on the basis

of a primary sequence similarity to Bcr136 (31), which revealed struc-tural, but not amino acid sequence, similarity to RRSP in our Dalisearch (fig. S2). Thus, transitive structure homology suggests thatRRSP may belong to a broader family of hydrolytic enzymes, withthe RRSP-C2B (residues 3882 to 4063) defined by the Structural Clas-sification of Proteins database as ana/b foldwith a b-strand core of theEreA/ChaN-like superfamily (33). Previous computational analysespredicted RRSP could belong to this family (34, 35).

The structural alignment of RRSPwithHopBA1,ChaN, andBcr135and identification of the known catalytic residues from EreA, EreB,and TIKI2, all of which were previously modeled using Bcr136, re-vealed residues Glu3900, His3902, Glu3930, and His4030 as putative cata-lytic residues for RRSP (Fig. 2B). Notably, all of the identified residuesare substituted in PMT (Fig. 2B), despite conservation of structure,

Fig. 1. Subdomain architecture of RRSP from V. vulnificus strain CMCP6. (A) Ribbon representation of RRSP struc-ture. The C1 subdomain includes a conserved MLD (blue). The C2 subdomain can be divided into C2A (green) andC2B (red), which are connected by a helical linker (yellow). The 23 a helices and nine b sheets are labeled. (B) Aminoacid sequence of RRSP color-coded to match the structure in (A) with secondary structure elements (a helices andb sheets) labeled.

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which is consistent with PMT lacking Ras protease activity (fig. S3B).The putative catalytic residues are located within a cleft on the proteinand are closely oriented, along with two nonconserved residues,Arg4001 and His3931 (Fig. 3A and fig. S4). We generated and purifiedrRRSPs in which alanine was substituted for each of the six identifiedresidues and tested for their ability to cleave rKRAS in vitro at equi-molar concentrations (Fig. 3, B and C). Replacing Glu3900 with alanine(E3900A) has no effect on the ability of rRRSP to cleave rKRAS,whereas replacing His3902 with alanine (H3902A) reduced rKRAS pro-cessing to nearly 50%. Substitution of the conserved Glu3930 (E3930A)and His4030 (H4030A) residues resulted in proteins that were unable tocleave rKRAS, suggesting a major role for these residues in the catalyticmechanism. Exchange of the nonconserved His3931 with alanine(H3931A) also reduced rKRAS-processing activity, whereas exchangeof the nonconserved proximal residue Arg4001 to alanine (R4001A)did not affect rKRAS processing.

RRSP is a unique protease in the EreA/ChaN-like familySome members of the EreA/ChaN-like enzyme family, including theerythromycin esterase EreA and the metalloprotease TIKI2, requiremetal cofactors for their hydrolytic activity (31, 36), and the crystalstructure of Bcr135 included a calcium ion in the putative active site(28). In contrast, EreB does not require a metal cofactor (31), and thestructures ofChaN, Bcr136, andHopBA1 lack a divalent ion (26, 27, 29).Given that TIKI2, the only known protease in the EreA/ChaN-likefamily, is a characterized Co2+/Mn2+-dependent metalloprotease (32),it was surprising that a metal ion was absent from the crystal structureof RRSP. To test whether RRSP activity depended on metal cofactors,we incubated rRRSP with increasing concentrations of the metal ion


chelators EDTA or 1,10-phenanthroline and then mixed it with anequimolar concentration of rKRAS. Chelator-treated rRRSP did notshow inhibition of activity, even at the highest molar ratio of 1:500(RRSP/chelator) (Fig. 4, A and B). As a positive control, the non-specific protease thermolysin was inhibited by both EDTA and 1,10-phenanthroline under the same conditions (Fig. 4C).

In addition, rRRSP pretreated with EDTA to chelate divalent cat-ions showed median effective concentration (EC50) value comparableto untreated rRRSP (Fig. 4D). Pretreatment of rRRSP with 1,10-phenanthroline did reduce the EC50 value about threefold, although100% of rKRAS was still processed within 10 min at an rRRSP/rKRAS ratio of 1:10 (Fig. 4E). The activity of chelator-treated rRRSPwas not stimulated by MgCl2 carried over with purified rKRAS, be-cause rKRAS that was not subjected to buffer exchange to removeMgCl2 before the addition of rRRSP showed identical EC50 values(fig. S5, A and B). Addition of 5 mMCaCl2, CoCl2, MgCl2, or MnCl2also did not stimulate chelated rRRSP, and in fact, addition of NiCl2,ZnCl2, or CuCl2 inhibited the reaction (Fig. 4F and fig. S6, A to C).These results demonstrate that RRSP is not a metal-dependent pro-tease and does not require divalent ions for its activity. Thus, RRSPuniquely represents a protease within the EreA/ChaN-like familythat is not a metal-dependent enzyme. This grouping could eventuallyalso include HopBA1 if this T3SS effector is found to cleave a plantprotein as its substrate and does so in the absence of a metal cofactor.

Cleaved KRAS has a disordered Switch I but retainsoverall structureThe processing catalyzed by RRSP is known to be a specific cleavagein the Switch I domain of Ras, and it is also known that the KRAS

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 (a helicesin teal, b sheets inmagenta, 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 ofamino acids comprising the putative catalytic site of RRSP with those of other knownmembers of the EreA/ChaN-like superfamily enzymes. The conserved residues (blackdots) and the nonconserved His3931 (asterisk) that were mutated to alanine in this study are noted. Multiple alignment was performed with ESPript 3.0 (72).

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N-terminal fragment is not released upon RRSP-mediated processingbut remains tethered to the protein (13) and dissociates only uponboiling in SDS-PAGE buffer (Fig. 3B). Thus, the functional and bio-


chemical properties of intact, cleaved KRAS (here called rKRAS*) canbe assessed in vitro under nondenaturing conditions. Thermal stabilityanalysis bydifferential scanning fluorimetry (DSF) showed that themeltingtemperature (Tm) of rKRAS* was negatively shifted by about 6.7°C com-pared to uncleaved rKRAS (fig. S7A), suggesting destabilization of the pro-tein structure. Superimposition of 1H-15N heteronuclear single-quantumcoherence (HSQC) NMR spectra of rKRAS before and after incubationwith rRRSP revealed structural differences concentrated in the Switch Iregion of rKRAS* (fig. S7B). The disordered Switch I loop region specifi-cally lacks signals for the 1HN protons of Switch I residues Ala29-Ser39,extending to the b-strand two residues Tyr40-Lys42 (Fig. 5A). This linebroadening surpasses the limit of detection, and hence, these signals donot appear in the spectra. In addition, residues Asp57-Gly60 of the DXXGmotif, which together with Thr35 are essential for interaction with the nu-cleotide phosphate groups or the Mg2+ ion (37), also showed linebroadening.The signal of several residues onb-strand 2 anda-helix 2wereaffected by RRSP cleavage as well (Fig. 5A and fig. S7B).

These data reveal that RRSP cleavage between the Tyr32 and Asp33

residues of Ras mainly affects the Switch I region and the backbonedynamics downstreamof the processing site.However, despite the dis-appearance of NMR signal around Switch I, rKRAS* is surprisinglynot disrupted in its overall tertiary structure, explaining why Ras thatis cleaved by RRSP in vivo is easily detected by Western blotting andrKRAS* routinely remains intact through manipulations required forin vitro biochemical assays (13).

