
==== Front
0370535
518
Anal Biochem
Anal Biochem
Analytical biochemistry
0003-2697
1096-0309

38825159
10.1016/j.ab.2024.115580
nihpa2019054
Article
A fluorescence anisotropy-based competition assay to identify inhibitors against ricin and Shiga toxin ribosome interactions
Dutta Arkajyoti a
Szekely Zoltan b
Guven Hakan b
Li Xiao-Ping a**
McLaughlin John E. a
Tumer Nilgun E. b*
a Department of Plant Biology, Rutgers, The State University of New Jersey, 59 Dudley Road, New Brunswick, NJ, 08901, USA
b Molecular Design and Synthesis Core, Rutgers University Biomolecular Innovations Cores, Office for Research, Rutgers University, 610 Taylor Rd, Piscataway, NJ, 08854, USA
* Corresponding author. Department of Plant Biology Rutgers, The State University of New Jersey, 59 Dudley Road, New Brunswick, NJ, 08901, USA. tumer@sebs.rutgers.edu (N.E. Tumer)
** Corresponding author. Department of Plant Biology Rutgers, The State University of New Jersey, 59 Dudley Road, New Brunswick, NJ, 08901, USA. xpli@sebs.rutgers.edu (X.-P. Li)
26 8 2024
9 2024
31 5 2024
23 9 2024
692 115580115580
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Ricin is one of the most toxic substances known and a type B biothreat agent. Shiga toxins (Stxs) produced by E. coli (STEC) and Shigella dysenteriae are foodborne pathogens. There is no effective therapy against ricin or STEC and there is an urgent need for inhibitors. Ricin toxin A subunit (RTA) and A1 subunit of Stx2a (Stx2A1) bind to the C-terminal domain (CTD) of the ribosomal P-stalk proteins to depurinate the sarcin/ricin loop. Modulation of toxin-ribosome interactions has not been explored as a strategy for inhibition. Therefore, development of assays that detect inhibitors targeting toxin-ribosome interactions remains a critical need. Here we describe a fluorescence anisotropy (FA)-based competitive binding assay using a BODIPY-TMR labeled 11-mer peptide (P11) derived from the P-stalk CTD to measure the binding affinity of peptides ranging from 3 to 11 amino acids for the P-stalk pocket of RTA and Stx2A1. Comparison of the affinity with the surface plasmon resonance (SPR) assay indicated that although the rank order was the same by both methods, the FA assay could differentiate better between peptides that show nonspecific interactions by SPR. The FA assay detects only interactions that compete with the labeled P11 and can validate inhibitor specificity and mechanism of action.

Fluorescence anisotropy
Surface plasmon resonance
Peptide inhibitors
Ricin
Shiga toxin
Ribosome binding
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pmc1. Introduction

Ricin is a type-II ribosome-inactivating protein (RIP) that is lethal, particularly when inhaled as an aerosol [1]. The toxin is produced from seeds of the castor bean plant (Ricinus communis), which is used worldwide to manufacture castor oil. Only one molecule of ricin is enough to kill a human cell [2]. Shiga toxins are the virulence factors of Shigella dysenteriae and Shiga toxin producing E. coli (STEC), which is the leading cause of renal failure in children worldwide [3–5]. Ricin and Shiga toxins (Stxs) are type II ribosome inactivating proteins (RIPs) with identical mechanism of action [6,7]. Currently, there are no therapeutic strategies available against either toxin. Ricin is an AB toxin, consisting of a B subunit (RTB) coupled by a disulfide linkage to an enzymatically active A subunit (RTA). RTB is a lectin, which binds to glycoproteins on the cell surface having either terminal β-1,4-linked galactose or N-ace-tylgalactosamine residues and causes endocytic uptake of the holotoxin. Only a small portion of the internalized ricin holotoxin reaches the trans-Golgi network and the endoplasmic reticulum (ER) through retrograde trafficking [8]. In the ER lumen, protein disulfide isomerase catalyzes the cleavage of the single disulfide bond between RTA and RTB and releases RTA into the cytosol [9]. Stx2a is an AB5 toxin that binds to the Gb3 receptor and is internalized by endocytosis [10]. Stx2A is proteolytically cleaved into a catalytic A1 subunit (Stx2A1), which is released from the A2-B5 complex in the ER and undergoes retro-translocation into the cytosol [10]. RTA and the A1 subunit of Stx2a (Stx2A1) depurinate a highly conserved adenine from the α-sarcin/ricin loop (SRL) of the 28S rRNA, resulting in the inhibition of protein synthesis [6,7], induction of the ribotoxic stress response and apoptosis in mammalian cells [11].

