Article Differences in Ribosome Binding and Sarcin/ Loop Depurination by Shiga and Ricin Holotoxins

Xiao-Ping Li * and Nilgun E. Tumer Department of Biology, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-848-932-6355

Academic Editors: Julien Barbier and Daniel Gillet Received: 3 January 2017; Accepted: 5 April 2017; Published: 11 April 2017

Abstract: Both ricin and Shiga holotoxins display no ribosomal activity in their native forms and need to be activated to inhibit translation in a -free translation inhibition assay. This is because the ribosome binding site of the ricin A chain (RTA) is blocked by the B subunit in ricin holotoxin. However, it is not clear why Shiga 1 (Stx1) or Shiga toxin 2 (Stx2) holotoxin is not active in a cell-free system. Here, we compare the ribosome binding and depurination activity of Stx1 and Stx2 holotoxins with the A1 subunits of Stx1 and Stx2 using either the ribosome or a 10-mer RNA mimic of the sarcin/ricin loop as substrates. Our results demonstrate that the active sites of Stx1 and Stx2 holotoxins are blocked by the A2 chain and the B subunit, while the ribosome binding sites are exposed to the solvent. Unlike ricin, which is enzymatically active, but cannot interact with the ribosome, Stx1 and Stx2 holotoxins are enzymatically inactive but can interact with the ribosome.

Keywords: Shiga toxin 1; Shiga toxin 2; Stx1; Stx2; ricin; ribosome binding; depurination activity

1. Introduction Shiga-toxin producing E. coli (STEC) infections can cause life threatening complications such as hemolytic uremic syndrome (HUS) or hemorrhagic (HC), and are the leading cause of death from foodborne bacterial infections in children [1]. Shiga toxins (Stxs) are the primary virulence factors of STEC and belong to a group of proteins called type II ribosome inactivating proteins (RIPs). There are two main Stx types, designated Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2), and within each are many subtypes. Stx1 and Stx2 contain a catalytically active A subunit and five copies of cell binding B subunits [2,3]. Stx1 and Stx2 share 55% and 57% amino sequence identity in the A and B subunits, respectively, and have similar molecular structures. The A subunits remove a universally conserved adenine from the sarcin/ricin loop (SRL) of the large ribosomal RNA and inhibit translation. The B subunits bind to a globotriaosylceramide (Gb3 or CD77) receptor on the cell surface and facilitate endocytosis. Ricin produced by the castor bean is another type II RIP. The A subunit of ricin (RTA) has similar enzymatic function and molecular structure to the A subunits of Stxs, while the B subunit of ricin (RTB) is functionally similar but structurally different and binds to different cellular receptors [2]. Residues important for the enzymatic activity of the A subunits are conserved among all RIPs [4]. Although Stx and ricin holotoxins are very toxic to eukaryotic cells, they are not active towards the ribosome and are activated after separation of the B subunits from the A subunits [5,6]. Stx1 and Stx2 are AB5 toxins consisting of one A subunit and a pentamer of B subunits. In E. coli, the genes encoding Stx1 or Stx2 A (A1 + A2) and B subunits are linked, but they are encoded by separate ORFs [7]. In ricin, the RTA and RTB are encoded by a single gene and synthesized as preproricin with a signal peptide preceding the A chain and a linker peptide between RTA and RTB [8]. The signal sequence and the linker peptide are cleaved during the maturation of ricin, but RTA and RTB remain linked together by a disulfide bond [9,10]. Stx1, Stx2 or ricin traffic from the endosome

Toxins 2017, 9, 133; doi:10.3390/toxins9040133 www.mdpi.com/journal/toxins ToxinsToxins2017 2017,, 99,, 133133 2 of 112 of 12

and RTB remain linked together by a disulfide bond [9,10]. Stx1, Stx2 or ricin traffic from the to the trans-Golgi network in a retrograde manner and reach the endoplasmic reticulum (ER) [5,6]. endosome to the trans-Golgi network in a retrograde manner and reach the endoplasmic reticulum During(ER) [5,6]. this During process, this A1 process, and A2 chainsA1 and of A2 Stxs chains are cleaved of Stxs byare furin cleaved between by furin R251 between and M252 R251 in Stx1and and betweenM252 in R250Stx1 and and between A251 in R250 Stx2 (Figureand A2511)[ in11 Stx2]. The (Figure furin 1) cleavage [11]. The sites furin are cleavage located sites in protease-sensitiveare located in loops.protease-sensitive After furin cleavage,loops. After the furin A1 subunit cleavage, and th A2e A1 chain subunit remain and connected A2 chain byremain a disulfide connected bond by and a the Bdisulfide subunit bond remains and connectedthe B subunit to the remains A1 subunit connected through to the the A1 A2 subunit chain. through In the ER, the theA2 disulfidechain. In the bond is reducedER, the disulfide by protein bond disulfide is reduced isomerase, by protein releasing disulf theide A1isomerase, subunit releasing of Stxs from the theA1 Bsubunit subunit, of andStxs RTA fromfrom RTB.the TheB subunit, A1 subunits and ofRTA Stxs from and RTARTB. unfold The A1 and subunits retrotranslocate of Stxs outand of theRTA ER unfold and refold and into anretrotranslocate active form in out the of cytosol the ER [and5,6]. refold into an active form in the cytosol [5,6].

Figure 1. TheThe sequences sequences of of mature mature A Achains chains of ofShigellaShigella StxStx and and Stx2. Stx2. Furin Furin recognition recognition sites sitesare shown are shown inin blue. The The sequence sequence before before the the furin furin cleavage cleavage site site corresponds corresponds to the to theA1 A1subunits subunits and andthe sequence the sequence after the furin cleavage cleavage site site corresponds corresponds to to the the A2 A2 subunits. subunits.