Cleaved KRAS retains its bound nucleotideThe observed destabilization of KRAS* Switch I could affect Ras bind-ing to nucleotides (14). To assess whether KRAS* retained the abilityto bind nucleotides, we performed an EDTA-catalyzed nucleotideexchange assay. We mixed rKRAS* with a fivefold molar excess offluorescent nucleotides, N-methylanthraniloyl (mant)–GDP, andthe nonhydrolyzable GTP analog mant-GppNHp. The nucleotideexchange assay was initiated by the addition of 20mMEDTA. KRAS*did not show a significantly higher kobs than rKRAS for mant-GDP ormant-GppNHp (Fig. 5, B andC). This demonstrates that the nucleotide-binding pocket of rKRAS* retains GDP and GTP, confirming that thetertiary structurewas not lost after RRSPprocessing and that bound gua-nine nucleotides are not expelled. Moreover, the ability of EDTA to cat-alyze the nucleotide exchange of rKRAS* indicates that, although theprocessing by RRSP affects the orientation of residues involved in Rasmetal binding, Mg2+ must be retained in the KRAS nucleotide-bindingpocket. In addition, residues Gly13 and Ser17, which bind to the a- andb-phosphates of the nucleotide, and residues Phe28, Asn116, and Asp119,which are critical for binding the guanosine moiety, are not affected byRRSP cleavage, explaining why rKRAS* retains its bound nucleotide(fig. S7B) (37).

Kinetic assays using nucleotide fluorescence detectionreveal additional residues that contribute to RRSP activityBecause rKRAS* preloaded with mant-GDP retains the nucleotideupon RRSP-mediated processing, we developed a novel kinetic assayfor KRAS processing in real time. Upon addition of rRRSP, the cleav-age of rKRAS–mant-GDP caused a loss in signal fluorescence (Fig. 6A)that was proportional to the amount of cleaved rKRAS (Fig. 6, B andC).On the basis of the NMR and fluorescence data, this shift is consistentwith processing of Switch I, resulting in disordered rearrangement ofthe Tyr32 and other Switch I residues around themant fluorophore. Asexpected, incubation of rRRSP E3930A and rRRSP H4030A with

Fig. 3. RRSP residues that contribute to KRAS processing. (A) Ribbon repre-sentation of the RRSP catalytic site. Sticks indicate the side groups of the residuessubstituted with alanine in our experiments. Amine groups and carboxyl groupsare colored in blue and red, respectively. Polar interactions between residues aredepicted by black dashed lines with distances indicated in Å. (B) RepresentativeSDS–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the reaction product(s)of 10 mM rKRAS and 10 mM of each indicated rRRSP mutant after a 30-min in vitroincubation (n = 3 independent experiments). (C) Quantification of rKRAS cleavageefficiency of rRRSP mutants shown in (B). Bars are color-coded by mutant. The bargraph reports means ± SD of three independent biological replicates [analysis ofvariance (ANOVA) followed by Dunnett’smultiple comparisons test, ****P ≤ 0.0001].WT, wild-type.

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rKRAS–mant-GDP did not show a change in fluorescence, indicatinga complete loss of the ability of rRRSP to process rKRAS (Fig. 6D).In this assay, rRRSP H3902A and rRRSP H3931A, both of which


showed partial-processing activity (Fig. 3, B and C), showed similarslow kobs rates (Fig. 6E). However, rRRSP E3900A and rRRSPR4001A, both of which completely cleaved rKRAS in the endpoint

Fig. 4. RRSP does not require a metal cofactor. (A and B) SDS-PAGE gels showing cleavage of 10 mM rKRAS that had been buffer-exchanged to removeMgCl2 by 10 mMRRSP that had been preincubatedwith increasing amounts of EDTA (A) or 1,10-phenanthroline (B) for 30min. (C) Cleavage of 10 mMMgCl2-free rKRAS by 10 mM thermolysin(T) that had been preincubatedwith EDTA (A) or 1,10-phenanthroline (B) for 30min. SDS-PAGE gels are representatives of three independent biological replicates. (D and E)EC50 curves for cleavage of 10 mMMgCl2-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/rRRSPwas 1:1 as in (A) and (B). (F) EC50 curves for cleavage of 10 mMMgCl2-free rKRAS byrRRSP 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 independentexperiments) 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 exchangecurves for rKRAS and RRSP-cleaved rKRAS (rKRAS*) in the presence of EDTA and either mant-GDP (mGDP) or mant-GppNHp (mGppNHp) (n = 3 independentexperiments). (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 GraphPadPrism 6.0 to a one-phase exponential association curve to determine kobs values. Data in (C) are means ± SD from three independent biological replicates (ANOVAfollowed by Dunnett’s multiple comparisons test; ns, nonsignificant).

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processing assay (Fig. 3, B and C), were significantly slower thanwild-type rRRSP (Fig. 6E). Thus, in addition to Glu3930 and His4030 beingrequired for catalysis, Glu3900 andArg4001 also contribute substantiallyto either catalysis or to the structural integrity of the active site.

Thermal stability analysis suggested that the substitution muta-tions did not affect the structural stability or RRSP (fig. S8, A and B).Moreover, surface plasmon resonance (SPR) revealed that rRRSP hasan affinity for rKRAS in the high micromolar range (fig. S9A). Thisaffinity was slightly increased by the E3930A and H4030A mutations(fig. S9B), suggesting that the loss of activity was not due to the absenceof protein-protein binding. In agreement,NMRalso demonstrated thatrRRSPH4030A retains the ability to interact with rKRAS, as evidencedby chemical shift perturbations in Ile46 and Asp54 of rKRAS that de-pended on the concentration of rRRSP H4030A (fig. S10). Binding torRRSP H4030A also induced substantial signal attenuation in Switch Iand Switch II of rKRAS, most likely due to the association with mu-tated RRSP reducing backbone dynamics in these regions. Overall,these results demonstrate that the substitutionmutations did not affectprotein stability but rather that RRSP Glu3930 and His4030 are essentialfor Ras processing and thus likely share a hydrolytic mechanism witherythromycin esterases that similarly use a Glu-His catalytic pair (31).

RRSP cleavage of KRAS Switch I prevents KRASfrom interacting with son of sevenlessThe sonof sevenless (SOS)protein is aRasGEF that opens thenucleotide-binding site of Ras by displacing Switch I and inserting a helical hairpin,


thus allowing GDP-to-GTP exchange (38). The phosphate-binding (P)loop (residues 10 to 17) and Switch I and Switch II regions of Ras di-rectly interact with SOS (38). NMR analysis revealed a certain degree ofdisorder within these regions in rKRAS* compared to unprocessedKRAS. Line broadening was also observed in the Switch I region andresiduesAsp57-Gly60,Met67, Arg73, Thr74, andGly75, all of which direct-ly interact with SOS (Fig. 7A and fig. S7B) (38). Such structural altera-tion should compromise SOS function by blocking SOS-catalyzednucleotide exchange. To test this prediction, we loaded rKRAS withmant-GDP and carried out the SOS-catalyzed nucleotide exchangeassay in the presence of GDP (39). SOS was not able to significantlyaccelerate the nucleotide exchange rate of rKRAS* compared to that ofuncleaved rKRAS (Fig. 7, B to D). The intrinsic nucleotide exchangerate of rKRAS* was threefold faster than that of uncleaved rKRAS,suggesting that rKRAS* has an open conformation that is favorablefor intrinsic nucleotide exchange (Fig. 7D). In addition, the EDTA-catalyzed nucleotide exchange rate of rKRAS*was slightly higher thanthat of uncleaved rKRAS (Fig. 7D), confirming the presence of Mg2+

in the nucleotide-binding site.