Our work has shown that RTA and Stx2A1 bind to the ribosomal P-stalk proteins to depurinate the SRL at a remote location on the large subunit of the ribosome [12,13]. RTA and Stx2A1 do not interact with yeast or human ribosomes lacking the P1–P2 proteins [13–15]. The P-stalk binding site and the active site are located on different faces of RTA and Stx2A1 [16]. The eukaryotic P-stalk is a pentameric protein complex formed by uL10 protein (P0) and two P1–P2 heterodimers attached to uL10 [17–19]. Each P protein has an unstructured C-termini (CTD) with identical 11 amino acids (P11: SDDDMGFGLFD) [18]. RTA and Stx2A1 bind to the CTD of P-stalk proteins with a hydrophobic cleft remote from the catalytic site [20–23]. The P-stalk proteins recruit the toxins to the SRL facilitating efficient depurination of the rRNA substrate. The function of the CTD of P-proteins is to bind to the translational GTPases, including the elongation factor 2 (eEF2) to activate their GTPase activity [17–19]. The entire structure of the P-stalk complex has not been solved because P-stalk CTD is flexible and can adopt diverse conformations.

The crystal structures of RTA bound with a peptide (P11) corresponding to the last 11 amino acids of the P-proteins (SDDDMGFGLFD) (PDB ID: 5GU4 and 5DD2) revealed that the last 6 amino acids (GFGLFD) are inserted into a hydrophobic pocket on RTA [20,21]. Mutation analysis indicated that Tyr183, Leu232 and Phe240 in the hydrophobic pocket along with Arg234 and Arg235 just outside the P-stalk pocket are critical for the activity and cytotoxicity of RTA [24, 25]. We solved the X-ray structure of Stx2a holotoxin with P11 (PDB ID:6×6H) [22] and the cryo-EM structure of Stx2a holotoxin with the P-stalk pentamer from yeast (PDB ID: 7U6V, EMD-26381) [26]. As observed with RTA only the last six amino acids of P11 interact with Stx2a in a shallow groove lined by Arg172, Arg176 and Arg179 on the surface of the A1 subunit. We recently solved the X-ray crystal structure of the A1 subunit of Stx2a (Stx2A1) with a peptide mimicking the last 8 amino acids of the P-stalk CTD (P8) (PDB ID: 8SZ2) [27] and showed that Stx2A1 interacts with P8 at a similar position as the Stx2a holotoxin and has distinct requirements for binding to the P-stalk CTD compared to RTA [22]. Although P-stalk binding sites of Stx2A1 and RTA are in different positions and show structural differences, inhibition of P-stalk binding can stop depurination by both toxins validating the P-stalk binding site as a druggable new target for the inhibition of RIPs [27,28].

Using fragment-based ligand discovery (FBLD) with surface plasmon resonance (SPR) we previously identified small molecules that bind at the P-stalk pocket of RTA and allosterically inhibit the catalytic activity of ricin [29–31]. Fluorescence polarization (FP) is a method widely used to evaluate the affinity of inhibitors of biomolecular interactions [32–34]. FP measures the change in polarization of emitted light upon excitation of a fluorescent probe with plane-polarized light after protein binding. The FP properties can be utilized to develop competition assays where a labeled probe bound to a protein is titrated in a dose-dependent manner with inhibitors designed to disrupt the interaction [35–37]. The FP values can be converted to fluorescence anisotropy (FA) values to estimate the equilibrium dissociation constant (KD) of the inhibitors [35]. The FA assay has been used to characterize small molecule inhibitors targeting the interaction of ricin with the P-stalk [31]. We describe here a FA-based competition assay that can measure the binding affinity of peptides derived from the P-stalk CTD for RTA and Stx2A1 to define the relative contribution of the conserved amino acids at the C-termini of P-proteins to the interaction with each RIP. We compare the affinities of the peptides obtained using the FA assay with the affinities obtained using surface plasmon resonance (SPR) assay with Biacore T200 [27,28] and demonstrate that the FA assay is more specific for ranking peptides that bind to the hydrophobic pocket at the P-stalk binding site of RTA and Stx2A1.