Stx2-producing E. coli are more often associated with HUS than Stx1-producing E. coli but the Stx2-producing E. coli are more often associated with HUS than Stx1-producing E. coli but the reason for this is unclear [12,13]. We showed that the A1 subunit of Stx2 (Stx2A1) bound ribosomes reason for this is unclear [12,13]. We showed that the A1 subunit of Stx2 (Stx2A1) bound ribosomes more tightly, had higher activity and was more toxic than the A1 subunit of Stx1 (Stx1A1) [14]. RTA, more tightly, had higher activity and was more toxic than the A1 subunit of Stx1 (Stx1A1) [14]. Stx1A1 and Stx2A1 interact with the C-terminus of the ribosomal P-protein stalk to access the SRL RTA, Stx1A1 and Stx2A1 interact with the C-terminus of the ribosomal P-protein stalk to access the [15–19]. The eukaryotic ribosomal P-protein stalk is a pentameric complex located on the 60S subunit SRLin close [15 –proximity19]. The eukaryoticto the SRL. It ribosomal consists of P-protein two P1/P2 stalk dimers, is a which pentameric bind to complex uL10 (previously located onP0) the to 60S subunit in close proximity to the SRL. It consists of two P1/P2 dimers, which bind to uL10 (previously form uL10-(P1/P2)2 structure. The C-terminal sequence of the five P-proteins is exactly the same and P0)is conserved to form uL10-(P1/P2) among all eukaryotes.2 structure. The The ribosomal C-terminal P-protein sequence stalk of together the five P-proteinswith the sarcin/ricin is exactly loop the same and(SRL), is conservedare part of among the GTPase all eukaryotes. associated The cent ribosomaler, which P-protein is responsible stalk together for the with recruitment the sarcin/ricin of looptranslational (SRL), areGTPases part ofand the stim GTPaseulation associatedof factor-dependent center, which GTP hydrolysis is responsible [20–23]. for the recruitment of translationalThe X-ray GTPases crystal structures and stimulation of Shiga of toxin factor-dependent from (Stx), GTP hydrolysiswhich differs [20 only–23]. in one residue fromThe E. coli X-ray Stx1, crystal and E. structurescoli Stx2 are of resolved Shiga toxin [24–28], from butShigella not the(Stx), structures which of differs the A1 onlysubunits in one alone. residue fromFigureE. 2 coli comparesStx1, and theE. structure coli Stx2 of are RTA resolved with the [24 structures–28], but notof StxA1 the structures and Stx2A1 of derived the A1 subunitsfrom their alone. Figureholotoxin2 compares structure. the On structure the left, the of residues RTA with in theStx structuresand in RTA, of which StxA1 interact and Stx2A1 with the derived C-termini from of their holotoxinribosomal structure.stalk P-proteins, On the are left, shown the residues in magenta in Stx and and in inlight RTA, blue. which Rotating interact 180° with along the the C-termini y-axis, of ribosomalon the right, stalk the P-proteins,active sites are shown in red. magenta Cys242/241 and in in light Stx/Stx2 blue. A1, Rotating which 180forms◦ along a disulfide the y-axis, onbond the with right, Cys261/260 the active in sites Stx/Stx2 are shown A2, and in Cys259 red. Cys242/241 in RTA, which in Stx/Stx2 forms a disulfide A1, which bond forms with a Cys2 disulfide bondin RTB, with areCys261/260 shown in yellow. in Stx/Stx2 A2, and Cys259 in RTA, which forms a disulfide bond with Cys2 in RTB, are shown in yellow. Toxins 2017, 9, 133 3 of 12 Toxins 2017, 9, 133 3 of 11

WeWe showed showed that that the the RTA-ribosomalRTA-ribosomal stalk stalk interaction interaction site site is located is located on the on opposite the opposite side of side the of theactive active site site [19]. [19 ].In In ricin ricin holotoxin, holotoxin, the the riboso ribosomeme binding binding site site of ofRTA RTA was was blocked blocked by byRTB RTB [19]. [19 ]. Consequently,Consequently, ricin ricin holotoxin holotoxin did did not notbind bind oror depurinatedepurinate the ribosome ribosome.. However, However, the the active active site site of of ricinricin was was not not blocked blocked by by RTB RTB and and ricin ricin holotoxin holotoxin couldcould depurinate the the naked naked RNA RNA [19]. [19 ].