The Ras-binding domain of RAF cannot bindto cleaved KRASThe direct physical interaction of Ras with its downstream effectorkinase Raf is essential for the activation of the Ras-MAPK pathway (6).Because the Ras Switch I domain is directly involved in the interactionwith Raf (40), RRSP cleavage of Ras between Tyr32 and Asp33 would be

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 wasloaded 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 incubatedwithwild-type RRSP at the indicated ratios (n = 3 independent experiments). (C) Bar graphs of the observed rKRAS cleavage rates (kobs) of wild-typerRRSP. (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 molarratio (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 arecolor-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) aremeans ± SD from three independent biological replicates (ANOVA followed by Dunnett’s multiple comparisons test, ****P ≤ 0.0001). ND, not detected.

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predicted to disrupt Raf binding (Fig. 7A and fig. S7B). To test thishypothesis, we used a proximity-based AlphaLISA binding assay(41). For this assay, biotinylated avidin-tagged KRAS (avi-KRAS)and the Ras-binding domain of RAF fused to glutathione S-transferase(GST-RAF-RBD) are attached to Alpha donor and acceptor beads,respectively, such that binding interaction results in singlet oxygenmolecule diffusion detected as light emission from the acceptor beadfluorophore after excitation of the donor bead (42). In this assay,binding of active GppNHp-loaded rKRAS to RAF was proportion-ally reduced by previous treatment of avi-KRAS with increasing con-centrations of rRRSP (Fig. 7E). This inhibition was not observed whenavi-KRAS was preincubated with rRRSP E3930A, rRRSP H3931A, orrRRSP H4030A mutant proteins (Fig. 7E) that do not cleave rKRAS(Fig. 3B). rRRSP E3900A and rRRSP H3902A were less efficient ininhibiting RAF-RBD–KRAS interaction, and the rRRSP R4001A mu-tant showed no inhibition (Fig. 7E).

We also used SPR to confirm that RRSP processing of KRAS re-duced the interaction between RAF-RBD (residues 55 to 131) andKRAS. The positive control, uncleaved GppNHp-loaded rKRASshowed strong interaction with RAF-RBD (Kd = 335 nM) (Fig. 7F).In contrast, we did not detect an interaction of RAF-RBDwith cleavedGppHNp-rKRAS* by SPR, even at the highest concentration of RAF-RBD (20 mM) (Fig. 7F). These data demonstrate that RRSP-mediatedprocessing of the Switch I domain of Ras prevents active KRAS from


binding to its downstream effector Raf and that this inhibition de-pends on the catalytic residues of RRSP.

RRSP processing of Ras in vitro and in cells requires catalyticresidues Glu3930 and His4030

To determine the impact of RRSP catalysis on cytotoxicity, we usedthe anthrax toxin protective antigen (PA) to deliver RRSP fused tothe anthrax toxin lethal factor N terminus (LFN-RRSP) into humanepithelial HeLa cells (fig. S11) (13, 20). In lysates from HeLa cellsintoxicated with LFN-RRSP+PA for 24 hours, an antibody recognizingthe G domain of all Ras isoforms detected a band of ~15 kDa cor-responding to the large fragment of cleavedRAS (Fig. 8A). In addition,phosphorylated ERK1 and ERK2 (pERK1/2) was reduced (Fig. 8B). Incontrast, only full-length (21 kDa) Ras and no change in ERK1/2 phos-phorylation were detected in HeLa cells intoxicated with catalyticallyinactive LFN-RRSP E3930A or LFN-RRSP H4030 (Fig. 8C). Thus,Glu3930 andHis4030 are essential for the Ras-processing activity of RRSPin cells and the downstream failure to activate ERK1/2.

Alanine substitution at His4030 in the MARTX holotoxinprevents Ras cleavage in cells treated with V. vulnificusTheRRSP in this studywas derived from the 5206 amino acidsMARTXtoxin from V. vulnificus strain CMCP6 (12, 16). To demonstrate therelevance of our biochemical findings in the context of the holotoxin

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 areindicated with dotted lines. (B and C) Representative curves of intrinsic and SOS-catalyzed nucleotide exchange for rKRAS (B) and rKRAS* (C) (n = 3 independentexperiments). (D) Bar graph of the intrinsic and SOS-catalyzed nucleotide exchange rates (kobs) of rKRAS (filled bars) and rKRAS* (open bars). Each bar represents themean ± 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 GraphPadPrism 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 pre-incubation of avi-KRAS with increasing concentrations of each rRRSP mutant. Emission data were normalized to control without addition of RRSP and plotted as 1/xto 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 threeindependent biological replicates. (F) Binding responses and dissociation constant (Kd) from Biacore SPR analysis of the interaction between immobilized RAF-RBD andinjected 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).

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during bacterial exposure, HeLa cells and clinically relevant T84 in-testinal cells, which express the activated mutant KRAS G13D, weretreated with live V. vulnificus that had been modified to alter the cat-alytic residues of RRSP in the holotoxin (Fig. 8D). Themodified strainswere confirmed to induce MARTX-associated cell rounding uponinfection (fig. S12A). To detect Ras processing, cells were treated withV. vulnificus for 1 hour, andwhole-cell lysateswere collected forWest-ern blotting. Lysates from cells treated with V. vulnificus carrying anintact copy of the gene that encodes the MARTXVv toxin (rtxA1+)showed detectable cleavage of Ras (Fig. 8E and fig. S12B). rtxA1+

strains both with and without the secondary virulence factor cytolysinVvhA showed equivalent processing of Ras. Cells treated with rtxA1-nullV. vulnificus (DrtxA1) or a strainwith an in-frame deletion in rtxA1to remove rrsp (Drrsp) did not cleave Ras, consistent with published ob-servations (13, 43). In contrast to previous studies, the use of a new pan-Ras antibody that recognizes the RasGdomain (rather than the Switch Idomain) facilitated detection of cleaved native Ras, as opposed to ecto-pically expressed taggedRas, indicating that this antibody can be used todetect cleavage of endogenous Ras without overexpression.

To test for contribution of the putative active site residues of RRSP,we used homologous recombination to mutate the His4030 codon to a


codon for alanine within the endogenousrtxA1 gene in the V. vulnificus chromo-some. This RRSP H4030A strain (rrsp*)retained the ability to induce cell round-ing, a phenotype of the multifunctionaltoxin that is independent of RRSP activity,thereby demonstrating that other por-tions of the large holotoxin remained in-tact. This single amino acid change did,however, completely abrogate MARTX-dependent Ras processing, demonstratingthat His4030 is an essential residue forMARTX toxin–mediated Ras cleavagein cells (Fig. 8, E and F.).