2. Materials and methods

2.1. Purification of ricin toxin A subunit (RTA)

Plasmid p64029 harboring the RTA gene was transformed to E. coli strain BL21 (DE3-pLysS) for overexpression. Single colonies obtained on 100 μg/mL ampicillin plates were transferred to 10 mL of 2xYT media and grown for overnight at 37 °C, followed by transfer into 1 L of 2xYT media and grown at 30 °C until OD595 reached 0.4 to 0.6. Expression was induced by 1 mM IPTG during further growth at 16 °C for 16 h. Cells were harvested at 5000 rpm for 30 min at 4 °C. The supernatant was bleached and discarded. The cell pellets were dissolved in lysis buffer containing 20 mM Tris-Cl pH 8.5, 0.1 mM EDTA, 0.1 mM DTT and 1 tablet of protease inhibitor cocktail from Roche by constant shaking at 4 °C for 1 h and then sonicated using the thick probe with 30 s pulses followed by incubation for 30 s on ice at 30 amplitude and the cycle was repeated for 5 to 7 times. The cell lysate was centrifuged at 16,000 rpm for 1 h at 4 °C and the supernatant was filtered using 0.22 μm Nalgene Rapid Flow filtration unit and loaded onto Hi Prep Q FF 16/10 column pre-equilibrated with 5 column volumes (CV) of Buffer A (20 mM Tris-Cl pH 8.5, 1 mM DTT) using the AKTA purifier 100/10 system (GE Healthcare Life Sciences). The column was washed with 5 CV of buffer A followed by elution using a high-salt linear gradient against buffer B (20 mM Tris-Cl pH 8.5, 1 mM DTT and 2 M NaCl). Eluted fractions were mixed with Laemmle Buffer, incubated at 95 °C for 2–5 min and separated on Express Plus PAGE (Genscript) along with pre-stained protein markers at a constant volt in 1X Tris-MOPS-SDS running buffer. The gel was stained with instant blue Coomassie protein stain for 15–20 min, washed with distilled water and scanned in Odyssey CLx image scanner and fractions containing RTA (32 kDa) were pooled together for overnight dialysis against buffer C (20 mM Tris-Cl pH 8.0, 0.1 mM EDTA, 1 mM DTT) at 4 °C. Hi-Trap Heparin column (CV = 5 mL, Pressure = 0.5 MPa) was washed with 5 CV of filtered and degassed double-distilled water followed by equilibration with buffer C (freshly prepared with 1 mM DTT). Samples were loaded at a flow rate of 1 mL/min, washed with 8 CV of buffer C and eluted with buffer D (20 mM Tris-Cl pH 8.0, 0.1 mM EDTA, 1 mM DTT, 1 M NaCl) using a linear gradient for 120 min. RTA eluted at ~300 mM NaCl. The fractions containing RTA were pooled together and separated on Hi Load 16/600 Superdex75 column pre-equilibrated in buffer C containing 0.1 M NaCl. Eluted fractions were separated on Express Plus PAGE and purified RTA was concentrated using Amicon 10 kDa filters and stored at −80 °C. The activity of the purified RTA was confirmed by an in vitro depurination assay [38, 39].