FigureFigure 2. 2.Structures Structures of of the the StxA1, StxA1, Stx2A1 Stx2A1 andand RTARTA shownshown as ribbon and and as as surface. surface. The The residues residues at at thethe active active site, site, Tyr76/Tyr77, Tyr76/Tyr77, Tyr114, Tyr114, Glu167Glu167 andand Arg170 in in Stx; Stx; Ty Tyr80,r80, Tyr123, Tyr123, Glu177 Glu177 and and Arg180 Arg180 in in RTA, are shown in red. The arginines that are critical for P protein interaction, Arg172, Arg176 and RTA, are shown in red. The arginines that are critical for P protein interaction, Arg172, Arg176 and Arg179 in Stxs and Arg189, Arg191, Arg193, Arg196, Arg197, Arg234 and Arg235 in RTA, are shown Arg179 in Stxs and Arg189, Arg191, Arg193, Arg196, Arg197, Arg234 and Arg235 in RTA, are shown in in magenta. The residues that form a hydrophobic pocket and interact with the last six residues of the magenta. The residues that form a hydrophobic pocket and interact with the last six residues of the P-proteins, Gln173, Leu198, Ile224/Ile223, Phe226/Phe225, Leu233/Leu232, Ser235/Thr234 in Stx/Stx2 P-proteins, Gln173, Leu198, Ile224/Ile223, Phe226/Phe225, Leu233/Leu232, Ser235/Thr234 in Stx/Stx2 and Tyr183, Leu207, Phe240, Ile247, Pro250 and Ile251 in RTA, are shown in cyan [29]. Cys242/241 in and Tyr183, Leu207, Phe240, Ile247, Pro250 and Ile251 in RTA, are shown in cyan [29]. Cys242/241 Stx/Stx2 A1, which form a disulfide bond with Cys261/260 in Stx/Stx2 A2, and Cys259 in RTA, which in Stx/Stx2 A1, which form a disulfide bond with Cys261/260 in Stx/Stx2 A2, and Cys259 in RTA, forms a disulfide bond with Cys2 in RTB, are shown in yellow. Stx PDB ID: 1R4Q, Stx2 PDB ID: 2RA4 which forms a disulfide bond with Cys2 in RTB, are shown in yellow. Stx PDB ID: 1R4Q, Stx2 PDB ID: and ricin PDB ID: 2AA1. The structures were generated using PyMOL. 2RA4 and ricin PDB ID: 2AA1. The structures were generated using PyMOL. The X-ray crystal structures of a peptide mimic of the conserved C-terminus of ribosomal P-proteinsThe X-ray in complex crystal structureswith trichosanthin, of a peptide a type mimicI RIP, and of RTA the conservedhave been resolved C-terminus [29–31]. of ribosomalThe last P-proteinssix amino in acids complex (P6) of with the trichosanthin,P-protein peptide a type bind I to RIP, a hydrophobic and RTA have pocket been on resolved RTA, which [29– 31is ].blocked The last sixby amino RTB acidsin the (P6) holotoxin. of the P-proteinSeveral ribosome peptide binding bind to aresidues hydrophobic on the pocketA1 subunits on RTA, of whichStxs have is blocked been byidentified RTB in the [32]. holotoxin. As shown Several in Figure ribosome 2, the bindingribosome residues binding onsites the of A1 Stxs subunits are also of located Stxs have on the been identifiedopposite [32face]. of As the shown active in site. Figure Stx12 ,holotoxin the ribosome displays binding almost sites no ribosomal of Stxs are activity also located in its native on the oppositeform. Why face ofStx1 the and active Stx2 site. holotoxins Stx1 holotoxin are not displaysactive on almostribosomes no ribosomalremains unknown. activity in Here itsnative we form.compare Why Stx1ribosome and Stx2 binding holotoxins and depurination are not active activity on ribosomes of Stx1 remains and Stx2 unknown. holotoxins Here with we the compare A1 ribosome binding and depurination activity of Stx1 and Stx2 holotoxins with the A1 subunits of Stx1 Toxins 2017, 9, 133 4 of 12

Toxins 2017, 9, 133 4 of 11 and Stx2 using intact yeast ribosomes or a 10-mer RNA mimic of the SRL. We demonstrate that the subunits of Stx1 and Stx2 using intact yeast ribosomes or a 10-mer RNA mimic of the SRL. We A2 chaindemonstrate and the that B subunit the A2 chain block and the the active B subunit site of block Stx1 the and active Stx2, site but of not Stx1 their andribosome Stx2, but not binding their site. Unlikeribosome ricin holotoxin, binding site. the Unli ribosomeke ricin holotoxin, binding sitesthe ribosome of Stx1 bindin and Stx2g sites holotoxins of Stx1 and are Stx2 accessible holotoxins to the ribosome.are accessible These differencesto the ribosome. have These implications differences for ha theve developmentimplications for of the antidotes development against of antidotes STEC. against STEC. 2. Results 2. Results 2.1. Stx1 and Stx2 Holotoxins Can Bind the Ribosome 2.1. Stx1 and Stx2 Holotoxins Can Bind the Ribosome Computer modeling of Stx1A1 in complex with a peptide that mimics the last 11 amino acids of the ribosomalComputer P-stalk modeling proteins of Stx1A1 (P11) in suggested complex with that a peptide the ribosomal that mimics stalk the binding last 11 amino site and acids the of active site arethe locatedribosomal on P-stalk opposite proteins sides (P11) of the suggested protein [that30]. the Our ribosomal recent study stalk binding showed site that and point the active mutations site are located on opposite sides of the protein [30]. Our recent study showed that point mutations at Arg172 or Arg176 located on the opposite face of the active site (Figures1 and3) disrupted the at Arg172 or Arg176 located on the opposite face of the active site (Figures 1 and 3) disrupted the ribosome and ribosomal stalk interaction of Stx1A1 and Stx2A1, but did not affect their depurination ribosome and ribosomal stalk interaction of Stx1A1 and Stx2A1, but did not affect their depurination activityactivity on RNA, on RNA, indicating indicating that that their their active sites sites we werere intact intact and and were were located located on the on opposite the opposite face of face of thethe ribosome ribosome binding binding sitesite [[32].32]. Figure 3 shows the the structure structure of of Stx Stx holotoxins holotoxins in comparison in comparison with with ricin.ricin. In Stxs, In Stxs, five five B-subunits B-subunits form form a a doughnut doughnut shape and and A2 A2 chains chains insert insert into into the thedoughnut doughnut hole. hole. The positionThe position of theof the B subunitsB subunits is is different differentbetween between Stxs and and ricin. ricin. The The pentameric pentameric B subunits B subunits are are dockeddocked on the on activethe active site site of of StxA1 StxA1 and and Stx2A1 Stx2A1 andand block the the active active site, site, wh whileile the theribosome ribosome binding binding site issite exposed is exposed in Stx in Stx and and Stx2 Stx2 holotoxins. holotoxins. However,However, in in ricin ricin holotoxin holotoxin the the RTB RTB covers covers the ribosome the ribosome bindingbinding site ofsite RTA of RTA while while the the active active site site of of RTA RTA is is exposed.exposed.