DISCUSSIONBacterial toxins have evolved to hijackcellular pathways to compromise the in-nate immune response and increase bac-terial invasion and systemic spread intothe host (1). Among many cellular tar-gets, inhibition or disregulation of smallGTPases is oneof themost commonmech-anisms for bacterial disruption of cellularprocesses (44). RRSP is a toxin effectordomain found inV. vulnificus,Aeromonashydrophila, and P. asymbiotica and hasbeen identified by sequence homology inother bacterial pathogens (13). RRSP spe-cifically processes the Switch I domain ofRas and Rap1A (13, 14), but the mecha-nism of cleavage remained elusive due tothe lack of RRSP sequence homology toknownprotease families.Wehave revealedthe mechanism of action of RRSP to be aproteolytic processing event that dislodgesSwitch I and alters backbone dynamics

downstream of the processing site without disturbing the overall struc-ture of Ras. We determined that this reaction is a metal-independentcatalysis mediated by Glu3930 and His4030, with contributions fromHis3902 and His3931 and, to a lesser extent, from Glu3900 and Arg4001.This study further demonstrates that RRSP cleavage of KRAS SwitchI between residues Tyr32 and Asp33 fully disconnects the Ras-MAPKpathway in human cells. The processing prevents the transition ofRas to an active conformation by blocking the interaction of Ras withGEFs and also impedes the interaction of Ras with the kinase Raf, suchthat proteins downstream of Raf in the signaling cascade, includingERK1/2, are not activated.

RRSP processing of Switch I does not affect the nucleotide-bindingcapacity of Ras. KRAS* showed strong affinity for both GDP andGTP, comparable to uncleaved KRAS. This result was surprising be-cause the nucleotide-binding pocket of Ras includes residues withinthe Switch I domain and the DXXG motif that were found by NMRanalysis to undergo conformational changes after RRSP-mediatedcleavage of Ras. The nucleotide-binding capacity of KRAS* can be ex-plained only by considering that a complex network of interactionsengages the nucleotide and its surrounding residues from Ras regionsthat interact with the guanosine moiety but were not affected by the

Fig. 8. Glu3930 and His4030 are essential for RRSP cytotoxicity. (A and B) Representative immunoblots of lysatesfrom HeLa cells incubated with LFNRRSP, the LFNRRSP E3930A and H4030A mutants, and anthrax toxin PA as indi-cated 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 mutantLFNRRSP as determined by densitometry analysis. (D) The ratio of pERK1/2 to total ERK1/2 in lysates from HeLa cellsafter treatment with wild-type and mutant LFNRRSP. (E) Schematic representation of MARTX toxin effector domainorganization in V. vulnificus CMCP6 strains. (F) Representative immunoblots showing RAS in lysates from T84 cells thathas been incubated with V. vulnificus producing the indicated MARTX toxins (n = 3). (G) Percentage of Ras that wascleaved in (E) as determined by densitometry analysis. All graphs (C, D, and G) showmeans ± SD from three independentbiological replicates (ANOVA followed by Dunnett’s multiple comparisons test, *P ≤ 0.05, **P ≤ 0.01, and ****P ≤ 0.0001).

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bisection of Switch I. In particular, the crystal structure ofHRAS in theactive state shows that Phe28 is positioned perpendicular to the aro-matic ring of the nucleotide guanosine moiety, making a tight hydro-phobic interaction (37). Amino acid substitution of Phe28 reduces theaffinity for GDP and GTP and increases the nucleotide exchange rate(45). Phe28maintains its orientation after RRSP processing, suggestingthat it is one of the crucial residues for the binding of nucleotides tocleaved Ras. However, despite retention of the nucleotide, cleaved Rascan no longer be activated by SOS or activate downstream effector ki-nases due to structural perturbation of the SOS-binding region.

The three-dimensional structure ofRRSP shares a protein foldwiththe C terminus of PMT, as previously predicted by primary sequencealignment (20). However, PMT-C1C2 does not cleave Ras, revealingthat PMT possibly evolved by sequence divergence to have a differentyet unknown function, potentially losing the ability to cleave Ras uponcombining with the C3 domain that converted PMT from a Ras-inactivating toxin to a Ras-activating toxin.

The structure ofRRSP also revealed that the RRSP-C2B subdomainshares an a/b fold withmembers of the EreA/ChaN-like family, whichGlu3930 andHis4030 have been shown to be critical for RRSPprocessingand cytotoxicity, consistent with the function of the corresponding res-idues from the erythromycin esterase EreB (31), Wnt metalloproteaseTIKI2 (32), and the plant cytotoxin HopBA1 (26). Mutant forms ofRRSP bearing substitutions in other modeled active site residues,E3900A and H3902A, showed reduced cleavage of KRAS. Unex-pectedly, His3931 was also important for RRSP activity. This histidine isnot conserved across the EreA/ChaN-like family but is 100% conservedin RRSP domains from other toxins. The identification of a conservedcatalytic site organization among different enzymes with different activ-ities might suggest a common catalytic mechanism that is preserved bythe similar structural fold. Morar et al. (31) proposed that EreA and EreBuse a His-Glu pair to activate a water molecule, which is involved in anucleophilic attack on the carbonyl carbon group of ester bonds. Wespeculate that RRSP would similarly use activation of water by its cat-alytic Glu-His pair to initiate the nucleophilic attack on the peptidebond in Ras. Zhang et al. (32) alternatively suggested that the putativeHis-Glu catalytic residues of TIKI2 and EreA are crucial for coordinat-ing catalytic divalent ions (Co2+ or Mn2+) to process the peptide bond.However, similar to RRSP, EreB and Bcr136 esterase activities are notinhibited by metal chelators. This suggests that the metal dependenceof EreA and Tiki proteins might be due to the requirement of a metalion for structural integrity but not for catalysis (31, 36). Cocrystalstructures of EreA/ChaN-like family members with their natural sub-strates will be critical to further probe the enzymatic activity of the pu-tative catalytic residues to determine whether all EreA/ChaN-likemembers have a sharedmechanismof nucleophilic attack for hydrolysisor whether some potentially use water whereas others use a metal ion.

Overall, this study reveals that the catalytic site residues used by theRRSP effector domain to promote pathogenesis are found within theC2B subdomain. The function of the C2A subdomain remains un-known. A previous study found that ectopic overexpression of C2A,but not ofC2B, inHeLa cells was sufficient to induce cell rounding andcytotoxicity (20). This finding is consistent with a proposed two-steprecognition of the substrate (14). In thismodel, C2Amight be requiredfor specific binding to the backbone of Ras before insertion of Switch Iinto the active site inC2B. These studies indicate that C2A overexpres-sion may inhibit Ras function by competitive binding, independent ofSwitch I processing, whereas C2B alone is not sufficient to cleave Ras(20). This may be due to improper folding of C2B when it is expressed


alone, to improper membrane targeting of C2B in the absence of theC1 subdomain, or to the absence of putative substrate binding byC2A.The interaction between the C2A and KRASmay account for some ofthe detected changes in backbone dynamics observed by NMR whenKRAS was incubated with increasing concentrations of catalyticallyinactive RRSP.