2.2. Purification of Shiga toxin 2 A1 subunit (Stx2A1)

Plasmid DNA isolated from glycerol stock of pTXB1-Stx2A1 plasmid (NT 1577 #4) was transformed into Rosetta 2 DE3 pLysS competent cells from Invitrogen and grown overnight at 37 °C on LB agar plates supplemented with ampicillin (100 μg/mL) and chloramphenicol (34 μg/mL). Single colonies obtained were transferred into 10 mL 2xYT media, followed by transfer into 1 L of 2xYT media with ampicillin and chloramphenicol and grown at 30 °C until OD595 reached 0.4 to 0.6. Expression of Stx2A1 was induced with 1 mM IPTG, and cells were incubated further for 16 h at 16 °C. Cells were harvested at 5000 rpm for 30 min at 4 °C. The supernatant was bleached and discarded. The pellet was resuspended in lysis buffer (20 mM HEPES pH 7.9, 500 mM NaCl and 1 tablet of protease inhibitor cocktail from Roche) very slowly for 1 h by gently rocking at 4 °C. Cell lysate was then sonicated using the thick probe at a 30 s pulse followed by an incubation of 30 s at 30 amplitude and repeating the cycle for 5 to 7 times. The cell lysate was centrifuged at 16,000 rpm for 1 h at 4 °C and the supernatant was filtered using 0.22 μm Nalgene Rapid Flow filtration unit and loaded onto 10 mL Chitin beads (NEB) pre-equilibrated with 5 CV of buffer A (20 mM HEPES pH 8.5, 500 mM NaCl). Washing was carried out with three different buffers to increase the salt stringency. First with 2 CV of buffer A, then with 1 CV of buffer B1 (20 mM HEPES pH 7.9, 750 mM NaCl) and then with 1 CV of buffer B2 (20 mM HEPES pH 7.9, 1 M NaCl). On-column cleavage of protein from the chitin resin was carried out by incubating the column overnight at room temperature in buffer A + 50 mM DTT. The eluted fractions were collected at 1.5 mL each and the column was washed with 10 mL of Buffer A. The eluted fractions were separated on Express Plus PAGE using 1X Tris-MOPS-SDS buffer at constant volts and the gel was stained with Instant Blue Coomassie Protein Stain from Abcam for 15 min, rinsed with water for 5 min and visualized using Odyssey Clx image viewer. The fractions containing Stx2A1 (27.5 kDa) were pooled and separated on Hi Load 16/600 Superdex75pg column pre-equilibrated with buffer C (20 mM HEPES pH 7.9, 150 mM NaCl, 0.5 mM DTT) using the AKTA purifier 100/10 system (GE Healthcare Life Sciences). The eluted fractions were separated on Express Plus PAGE and purified Stx2A1 was concentrated using Amicon 10 kDa filters and stored at −80 °C. The molar extinction coefficient of Stx2A1 is 0.674 M−1cm−1 and thus absorption correction is required while measuring the concentration of the protein. The activity of the purified Stx2A1 was confirmed by performing the in vitro depurination assay [38].

2.3. Analytical and purification methods for the native and labeled peptides

A Shimadzu LC-40D XR UPLC system equipped with a PDA detector and an XBridge C18 column (4.6 × 50 mm, 3.5 μm particle size, from Waters Inc) and connected to an LCMS-2020 mass spectrometer was utilized for both reaction monitoring and fraction analysis. For analytical UPLC applications the flow rate was set at 2 mL/min and the eluents were A: 0.1 % TFA in water, eluent B: 0.1 % TFA in ACN. A linear gradient: B: 5–100 % in 4 min was applied.

The precipitated peptides, as well as the fluorescently labeled P11 after conjugation reaction were purified by AccqPrep semi-preparative HPLC instrument equipped with a Gemini 5 μm NX-C18 30 × 150 mm column under the following conditions: 42.5 mL/min flow rate, eluent A: 0.1 % TFA in water, eluent B 0.1 % TFA in ACN; linear gradient: B: 5–100 % in 40 min. The fractions of interest were analyzed by the Shimadzu 2020 UPLC-MS system. The LC-MS data for P3–P11 are shown in Figs. S1–S9. Fractions over 98 % purity were pooled and lyophilized overnight. High-resolution mass spectroscopy was performed by an Agilent 6546 qTOF instrument, and the data analysis is shown in Table S1.