Figure 3. Structures of Stx, Stx2 and ricin holotoxin shown in ribbon and surface. The A1 subunits are Figure 3. Structures of Stx, Stx2 and ricin holotoxin shown in ribbon and surface. The A1 subunits are shown in green, the A2 chains are shown in gray and the B subunits are shown in yellow. The shown in green, the A2 chains are shown in gray and the B subunits are shown in yellow. The labeling labeling of the residues is the same as in Figure 1. of the residues is the same as in Figure1.

To determine if Stx1 and Stx2 holotoxins can bind ribosomes, we immobilized Stx1, Stx2 and ricin on a CM5 chip of Biacore T200 and passed yeast ribosomes over the surface. As shown in Figure4, both Stx1 and Stx2 could interact with yeast ribosomes. The binding levels of the interaction were Toxins 2017, 9, 133 5 of 11

To determine if Stx1 and Stx2 holotoxins can bind ribosomes, we immobilized Stx1, Stx2 and Toxins 2017, 9, 133 5 of 12 ricin on a CM5 chip of Biacore T200 and passed yeast ribosomes over the surface. As shown in Figure 4, both Stx1 and Stx2 could interact with yeast ribosomes. The binding levels of the interactionribosome concentration-dependent. were ribosome concentration-dependent. In order to observe In order the interaction to observe signal the interaction of Stx1, we signal immobilized of Stx1, we37% immobilized more Stx1 (254137% more RU for Stx1 Stx1 (2541 and RU 1853 for RUStx1 for and Stx2) 1853 on RU the for chip. Stx2) When on the the chip. same When concentration the same concentrationof ribosomes of was ribosomes passed over was thepassed surface, over thethe bindingsurface, levelthe binding of Stx2 level was stillof Stx2 about was 90% still higherabout 90% than higherStx1 at than 2.5 nMStx1 andat 2.5 66% nM higher and 66% than higher Stx1 atthan 40 Stx1 nM ribosomeat 40 nM ribosome concentration. concentration. These data These indicate data indicatethat Stx1 that and Stx1 Stx2 and holotoxins Stx2 holotoxins can bind can ribosomes,bind ribosomes, and Stx2and Stx2 holotoxin holotoxin has has higher higher affinity affinity for for the theribosome ribosome than than Stx1 Stx1 holotoxin. holotoxin. Similar Similar results results were were observed observed when when Stx1A1 Stx1A1 and and Stx2A1 Stx2A1 interacted interacted with withthe ribosomethe ribosome [14]. There[14]. There was no was detectable no detectab interactionle interaction between between the ribosome the ribosome and ricin and holotoxin, ricin holotoxin,as previously as previously observed [observed19]. [19].

FigureFigure 4. 4. InteractionInteraction of of yeast yeast ribosomes ribosomes with with Stx1 Stx1 (magen (magenta),ta), Stx2 Stx2 (blue) (blue) and and ricin ricin (green) (green) holotoxins. holotoxins. ToxinsToxins were were immobilized immobilized on on a a CM5 CM5 chip. chip. Ricin Ricin was was immobilized immobilized on on Fc2 Fc2 at at 1873 1873 RU, RU, Stx1 Stx1 was was immobilizedimmobilized on on Fc3 Fc3 at at 2541 2541 RU, RU, and and Stx2 Stx2 was was immo immobilizedbilized on Fc4 on Fc4at 1853 at 1853 RU. RU.Fc1 was Fc1 activated was activated and blockedand blocked as a control. as a control. Ribosomes Ribosomes were passed were passed over the over surfaces the surfaces at 2.5, at5, 2.5,10, 5,20 10,and 20 40 and nM. 40 The nM.

runningThe running buffer buffer contained contained 10 mM 10 Hepes, mM Hepes, pH 7.4, pH 150 7.4, mM 150 NaCl, mM NaCl, 5 mM 5 MgCl mM MgCl2, 50 µM2, 50 EDTAµM EDTA and 0.003%and 0.003% Surfactant Surfactant P20. The P20. surface The surface was regenerated was regenerated with one-minute-injection with one-minute-injection of 3 M ofNaCl 3 M for NaCl three for times.three times.The interaction The interaction was measured was measured at 25 °C. at 25The◦C. signals The signals were normalized were normalized to the toligand the ligand level of level 1000 of RU.1000 The RU. experiment The experiment was replicated was replicated three threetimes. times.