During V. vulnificus infection, the loss of signaling through the Ras-ERK pathway would result in reduced innate immune responses es-sential for host protection, thus enhancing bacterial infection (46–48).Thepresence of RRSP increases the potency of theV. vulnificusMARTXtoxin (12). Furthermore, a study found that infection of mice with aV. vulnificus strain that delivers an RRSP+MARTX toxin induced a lessrobust immune defense compared to a strain that delivers a MARTXtoxin with a different collection of effectors that does not include RRSP(49). Furthermore, toxin modifications to delete RRSP from thisMARTX toxin, alone or in combination with another effector, affectednot only ERK but also AKT signaling (50).

These studies may affect our understanding of tumorigenesis andanticancer drug discovery in the future. There are currently no directinhibitors of Ras in clinical use, and there are no chemical inhibitorsuseful for either in vitro or in vivo studies of Ras. Bacterial toxins suchas the clostridial toxin TpeL that glucosylates Thr35 in Ras have beenused to inhibit Ras to study downstream signaling (24). Here, rRRSPprocessing of KRAS in the AlphaLISA proximity assay not only dem-onstrates that RRSP blocks Raf recruitment but also validates this as-say as a screening tool for identifying chemical inhibitors that couldinhibit Ras-Raf binding. There are no specific inhibitors of the Ras-Rafinteraction available, but blocking this interaction would be a usefulstrategy for targeting Ras activation in cancer. Furthermore, our find-ing that LFN-RRSP cleaves Ras in vivo creates a tool for inhibiting RASand pERK in cells to probe other aspects of cell biology. LFN-RRSP+PAhas already been used to demonstrate that desmoplakin activation ofERK1/2 depends onKRAS signaling in cardiomyocytes (51). It has beendemonstrated that LFN-RRSP can cleave mutant KRAS G13D fromHCT116 colorectal and MDA-MB-231 breast cancer cells (13), andnow, we report detection of LFN-RRSP–mediated processing of KRASG13D in T84 colorectal cancer cells. Future studies will address thebroad applicability of RRSP as a tool to study the role of KRAS in cellbiological processes and cancer and may inform strategies for attenuat-ing Ras signaling in cancer cells.

MATERIALS AND METHODSReagentsEscherichia coli TOP10 cells were obtained from Thermo Life Tech-nologies, and E. coli BL21(DE3)(pMagic) (52) and pMCSG7 bacterialexpression vector (53) were provided by A. Joachimiak (Argonne Na-tional Laboratory). Common reagents were obtained from Sigma-Aldrich, Fisher Scientific, or VWR. Restriction enzymes Stu I, Eco RI,Ssp I, and Bam HI and Gibson Assembly Master Mix were obtainedfrom New England Biolabs. Custom DNA oligonucleotide primersand gBlocks as listed in tables S1 and S2were purchased from IntegratedDNA Technologies. Plasmids were purified by using Epoch spincolumns or Qiagen Midiprep kit according to the manufacturer’s re-commended protocol. Plasmids were introduced into E. coli BL21(DE3)(pMagic) by electroporation. Recombinant proteinswere purifiedusing ÄKTAXpress Purifier system (GEHealthcare Life Sciences). Pre-packed HisTrap FF nickel-Sepharose for affinity chromatography orSuperdex 200 (26/60) for size exclusion chromatography (SEC) were

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purchased from GE Healthcare Life Sciences. Anthrax PA was pur-chased from List Biological Laboratories Inc.

Crystallization and structure determination of RRSPrRRSP construct previously designed (13) was expressed in M9 seleno-methionine high-yield media according to the vendor’s instructions(MD045004-50L, Orion Enterprise Inc.). Protein expression was in-duced by adding 1 mM isopropyl-b-D-thiogalactoside (IPTG) at anOD600 (optical density of 600 nm) of 0.8, and growth was continuedat 18°C cells for 18 hours. Cells were harvested by centrifugation, andrRRSP was purified as previously described (13). The purified proteinwas concentrated to 15 mg/ml. Sitting drop crystallization plates wereset up at room temperature by mixing 1 ml of rRRSP and 1 ml of res-ervoir solution with 90 ml of reservoir solution. Crystals were grown at19°C using a reservoir of 0.1 M 3-(N-morpholino)propanesulfonicacid) (Mops) (pH 6.5), 5% (w/v) polyethylene glycol 400, and 2 MNH4SO4. Harvested crystals were transferred to 4 M formate beforebeing frozen in liquid nitrogen. Diffraction data were collected at 100 Kfrom a single crystal at experimental station 21ID-F of the Life ScienceCollaborative Access Team (LS-CAT) at the Advanced Photon Source(APS). Crystals belong to the cubic space group I23 with unit cellparameters 247.1, 247.1, 247.1, 90.0, 90.0, and 90.0. Two hundredframes, which correspond to 120° of spindle axes rotation, werecollected from a single crystal at the selenium peak (l = 0.97872).Data were indexed, integrated, and scaled using the HKL3000 suite(54). The structure was solved with PHENIX (55), using the singleanomalous dispersion technique. The initial figure of merit was0.40, and after density modification, 975 of 1036 residues (two proteinchains of 494 each and 24 residues of the purification tag) were auto-matically built into the experimentally obtained electron density maps.The initial model was checked and manually corrected in Coot (56).Another data set was generated for refinement by scaling and mergingonly the first 100 frames, thus reducing radiation damage andincreasing the resolution of the data. The corrected model was refinedin Refmac (57), and solvent molecules were added to the model usingARP/wARP (58) followed by several rounds of refinement in Coot. TheTLS group correction to the B factors (59) was applied at the final stepsof refinement. The structure factors and coordinates of the final modelwere validated using SFCHECK (60) and MolProbity (61) anddeposited to the PDB with PDB ID 5W6L. The data quality and thequality of the model are summarized in Table 1.

Cloning, expression, and purification of RRSP andLFNRRSP proteinsCustom gBlocks designed to introduce mutations into C2B domain ofRRSP sequence were assembled into the Stu I/Eco RI–6xHis Tag–RRSP-pMCSG7 (13) digested vector using Gibson Assembly (NewEnglandBiolabs), following themanufacturer’s recommendedprotocol.For LFNRRSP proteins, DNA sequences corresponding to wild-typeRRSP, RRSP E3930A, and RRSPH4030Awere amplified from the tem-plates described above using primers designed for Gibson Assembly.Polymerase chain reaction (PCR) products were cloned into the expres-sion vector pABII previously digested with BamHI in frame with N-terminal 6xHis-tagged LFN domain (62). Plasmids were confirmed tobe accurate by DNA sequencing and then transformed into BL21(DE3)(pMagic) cells. All N-terminal 6xHis-tagged RRSP and LFNRRSPproteins were purified over a Ni-NTA HisTrap column followed bySEC using the ÄKTA protein purification system, as previously de-scribed (13).


Purification of KRAS 1 to 169The N-terminal portion of human KRAS (amino acids 1 to 169) wastagged with 6xHis and purified over a HisTrap FF and SEC followedby 6xHis-tag removal by Tobacco Etch Virus protease cleavage usingthe ÄKTA protein purification system, as previously described (63).