2.4. Synthesis of P3, P4, P5, P6, P7, P8, P9, P10 and P11 peptides

Peptides were synthesized using Fmoc-based solid supported synthesis protocol. The Fmoc-Asp (OtBu)-Wang Resin was from Novabiochem with an original substitution of 0.31 mmol/g. Fmoc amino acids were purchased from Anaspec, HATU and HOAt coupling reagents were purchased from GeneScript. The amino acid coupling cycles included 1-h coupling step with 4 eq. amino acid, 4 eq. HATU/HOAt, 8 eq. diisopropylethylamine in N-methylpyrolidone (NMP), followed by two Fmoc deprotection steps for 10 min each with 20 % (v/v) piperidine in NMP. To suppress aspartamide formation 1 % (v/v) formic acid was added to the deprotection mixture. The cleavage was performed by using trifluoroacetic acid/water/triisopropylsilane/dithiothreitol (94/2/2/2, v/v/v/w) for 1 h. Peptides were precipitated by cold methyl tertbutylether/hexane mixture (1/1 v/v). After two consecutive cycles of centrifugation and washing the precipitates were dissolved in acetonitrile-water mixtures for purification.

2.5. Labeling of the P11 peptide

The P11 peptide was purchased from Genscript Inc. For our experiments, we have used BODIPY™ TMR-X NHS ester dye from Thermo Scientific (Catalog #D6117). The peptide was labeled with BODIPY™ TMR-X NHS ester dye (Thermo Scientific) at the N-terminus. Five mg of free dye was dissolved in 500 μl of DMF followed by the addition of 5 mg of P11 peptide and 5 μL of diisopropylethylamine. The reaction was incubated for 16 h with constant shaking at room temperature.

2.6. Fluorescence anisotropy assay to measure the binding affinity of P-stalk peptides

A fluorescence anisotropy (FA) assay was developed to examine the interaction of RTA and Stx2A1 with peptides mimicking the identical sequence at the C-termini of the P-stalk proteins by competition with the fluorescently labeled P11 peptide. To determine the appropriate concentration of RTA and Stx2A1, the labeled P11 peptide (1 μM) was incubated with RTA (0.08, 0.16, 0.32, 0.625, 1.25, 2.5, 5, 10, 20 and 40 μM) and Stx2A1 (0.625, 1.25, 2.5, 5, 7.5, 10, 12.5, 15 and 18 μM) in a dose dependent manner in 1X FA Buffer (25 mM Tris-Cl pH 8.0 and 100 mM NaCl) for half an hour at room temperature in the dark followed by centrifugation at 400×g for 2 min at 25 °C. The 96 well plates (Corning #3993) were scanned using a microplate reader (BioTek Synergy 4) having an excitation and emission filter of 530/25 nm and 590/35 nm, respectively. The fluorescence polarization (FP) and the anisotropy (A) values were obtained from equations (Eq) 1 and 2, respectively. Eq (1) FP=F‖−F⊥F‖+F⊥

Eq (2) A=F‖−F⊥F‖+2F⊥

Eq (3) A=2FP3−FP

Eq (4) Fb=Aobs−Afree(Abound−Aobs)Q+(Aobs−Afree)

F‖ and F⊥ represent the parallel and perpendicular intensities of emitted light, respectively. The anisotropy values are related to the FP values by equation (3). The parallel and perpendicular intensities of emitted polarized light were used to calculate the polarization values using equation (1) and multiplied by 1000 to express in millipolarization (mP) units. For quantitative analysis, we have used the anisotropy values to determine the fraction of labeled P11 peptide bound to the RIPs [40,41]. The fraction bound (Fb) is related to the observed anisotropy values (Aobs) at a given concentration of RTA or Stx2A1 as shown in equation (4). The anisotropy values obtained from labeled P11 peptide (Afree) and from peptide bound to the RIPs (Abound) along with the quantum yield of the fluorophore (Q) were substituted in equation (4) to calculate the fraction of the labeled P11 peptide bound to each RIP. The Fb values were plotted against the concentration of RTA and Stx2A1. The equilibrium dissociation constant (KD), which represents the binding affinity of labeled P11 peptide for the RIPs, was derived by non-linear regression analysis as shown in equation (5) using OriginPro graphical software [40,41]. Lt is the total concentration of the ligand (labeled P11 peptide) and Rt represents the concentration of RTA or Stx2A1.