2.2.2.2. Stx Stx Holotoxins Holotoxins are Are Inactive Inactive Towards Towards the the Ribosome, Ribosome, Bu butt Are Are Active Active After After Separation Separation of of the the A1 A1 from from the the A2 A2 ChainChain and and the the B B Subunit Subunit WeWe measured thethe depurinationdepurination activities activities of Stxof St holotoxinsx holotoxins before before and afterand treatmentafter treatment with trypsin with trypsinand TCEP and (TrisTCEP (2-carboxyethyl) (Tris (2-carboxyethyl) phosphine phosphine hydrochloride) hydrochloride) to separate to separate the A1 the from A1 thefrom A2 the chain A2 chainand theand B subunit.the B subunit. Stx1 and Stx1 Stx2 wereand Stx2 treated were with treated TPCK (withN-tosyl- TPCKL-phenylalanine (N-tosyl-L-phenylalanine chloromethyl chloromethylketone)-treated ketone)-treated trypsin, which trypsi cann, cleave which at can the cleave furin at sites, the withfurin orsites, without with or TCEP, without to reduce TCEP, theto reducedisulfide the bond. disulfide Ricin bond. was treatedRicin was with treated TCEP only.with TCEP After treatment,only. After the treatment, toxins were the separatedtoxins were on separatednon-reducing on non-reducing SDS-PAGE. As SDS-PAGE. shown in FigureAs shown5, the in A Figure (A1 + 5, A2) the subunits A (A1 + migrated A2) subunits at 31 migrated and 32 kDa at 31for and Stx1 32 and kDa Stx2, for Stx1 respectively, and Stx2, and respectively, the B subunits and migratedthe B subunits at about migrated 8 kDa withoutat about TCEP 8 kDa treatment. without TCEPAfter treatment. TCEP treatment, After TCEP the A1 treatment, and A2 subunits the A1 wereand A2 separated subunits and were the separa A1 subunitsted and migrated the A1 atsubunits 28 kDa. migratedThe A2 subunits at 28 kDa. of 5The kDa A2 were subunits not observed of 5 kDa on were SDS-PAGE, not observed possibly on because SDS-PAGE, their amountspossibly werebecause low. theirRicin amounts holotoxin were migrated low. toRicin about holotoxin 60 kDa. Themigrated A and to B subunitsabout 60 were kDa. separated The A and after B TCEPsubunits treatment were separatedwith molecular after TCEP weights treatment around with 30 and molecular 32 kDa, weights respectively. around 30 and 32 kDa, respectively. WeWe previously previously measured thethe depurinationdepurination kinetics kinetics of of recombinant recombinant Stx1A1 Stx1A1 and and Stx2A1 Stx2A1 from fromE. coliE. colion on ribosomes ribosomes and and on on an an SRL SRL mimic mimic RNA RNA [ 14[14].]. Both Both StxA1StxA1 andand RTA cancan depurinate ribosomes ribosomes at at physiologicalphysiological pH pH but but they they depurinate depurinate RNA RNA only only at at an an acidic acidic pH pH [14]. [14]. Using Using previously previously established established conditionsconditions to to measure measure depurination depurination kinetics kinetics [14], [14], we we compared compared the the depurination depurination activities activities of of Stx1 Stx1 andand Stx2 Stx2 holotoxins holotoxins with with their their activated activated forms forms wh whereere the the catalytic catalytic A1 A1 subunit subunit was was separated separated from from thethe A2 A2 chain chain and and the the B B subunit. subunit. Stx1 Stx1 and and Stx2 Stx2 ho holotoxinslotoxins were were not not active active towards towards the the ribosome. ribosome. However, after trypsin and TCEP treatment to release the A1 subunits, Stx1A1 and Stx2A1 were able Toxins 2017, 9, 133 6 of 11 Toxins 2017, 9, 133 6 of 12

However, after trypsin and TCEP treatment to release the A1 subunits, Stx1A1 and Stx2A1 were able to depurinate the the yeast yeast ribosome ribosome at at 374-fold 374-fold and and 92 92-fold-fold higher higher levels levels than than Stx1 Stx1 and and Stx2 Stx2 holotoxins, holotoxins, respectively, at 0.5 µµMM ribosomeribosome concentrationconcentration (Figure(Figure6 6a).a).A A previous previous study study examined examined the the kkcatcat and Km ofof Stx1 Stx1 holotoxin holotoxin on on Artemia salina salina ribosomesribosomes before before and and after after treatment with trypsin, urea and DTT [33]. [33]. Our results are consistent with this study in the requirement for activation, although this study used a different different assay, different source of ribosomes,ribosomes, different method of activation, and much higher toxin concentrations [33]. [33].

Figure 5. 5. MolecularMolecular weight weight of of the the subunits subunits of Stx1, of Stx1, Stx2 Stx2 and and ricin ricin (a) and (a) andSDS-PAGE SDS-PAGE analysis analysis (b); Stx1 (b); andStx1 Stx2 and (7 Stx2 µg) (7 wereµg) weretreated treated with withtrypsin trypsin with withor without or without TCEP, TCEP, and ricin and (7 ricin µg) (7 wasµg) treated was treated with TCEPwith TCEP at 25 at°C 25for◦ C2 h for and 2 h separated and separated on a 12% on a SDS-PAGE. 12% SDS-PAGE. The gel The was gel stained was stained with Coomassie with Coomassie blue. Theblue. protein The protein marker marker is Invitrogen is Invitrogen Novex® Novex sharp®sharp pre-stained pre-stained protein protein marker. marker. * *Molecular Molecular weight weight is affected by glycosylation in both A and B subunits of ricin.