Cloning, expression, and purification of PMT-C1C2The codon-optimized DNA sequence corresponding to PMT (residues561 to 1088) was amplified from template plasmid previously described(20) using primers designed for ligation-independent cloning. Theproducts were cloned into the pMCSG7 expression vector previouslydigested with Ssp I. Plasmid was confirmed to be accurate by DNAsequencing and then transformed into BL21(DE3)(pMagic) cells. Re-combinant PMT-C1C2 was purified as described above for rRRSP.

Cloning, expression, and purification of SOSDNA sequence corresponding to SOS1 (residues 566 to 1049)was am-plified from the template plasmid obtained from the National CancerInstitute (NCI)-RAS Initiative RAS Reagents Core fully sequence-validated ORFeome clone set (41) using primers designed for GibsonAssembly (New England Biolabs). PCR products were cloned into thepMCSG7 expression vector previously digested with Ssp I. The plas-mid nucleotide sequence was confirmed byDNA sequencing and thentransformed into BL21(DE3)(pMagic) cells. Recombinant SOS waspurified as described above for rRRSP.

Expression and purification of 15N KRAS 1 to 169 for NMRKRAS 1 to 169 fused to the C-terminal 6xHis-tagged cysteine proteasedomain (CPD) (63) was expressed in M9 media containing 0.4 M15NH4Cl (CIL Inc.), 10 mM CaCl2, 10 mMMgCl2, and trace elements.Protein expression was induced by adding 1 mM IPTG at an OD600

of 0.8, and growth was continued at 25°C cells for 18 hours. Cellswere harvested by centrifugation, and 15N KRAS 1 to 169 was puri-fied as previously described (63). Briefly, KRAS 1 to 169 fused to theC-terminal 6xHis-tagged CPD was purified over a HisTrap FF col-umn and by SEC using the ÄKTA protein purification system,followed by the incubation of 100 mMof InsP6 (phytic acid) to triggerCPD autocleavage. Tagless 15N KRAS 1 to 169 was recovered by re-moving 6xHis-tagged CPD over a HisTrap FF column (63). NMRexperiments were performed at 25°C on a Bruker Avance 900-MHzspectrometer equipped with a high-sensitivity cryogenic probe. Theprotein was dissolved in 50 mM tris citrate (pH 6.5), 5 mMMgCl2,50 mM NaCl, 10 mM b-mercaptoethanol, 20 mM CaCl2, and 10%D2O to achieve the final concentration of 400 mM. Unlabeled wild-type rRRSP was added to KRAS at a 1:1 molar ratio. 15N HSQCexperiments were performed, and spectra of KRAS before and afterthe addition of rRRSP were compared. Experiments with rRRSPH4030A were performed similarly to the experiments with wild-typerRRSP, except rRRSP H4030A was added to KRAS at 0.01:1, 0.5:1, and1:1molar ratios. Sparky (University of California, San Francisco) (64) wasused for KRAS and cleaved KRAS assignments based on HC-NH NOEand Ca-NH (HNCA) (65), CBCACONH, and HNCACB experiments(66). NMRPipe software was used for processing and analyzing thedata (67).

In vitro RRSP cleavage assayrKRASwas incubatedwith rRRSP or rRRSPmutants or PMT-C1C2 atequimolar concentrations (10 mM) in 10 mM tris (pH 7.5), 500 mMNaCl, and 10 mMMgCl2 at 37°C for 30 min. Reactions were stopped

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by adding 6× Laemmli sample buffer and incubating the samples at90°C for 5 min. Proteins were separated by SDS-PAGE on 18% gelsand visualized using Coomassie stain.

For assays in the presence of chelators, 10 mM rRRSP was preincu-bated with 0.625, 1.25, 2.5, and 5mM of EDTA or 1,10-phenanthrolineat 37°C. After 30 min, 10 mM of rKRAS, previously exchanged in abuffer without MgCl2, was added, and the mixture was incubated at37°C for 30 min. As control, 0.1 mM thermolysin (Sigma) was pre-incubated with 5 mM of EDTA or 1,10-phenanthroline at 37°C. Forsome assays, rRRSP was first treated in 5 mM EDTA or 5 mM 1,10-phenanthroline for 30min and then passaged over a PD10 columnwithexchange to buffer A [10 mM tris-HCl and 500 mM NaCl (pH 8.0)].The treated rRRSP was then added at increasing concentrations to thebuffer-exchanged rKRAS. For some assay, rKRAS that was not buffer-exchanged was used to show the impact ofMgCl2 carryover. For someassays, divalent cations as indicated in the legendwere added to a finalconcentration of 5 mM. These reactions were incubated at 37°C for10 min and then analyzed by SDS-PAGE as above.

SDS-PAGE analysis and quantificationCleavage products of rKRAS were quantified from scanned SDS-polyacrylamide gels using National Institutes of Health (NIH) ImageJ1.64 (68). The percentage of cleavage product was calculated by thefollowing formula

%Cleavage product ¼ cleaved band=ðcleaved rKRAS þuncleaved rKRASÞ � 100

where cleaved rKRAS is the band running at 19 KDa. Each percentagevalue reported represents the mean ± SD of results from three in-dependent experiments.

Differential scanning fluorimetryDSF experiments were performed using a 96-well thin-wall PCR plate(Axigen). Twenty-microliter reactions consisted of 2mMrKRAS, cleavedrKRAS, or rRRSP proteins in a solution of 5× SYPRO orange dye(Thermo Life Technologies); 0.1 mM tris; and 150 mM NaCl (pH 7.5).Fluorescence intensity was monitored using the StepOnePlusTM Real-Time PCR Systems (Thermo Life Technologies) instrument. Sampleswere heated from 25° to 95°C at a scan rate of 1°C/min. Data were fitin GraphPad Prism 6.0 (GraphPad Software Inc.) to Boltzmann equa-tion to extrapolateTmvalues that are reported asmeans± SD from threeindependent biological replicates.

Nucleotide exchange assayrKRASwas exchanged in 20mMtris (pH7.5), 150mMNaCl, and5mMb-mercaptoethanol using PD10 desalting columns (GE Healthcare).rKRAS was incubated for 1 hour at 37°C with rRRSP at a molar ratioof 1:100 (rKRAS/rRSSP). For the EDTA-mediated nucleotide ex-change assay, 2 mM rKRAS and 2 mM rKRAS* were mixed with 4 mMmant-GTP (Thermo Life Technologies) or mant-GppNHp (JenaBioscience) in assay buffer [20 mM tris (pH 7.5), 150 mM NaCl,5 mM b-mercaptoethanol, 10 mM MgCl2, and 20 mM EDTA] anddispensed into a 384-well plate. Fluorescence was measured every10 s for 5 min at excitation/emission set to 360 nm/440 nm at 25°Cin a SpectraMaxM3 plate reader (Molecular Devices). Data were fitin GraphPad Prism 6.0 (GraphPad Software Inc.) to a one-phaseexponential association curve to determine the observed nucleotide


exchange rate kobs. Data were reported as means ± SD from threeindependent biological replicates.

Fluorescence real-time cleavage assayrKRAS was incubated with 20-fold excess mant-GDP in the presenceof 20mMEDTA. After 1 hour at room temperature,MgCl2 was addedto a final concentration of 40 mM, and the mix was incubated for30 min at 25°C. The protein was exchanged in 20 mM tris (pH 7.5),150 mM NaCl, 10 mM MgCl2, and 5 mM b-mercaptoethanol usingPD10 desalting columns (GE Healthcare); 1 mM of mant-GDP–loaded rKRAS was dispensed in a 384-well plate, and the cleavagereaction was started by adding each rRRSP at equimolar concentra-tion with rKRAS. Fluorescence wasmeasured every 25 s for 30min atexcitation/emission set to 360 nm/440 nm at 25°C in a SpectraMaxM3plate reader (MolecularDevices). Datawere fit inGraphPadPrism6.0 (GraphPad Software Inc.) to a single-exponential decay curve todetermine the observed rKRAS processing kobs. Data were reportedas means ± SD from three independent biological replicates.

SOS-mediated nucleotide exchange assayrKRAS was loaded with mant-GDP, as described above. For the assay,1 mM rKRAS and 1 mM rKRAS* were in the assay buffer [20 mM tris(pH 7.5), 150 mM NaCl, 5 mM b-mercaptoethanol, 10 mM MgCl2,and 5 mM GDP] and dispensed into a 384-well plate. The SOS-mediated nucleotide exchange assay was initiated by the addition of1 and 10 mM SOS to the reaction mix. Fluorescence was measuredevery minute for 5 hours at excitation/emission set to 360 nm/440 nmat 25°C in a SpectraMax M3 plate reader (Molecular Devices). Datawere fit in GraphPad Prism 6.0 (GraphPad Software Inc.) to a singleexponential decay curve to determine the observed rKRAS processingkobs. Data were reported as means ± SD from three independentbiological replicates.

SPR measurementsSPR binding experiments were performed on a Biacore T200 instru-ment (GE). Human RAF1-RBD (RAF1 residues 51 to 131) and bio-tinylated avi-KRAS [KRAS with a C-terminal 15 amino acid AviTag(GLNDIFEAQKIEWHE)] proteins for this assay are described byEsposito et al. (41). Avi-KRASwas loadedwithGppNHp and incubatedwith rRRSP (1:1 molar ratio) for 30 min at 37°C. Binding measure-ments of RAF1-RBD on avi-KRAS–GppNHp and cleaved avi-KRAS–GppNHp were carried out as follows. Neutravidin (Pierce) wasamine-coupled to the carboxymethylated dextran surface of a CM5sensor chip (GE) using standard amine coupling chemistry. The sur-face was activated with 0.1 M N-hydroxysuccinimide and 0.4 MN-ethyl-N′-(3-dimethylaminopropyl) carbodiimide at a flow rate of20 ml/min. Neutravidin was diluted to 20 mg/ml in 10 mM sodiumacetate (pH 4.5) and injected on all four flow cells until a density ofabout 5000 RU was attached. Activated amine groups were quenchedwith an injection of 1M ethanolamine (pH 8.0). Avi-KRAS-GppNHp,and the cleaved avi-KRAS–GppNHp were captured on flow cells 2and 4, and flow cells 1 and 3 were used for referencing purposes.After the protein capture, a series of buffer injections were performedin the running buffer [20 mM Hepes (pH 7.5), 150 mM NaCl, 5 mMMgCl2, 1 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP),and 5 mMGppNHp] to establish a stable baseline. RBD was diluted inrunning buffer from 20 mM to 4 nM and injected over the capturedavi-KRAS protein surface for 1min at 30 ml/min at 25°C. At the end ofthe injection, bound RAF1-RBD was removed by a 30-s injection of

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1 M NaCl. The data were fit using the Biacore T200 evaluationsoftware. Binding of rKRAS to rRRSP was performed by amine cou-pling wild-type rRRSP, E3930A, or H4030A protein on a CM5 sensorchip as outlined above. Serial dilutions of rKRAS-GDP (500 to 3.9 mM)were injectedover the rRRSPproteins in 20mMHepes (pH7.5), 150mMNaCl, 5 mMMgCl2, 1 mM TCEP, and 5 mMGDP. The RRSP surfaceswere regenerated with an injection of 1 M NaCl. Data were analyzedusing Biacore T200 evaluation software, and steady-state binding wasfit using Origin software.

GST-RAF-RBD/rKRAS AlphaLISA binding assayThe effect of KRAS cleavage by rRRSP on effector binding wasstudied by in vitro protein-protein interaction using PerkinElmer’sAlphaLISA Technology as previously described (41, 42). RAF1-RBD(RAF residues 51 to 131) and biotinylated avi-KRAS proteins for thisassay are described by Esposito et al. (41). Briefly, avi-KRAS bindsthe donor bead, and GST-RAF-RBD binds the acceptor bead. Uponlaser excitation of the donor bead, donor bead–generated singlet-state oxygen molecules react with the acceptor bead to generatechemiluminescence that then activates a fluorophore in the samebead for detectable emission at 520 to 620 nm (42). To determinethe impact of rRRSP processing on KRAS interaction with RAF,avi-KRAS–GppNHp was incubated with rRRSP proteins (titration,1 mMto 0.4 nM) for 1 hour at 37°C in a shaker (300 rpm), in the assaybuffer [50mM tris (pH 7.5), 5mMNaCl, and 0.1mMdithiothreitol],followed by the addition of GST-RAF-RBD, and the binding reac-tions were carried out for 1 hour at room temperature with shaking(700 rpm). In the final reaction, avi-KRAS–GppNHp andGST-GST-RAF-RBD were 300 nM. After incubation, 30-ml mix of streptavidinalpha donor beads (PerkinElmer) and glutathione acceptor beads(PerkinElmer) in bead buffer [50mM tris (pH 7.5) and 0.01%Tween20] were added to each reaction, and the mix was incubated for1 hour at room temperature and protected from light. The reactionswere read on a PerkinElmer EnVisionMultilabel Reader. Emission datawere normalized to control sample without addition of RRSP and thenplotted versus concentration using GraphPad Prism 6.0 GraphPadSoftware Inc.) as log(inhibitor) versus normalized response curve todetermine median inhibitory concentration (IC50) values. Data werereported as means ± SD from three independent biological replicates.

HeLa cells intoxicationHuman HeLa female epithelial cells (American Type Culture Collec-tion, CCL-2) were seeded into six-well cell culture-treated plate andgrown to 70%, and at that time, cells were intoxicated with 3 nM ofLFNRRSPproteins in combinationwith 7 nMof PA for 24 hours. Cellswere collected in the lysis buffer, and total protein content was deter-mined by a BCA kit. Cell lysates were separated on 18% SDS-PAGEfollowed by immunoblotting using an antibody that recognizes allforms of Ras (CPTC-KRAS4b-2, Developmental Studies HybridomaBank at IowaUniversity) and antibodies that recognize ERK1/2 (catalogno. 4696S, Cell Signaling Technology), pERK1/2 (catalog no. 4377S,Cell Signaling Technology), and vinculin (Abcam). Band quantificationwas performed by NIH ImageJ 1.64 (68), and band intensities valueswere normalized with vinculin.