We further examined the activity of Stx1 and Stx2 holotoxins with their activated forms toward a 10-mer10-mer RNARNA stem stem loop loop mimic mimic of theof the SRL. SRL. Activated Activated Stx holotoxinsStx holotoxins could could depurinate depurinate the SRL the mimic SRL mimicRNA, whileRNA, the while holotoxins the holotoxins were inactive were inactive (Figure6 b).(Figure These 6b). results These are results consistent are consistent with the structural with the structuraldata, indicating data, indicating that the A2 that chain the A2 and chain the B and subunit the B blocksubunit the block active the sites active of Stx1sites andof Stx1 Stx2 and [24 –Stx228]. [24–28].We have We previously have previously shown that shown ricin holotoxinthat ricin holo couldtoxin not depurinatecould not depurinate the ribosome the but ribosome can depurinate but can depurinatethe naked RNA the naked using aRNA qRT-PCR using assaya qRT-PCR to measure assay depurination to measure depurination [19]. Here, as [19]. a control, Here, we as measureda control, we measured the activities of ricin holotoxin toward the yeast ribosome and the SRL using a highly the activities of ricin holotoxin toward the yeast ribosome and the SRL using a highly sensitive sensitive luminescent assay [34]. Consistent with the previously published data, ricin holotoxin luminescent assay [34]. Consistent with the previously published data, ricin holotoxin could not could not depurinate yeast ribosomes unless it was reduced to separate the catalytic A subunit from depurinate yeast ribosomes unless it was reduced to separate the catalytic A subunit from the B the B subunit (Figure 6c). In contrast, both unreduced and reduced ricin holotoxin could depurinate subunit (Figure6c). In contrast, both unreduced and reduced ricin holotoxin could depurinate the the SRL mimic RNA with similar activity (Figure 6d). Stx1A1, Stx2A1 (pH 4.5) and ricin (pH 4.0) SRL mimic RNA with similar activity (Figure6d). Stx1A1, Stx2A1 (pH 4.5) and ricin (pH 4.0) have have different optimum catalytic pH on nucleic acid substrates [14,34,35]. We examined different optimum catalytic pH on nucleic acid substrates [14,34,35]. We examined depurination of depurination of the SRL mimic RNA at the same pH (pH 4.5) in order to compare the activity of the SRL mimic RNA at the same pH (pH 4.5) in order to compare the activity of activated Stx1 and activated Stx1 and Stx2 holotoxins with ricin. At pH 4.5, A1 subunits of Stxs are about 100-fold more Stx2 holotoxins with ricin. At pH 4.5, A1 subunits of Stxs are about 100-fold more active than ricin active than ricin holotoxin or RTA (Figure 6b,d). To determine if Stx1 and Stx2 holotoxins have any holotoxin or RTA (Figure6b,d). To determine if Stx1 and Stx2 holotoxins have any activity on the SRL activity on the SRL mimic RNA at a very high concentration, we compared their activity with ricin

Toxins 2017, 9, 133 7 of 12

Toxins 2017, 9, 133 7 of 11 mimic RNA at a very high concentration, we compared their activity with ricin holotoxin at 1 µM toxinholotoxin concentration at 1 µM usingtoxin theconcentration same conditions using the as sa inme Figure conditions6b,d. As as shownin Figures in Figure6b,d. As6e, shown we could in notFigure detect 6e, any we depurination could not detect activity any of depurination Stx1 and Stx2 acti holotoxins,vity of Stx1 even and atStx2 this holotoxins, higher dose. even In contrast,at this higher dose. In contrast, ricin holotoxin could depurinate the SRL at a similar level as that in Figure ricin holotoxin could depurinate the SRL at a similar level as that in Figure6d. Ricin holotoxin could 6d. Ricin holotoxin could depurinate the SRL mimic RNA at 252-, 326-, and 364-fold higher levels depurinate the SRL mimic RNA at 252-, 326-, and 364-fold higher levels than Stx1 and at 51-, 76-, than Stx1 and at 51-, 76-, and 114-fold higher levels than Stx2 at 1, 2 and 4 min, respectively (Figure 6e). and 114-fold higher levels than Stx2 at 1, 2 and 4 min, respectively (Figure6e).

FigureFigure 6. Depurination6. Depurination of of yeast yeast ribosomes ribosomes ((aa,,cc)) andand SRL mimic RNA RNA (b (b,d,d,e,e) )by by Stxs Stxs (a (,ab,,be,)e and) and ricin ricin (c,d(c,e,d).,e ().a ,(ca),c Stx) Stx holotoxins holotoxins (I (I for for inactive) inactive) werewere activatedactivated by by trypsin trypsin and and TCEP TCEP (Tris (Tris (2-carboxyethyl) (2-carboxyethyl) phosphinephosphine hydrochloride) hydrochloride) treatment treatment toto releaserelease thethe A1 subunit from from the the A2 A2 chain chain and and the the B Bsubunit subunit (A(A for for active) active) and and ricin ricin holotoxin holotoxin (I for inactive) inactive) was was activated activated by by TCEP TCEP treatment treatment to release to release the A the A subunitsubunit fromfrom the B subunit (A for for active). active). Ribosomes Ribosomes were were used used at at 0.5 0.5 µM,µM, Stxs Stxs were were used used at at0.2 0.2 nM nM andand ricin ricin was was used used at at 1 1 nM. nM. The The reaction reaction was was incubatedincubated at 25 °C.◦C. The The adenine adenine released released was was measured measured byby the the enzyme enzyme coupled coupled fluorescence fluorescence assayassay forfor 1515 min.min. The linear linear part part of of the the reactions reactions was was used used to to calculatecalculate the the activities. activities. The The depurination depurination raterate isis shownshown as pmol of of adenine adenine released released per per pmol pmol of oftoxin toxin in onein one minute. minute. (b ,(db),d The) The SRL SRL mimic mimic RNA RNA was was used used at 2atµ 2M, µM, Stxs Stxs were were used used at 5at nM 5 nM and and ricin ricin was was used at 40used nM. at The40 nM. reactions The reactions were incubated were incubated at 20 ◦atC 20 at °C pH at 4.5. pH The4.5. The depurination depurination was was stopped stopped at 1,at 21, and2 4 min.and The4 min. adenine The adenine released released was measured was measured by the enzymeby the enzyme coupled coupled fluorescence fluorescence assay. assay. The amount The of adenineamount of released adenine isreleased shown is as show pmoln as of pmol adenine of adenine released released per pmol per pmol of toxins of toxins at the at samplingthe sampling time point.time ( epoint.) The ( SRLe) The mimic SRL mimic RNA concentrationRNA concentration was 2wasµM 2 andµM and the holotoxinthe holotoxin concentrations concentrations were were 1 µ1M. µM. The reactions were incubated at 20 °C at pH 4.5. The depurination was stopped at 1, 2 and 4 min. The reactions were incubated at 20 ◦C at pH 4.5. The depurination was stopped at 1, 2 and 4 min. The adenine released was measured by the enzyme coupled fluorescence assay. The amount of The adenine released was measured by the enzyme coupled fluorescence assay. The amount of adenine adenine released is shown as pmol of adenine released per pmol of toxins. released is shown as pmol of adenine released per pmol of toxins. In summary, the A2 chain and the B subunit block the active site in the Stx holotoxins, but leave theIn ribosome summary, binding the A2 sites chain open. and As the a result, B subunit Stx holotoxins block the activecould not site depurinate in the Stx holotoxins,either the ribosome but leave theor ribosome the 10-mer binding RNA stem sites loop open. mimic As a result,of the SRL, Stx holotoxinseven though could they not could depurinate interact with either the the ribosome. ribosome orIn the contrast, 10-mer in RNA ricin stem holotoxin loop mimicRTB covers of the the SRL, ribosome even thoughbinding theyside couldof RTA interact and leaves with the the active ribosome. site Inof contrast, RTA exposed. in ricin Ricin holotoxin holotoxin RTB could covers depurina the ribosomete the SRL binding mimic side RNA of at RTA low and pH, leaves but could the activenot siteinteract of RTA with exposed. the ribosome Ricin or holotoxin depurinate could the depurinateribosome. These the SRLresults mimic explain RNA why at Stx1, low Stx2 pH, and but ricin could notholotoxins interact with are theinactive ribosome towards or depurinateribosomes and the ribosome.show that the These molecular results explainbasis for why the Stx1,inactivity Stx2 of and ricinStx1/Stx2 holotoxins holotoxins are inactive differs towards from that ribosomes of the ricin and holotoxin. show that the molecular basis for the inactivity of Stx1/Stx2 holotoxins differs from that of the ricin holotoxin.