Generation of RRSP H4030A (rrsp*) in V. vulnificus strainsCustomgBlock corresponding to regions upstream anddownstreamofRRSPH4030Awas designed and assembled into digested pDS132 (69)usingGibsonAssembly according to themanufacturer’s protocols. The


resulting plasmid was transformed to DH5a, and it was purified byminiprep. It was then transformed to SM10lpir (70). The RRSPH4030A plasmidwas transferred toV. vulnificusDvvhACMCP6 strainby conjugation followed by selection for double homologous recombi-nation using sucrose counter selection to isolate recombinants as pre-viously described (71). The strainwas validated to acquire themutationby sequencing of amplified DNA.

Bacterial challenge of T84 and HeLa cellsV. vulnificus rifampicin-resistant isolates of strains CMCP6, CMCP6DvvhA, CMCP6 DvvhADrtxA1 (12), CMCP6 DvvhADrrsp (43), andCMCP6 DvvhArrsp* were grown at 30°C in Luria-Bertani mediumwith rifampicin (50 mg/ml). Overnight cultures were diluted 1:1000and grown at 30°C with shaking until the OD600 reached 0.5 to 0.6.Bacteria from1 to 2mlwere pelleted by centrifugation and resuspendedin phosphate-buffered saline (PBS) to equal bacterial concentrations.Media were exchanged over 1 × 106 human T84 male intestinal epithe-lial cells (American Type Culture Collection, CCL-248) or 2 × 105HeLacells previously seeded into six-well cell culture-treated plate overnightfor antibiotic-free media. V. vulnificus strains in PBS (multiplicity ofinfection = 100) or an equal volume of buffer was added to mediaover cells and plates. After 60min, bothHeLa cells and T84 cells werecollected for immunoblotting analysis, as described above. HeLacells were also photographed before collection to validate that intro-duced mutations did not affect other biological activities of the toxinincluding cell rounding.

SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/11/550/eaat8335/DC1Fig. 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 RRSPprocessing in HeLa cells.Table S1. Oligonucleotides used in the study.Table S2. Sequence of gBlocks used in the study.References (74–80)

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Acknowledgments: We thank Z. Wawrzak, M. Lam, H. Gavin, S. F. Dunn, and C.-H. Luan forassistance in technical development of the project. X-ray data collection was done at theLS-CAT beamlines 21ID-F at the APS Science User Facility operated for the U.S. Department ofEnergy. Services were provided by the Northwestern Genomics Core, Structural Biology Core,and High Throughput Analytical Laboratory and by the Nuclear Magnetic ResonanceLaboratory of the University of Illinois at Chicago. Funding: This work has been funded inwhole or in part with Federal funds under NCI/NIH contract number HHSN261200800001E forthe Frederick National Laboratory for Cancer Research (FNLCR) and National Institute ofAllergy and Infectious Diseases (NIAID)/NIH contract numbers HHSN272201200026C (to W.F.A.)and HHSN272201700060C (to K.J.F.S.) for the Center for Structural Genomics of InfectiousDiseases. Additional funding came from PanCan/FNLCR KRAS and Chicago BiomedicalConsortium Fellowships (to M.B.), U.S. Department of Defense Congressionally DirectedMedical Research Programs grant CA160954 (to A.B.), NIAID/NIH fellowship T32AI007476(to P.J.W.), NIH grants R01AI092825 and R01AI098369 (to K.J.F.S.) and R01CA188427 (to V.G.),the NCI-RAS Initiative at FNLCR (to A.G.S. and M.H.), the Robert H. Lurie Cancer ResearchCenter, the Northwestern Medicine Catalyst Fund, and a pilot grant from the NorthwesternUniversity Skin Diseases Research Center funded by NIH/National Institute of Arthritis andMusculoskeletal and Skin Diseases (grant P30 AR057216). The content of this publicationdoes not necessarily reflect the views or policies of the Department of Health and HumanServices, nor does the mention of trade names, commercial products, or organizations implyendorsement by the U.S. Government. Author contributions: M.B. designed and conductedexperiments, analyzed data, prepared figures, and wrote the manuscript. G.M. collectedx-ray data and refined the crystal structure. A.B. collected and analyzed NMR data. A.H. andM.B.K. collected and analyzed EC50 data for RRSP cleavage. P.J.W. collected and analyzeddata using T84 cells. L.B. and M.A.-B. collected and analyzed SPR and AlphaLISA data, respectively.W.F.A. provided insight into the crystallization of RRSP and its catalytic mechanism. V.G.provided insight into the NMR studies and analyzed data. A.G.S. and M.H. provided insightinto design, conduct, and analysis of the KRAS-RAF interaction studies. K.J.F.S. coordinated allaspects of the design and analysis of experiments and wrote the manuscript. Competinginterests: M.B. and K.J.F.S. have a patent pending related to use of RRSP in study andtreatment of cancer and other Ras-related syndromes. All other authors declare that theyhave no competing interest. Data and materials availability: The structure of RRSP hasbeen reviewed and deposited into PDB (www.rcsg.org) with PDB ID 5W6L. Recombinantoverexpression plasmids for proteins described herein and newly created bacterial strains areavailable from Northwestern University with a material transfer agreement. Reagents relatedto the AlphaLISA and SPR are available from the NCI-RAS Initiative with a materials transferagreement. All other data needed to evaluate the conclusions in the study are present in thepaper or the Supplementary Materials.

Submitted 9 April 2018Accepted 4 September 2018Published 2 October 201810.1126/scisignal.aat8335

Citation: M. Biancucci, G. Minasov, A. Banerjee, A. Herrera, P. J. Woida, M. B. Kieffer, L. Bindu,M. Abreu-Blanco, W. F. Anderson, V. Gaponenko, A. G. Stephen, M. Holderfield, K. J. F. Satchell,The bacterial Ras/Rap1 site-specific endopeptidase RRSP cleaves Ras through an atypicalmechanism to disrupt Ras-ERK signaling. Sci. Signal. 11, eaat8335 (2018).

15 of 15

http://www.rcsg.org


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

Abreu-Blanco, Wayne F. Anderson, Vadim Gaponenko, Andrew G. Stephen, Matthew Holderfield and Karla J. F. SatchellMarco Biancucci, George Minasov, Avik Banerjee, Alfa Herrera, Patrick J. Woida, Matthew B. Kieffer, Lakshman Bindu, Maria

DOI: 10.1126/scisignal.aat8335 (550), eaat8335.11Sci. Signal.

activated RAS proteins.mechanism of an unusual protease and may prove useful for developing new strategies for targeting cancer-associatedexchange was inhibited and it could not bind to its downstream effector RAF. These findings provide insight into the not release any fragments or bound nucleotides from KRAS but altered the structure of KRAS so that nucleotideof hydrolytic enzymes, and identified the catalytic residues required for RRSP to cleave KRAS. Cleavage by RRSP did

. solved the crystal structure of RRSP, which showed similarity to a familyet alhomology to known proteases. Biancucci toxin that includes the Ras/Rap1-specific endopeptidase (RRSP) effector domain, which cleaves Ras but has no

produce aVibrio vulnificushost cell biology and innate defenses. Highly virulent strains of the opportunistic pathogen Many pathogenic bacteria target the small GTPase Ras because it is critical for signaling pathways that control

disrupts RAS signalingVibrioHow

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