Toxins 2017, 9, 133 8 of 12

3. Discussion We previously showed that ricin holotoxin is not active on ribosomes because the ribosome binding site of RTA is blocked by the B subunit [19]. However, the molecular basis for the inactivity of Stx1 and Stx2 holotoxins towards ribosomes remained unknown. Here, we compared the ribosome binding and depurination activity of Stx1 and Stx2 holotoxins with the A1 subunits of Stx1 and Stx2 and demonstrated that the active sites of Stx1 and Stx2 are blocked by the A2 chain and the B subunit, while the ribosome binding sites are exposed. Although Stx1 and Stx2 holotoxins are enzymatically inactive, they are able to interact with the ribosome. Previous results indicated that the A1 subunit of Stx2 interacts with the ribosome with higher affinity than the A1 subunit of Stx1 [14]. Conserved arginines at the P-protein stalk binding site did not contribute to the differences in the affinity of the A1 subunits for the ribosome [32]. We show here that Stx2 holotoxin also interacts with the ribosome with higher affinity than Stx1 holotoxin (Figure4). Since the difference in ribosome binding previously observed for the A1 subunits [14] is also observed with the Stx1 and Stx2 holotoxins, the regions of the A1 subunits, which are covered by the A2 chain and the B subunit, are not likely responsible for the differences in the ribosome binding. These results suggest that residues at the active site of Stx2A1 and Stx1A1 do not contribute to the higher affinity of Stx2 for the ribosome. The surface charge and the distribution of the surface charge are different between Stx1A1 and Stx2A1 [32]. Our results suggest that surface charge differences in regions other that the active site or the stalk binding site contributes to the higher affinity of Stx2 for the ribosome. Although Stx2A1 is more active in depurination of the ribosome and the RNA than Stx1A1 [32], we did not observe this difference when the holotoxins were activated by trypsin and TCEP (Figure6). As previously observed [14], the holotoxin is not a good choice for comparison of the depurination activities of Stx1 and Stx2. Stx1 and Stx2 holotoxins have to be activated by trypsin and TCEP treatment to separate the A2 chains and the B subunits from the A1 subunits. It is impossible to digest Stx1 and Stx2 equally with trypsin. Since trypsin cleaves the peptide chains at the carboxyl side of lysine and arginine, trypsin can cleave the toxins at other sites besides the furin cleavage site between A1 and A2, and inactivate them. Furthermore, during toxin purification, some of the toxin molecules are already cleaved between A1 and A2 subunits and the holotoxins can be activated to up to 80% of their maximal activity after treatment with only TCEP. Therefore, the activity measured after trypsin plus TCEP (or DTT) treatment does not necessarily reflect the total activity of the A1 subunits in the Stx holotoxins, but the activity of the A1 subunits remaining after the trypsin digestion. The X-ray crystal structure comparison of Stx2 holotoxin with Shigella Stx indicated some differences. While the active site of the A subunit was blocked by the A2 chain in the Shigella Stx, the active site of Stx2 holotoxin was accessible to a small substrate, adenosine [26]. However, it was predicted that while Stx2 holotoxin was active against adenosine, it was unlikely to bind to the SRL because the A2 chain and the B subunit would interfere with binding to the 28S rRNA [26]. Our results (Figure6c,e) provide direct experimental evidence that the Stx1 and Stx2 holotoxins cannot depurinate the SRL or the ribosome because their active sites are blocked by the A2 chain and the B subunit. In contrast, ricin holotoxin could depurinate the SRL mimic RNA. However, depurination of the RNA occurs only at the low pH, but not at the physiologic pH. Ricin holotoxin could not interact with the ribosome or depurinate the ribosome due to the blockage of the ribosome binding site by RTB. These results demonstrate the molecular basis for the inactivity of ricin, Stx1 and Stx2 holotoxins towards ribosomes and suggest that deactivation of the toxins could be achieved by blocking the ribosome binding site as an alternative to the active site, since targeting the active site has not yielded an inhibitor with sufficiently high activity to date [36]. Although Stx1A1, Stx2A1 and RTA depurinate small RNA substrates at a low pH [14,34,35], at the physiologic pH, Stx1A1, Stx2A1 and RTA need to interact with the ribosomal P-proteins to catalyze depurination of the SRL. Stx1A1, Stx2A1 and RTA can depurinate the ribosome with high affinity. However, since the ribosome and the A subunits interact with each other electrostatically [32,37], the local concentration of the toxins around the ribosome can be high. In order to block ribosome Toxins 2017, 9, 133 9 of 12 interaction inhibitors that can bind either to the P-protein stalk interaction site or to the active site would require very high affinity. The molecular details of how Stx1A1 and Stx2A1 interact with ribosomes and the structures of the C-termini of P-proteins in complex with the toxins could lead to the identification of inhibitors that can disrupt ribosome interactions and thereby offer new opportunities for antidote development.

4. Materials and Methods

4.1. Materials Stx1 and Stx2 holotoxins were from Phoenix Laboratory at the Tufts Medical Center, Boston, MA, USA. They are purified by receptor analog affinity chromatography [38]. Ricin holotoxin was from Vector Laboratory, Burlingame, CA, USA. It was purified from castor bean (catalog number L-1090). Yeast monomeric ribosomes were purified as described previously [19]. The 10-mer SRL mimic oligo (50-rCrGrCrGrArGrArGrCrG-30) was purchased from Integrated DNA Technologies, Coralville, Iowa, IA, USA.

4.2. Toxin Structures Stx (1R4Q), Stx2 (2GA4) and ricin (2AAI) holotoxins were downloaded from PDB. The A1 subunits of Stx and Stx2 or the A subunit of ricin were obtained from the holotoxins. The structures were visualized using PyMOL (The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC, Portland, OR, USA).

4.3. SDS-PAGE Analysis Stx1 or Stx2 holotoxin (7 µg) was treated with 50 ng TPCK-treated trypsin or trypsin plus 50 mM of TCEP at 25 ◦C for 2 h in a total volume of 10 µL in trypsin reaction buffer (50 mM Tris-HCl pH 8.0, 20 mM CaCl2). TPCK-treated trypsin was from Biolabs (Ipswich, MA, USA). Trypsin was reconstituted following the manufacturer’s instructions and used within a week. Ricin holotoxin (7 µg) was treated with 50 mM of TCEP at 25 ◦C for 2 h in 10 µL in its original buffer (10 mM of phosphate pH 7.8, 150 mM NaCl, and 0.08% sodium azide). 5 µL of 5X SDS-PAGE loading buffer was added to the treated and untreated Stx1, Stx2 or ricin. The samples were heated at 95 ◦C for 5 min and separated on 12% SDS-PAGE. The gel was stained with Coomassie blue.

4.4. Holotoxin-Ribosome Interaction Holotoxins were immobilized on a CM5 chip of a Biacore T200. Ricin was immobilized on Fc2 at 1873 RU, Stx1 on Fc3 at 2541 RU and Stx2 on Fc4 at 1853 RU. Fc1 was activated and blocked as a control. Yeast ribosomes at 2.5, 5, 10, 20 and 40 nM were passed over the surface using single kinetic injection method. The running buffer contained 10 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 50 µM EDTA and 0.003% surfactant P20. The surface was regenerated by three times 1 min injection of 3 M NaCl. The interaction was measured at 25 ◦C.

4.5. Toxin Activation The activation conditions were the same as used in the SDS-PAGE analysis described in Section 4.3. Stx1 and Stx2 were activated by treatment with trypsin and TCEP. At the end of the reaction 1 mM of PMSF was added to stop the reaction. The activated Stx1 or Stx2 was stored at 4 ◦C and used within two days. The activated ricin was also stored at 4 ◦C and used within two days.

4.6. Ribosome Depurination Ribosome depurination was measured using continuous enzyme coupled luminescent assay [34]. Ribosomes were used at 0.5 µM, Stx1 and Stx2 holotoxins were used at 0.2 nM and ricin was used at 1 nM. The reaction was started by adding the toxin, and luminescence intensity was Toxins 2017, 9, 133 10 of 12 recorded continuously for 15 min using a BioTek Synergy 4 Microplate reader (Winooski, VT, USA). Adenine standards were assayed at same time. The rates were determined from the linear region of the luminescence intensity. The adenine generated from ribosomes alone was subtracted.

4.7. SRL Depurination Stem-loop depurination was performed using a synthetic 10-mer RNA mimic of the SRL. The discontinuous luminescence assay described by Sturm and Schramm was used [34]. In the reaction mixture, the SRL was used at 2 µM, Stxs at 5 nM or 1 µM and ricin at 40 nM or 1 µM. The reaction was incubated at 20 ◦C in 50 µL of 10 mM potassium citrate-KOH and 1 mM EDTA at pH 4.5 for both Stxs and ricin. The reaction was started by adding the toxins. Samples were taken at 1, 2 and 4 min and mixed immediately with 2X coupling buffer to stop the reaction. Adenine standards were assayed at the same time. Luminescence intensity was measured using a BioTek Synergy 4 Microplate reader.

Acknowledgments: The authors thank Jennifer Kahn for reading the manuscript. The work was supported by National Institutes of Health grants, AI092011 and AI072425 to Nilgun E. Tumer. Author Contributions: Both authors designed the experiments and wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

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