Eq (5) Fb=(KD+Lt+Rt)−(KD+Lt+Rt)2−4LtRt2Lt

The P-stalk peptides, P3, P4, P5, P6, P7, P8, P9, P10 and P11 were used in a fluorescence anisotropy-based competition assay to determine if the unlabeled peptides can displace the fluorescently labeled P11 peptide bound to the RIPs. Labeled P11 peptide (1 μM) was incubated with 3 μM of RTA or 2.5 μM of Stx2A1 and competition reactions were initiated by adding varying concentrations of peptides (1.25, 2.5, 5, 10, 20, 31.25, 40, 62.5, 125 μM) in 40 μl of 1X FA buffer. The bound state anisotropy values were calculated by preparing a control set of experiments with 3 μM of RTA or 2.5 μM of Stx2A1 and 1 μM of labeled P11 peptide, whereas the anisotropy values of the free state were estimated by incubating only 1 μM of labeled P11 peptide in the reaction buffer. The bound and free state observables were used to calculate the Q factor for the competition experiments. The samples were kept in the dark for half an hour and scanned using BioTek Synergy 4 microplate reader. Eq (6) NormalizedBinding(%)=Abound−Aobs(Abound−Aobs)Q+(Aobs−Afree)×100

Eq (7) Aobs=Afree+Abound−Afree1+10(Logx−LogIC50)

In a competitive binding assay, the concentration of the inhibitor required to displace 50 % of bound ligand from the RIPs is defined as the IC50 of the inhibitor and is used to calculate the inhibition constant (Ki) of the inhibitor. The anisotropy values obtained from the competition of labeled P11 peptide by the unlabeled peptides (P3 – P11) were used to calculate the normalized percentage of binding of the unlabeled peptides using equation (6) and non-linear regression analysis of the curve using equation (7) was employed to estimate the IC50 values [31]. The reaction buffer for small molecules contained 1 % DMSO in 1X FA Buffer as the compounds were dissolved in 100 % DMSO. The free and bound state control sets for the small molecules were also carried out in 1X FA + 1 % DMSO buffer to adjust for DMSO tolerance.

Eq (8) IC50=F0×KD(1−F0)⋅(2−F0)+F0×Lt2Ki(2−F0)KD×F0+1

Eq (9) Ki=IC50F0×KD(1−F0)(2−F0)+F0×Lt2−1KD×F02−F0

For FA-based competition assays, RIP concentration must be chosen such that the fraction of labeled P11 bound over total (F0) is between 0.5 and 0.8, indicating that the bound ligand accounts for 50–80 % of the total ligand [42]. In our experiments, RTA concentration (Rt) was kept constant at 3 μM and Stx2A1 was maintained at 2.5 μM, while the labeled P11 (Lt) was 1 μM and the KD value was 1 μM for both RIPs. Substituting these values in equation (5), the F0 value is 0.69 for RTA and 0.65 for Stx2A1, which is in accordance with these criteria. The inhibition constant, Ki, describes the equilibrium binding affinity between the inhibitor and each toxin and is defined as the concentration of the inhibitor that will bind to half the binding sites on each RIP at equilibrium in the absence of labeled P11 [43]. The Ki value for each compound was calculated by solving equation (8) for Ki using equation (9) where F0 is 0.69 for RTA and 0.65 for Stx2A1, KD is 1.06 μM for RTA and 0.98 μM for Stx2A1, Lt is 1 μM and the IC50 values are obtained from equation (7) [31]. For all our experiments, the fluorescence intensity values corresponding to the highest and lowest concentration of the inhibitors representing the dynamic range of the assay was greater than 100 millipolarization units.

3. Results and discussion

3.1. Affinity of P11 for RTA and Stx2A1 by fluorescence anisotropy

We set up an FA assay to determine the affinity of the P-stalk peptides ranging in size from 3 to 11 amino acids for RTA and Stx2A1. P11 (SDDDMGFGLFD) was chosen as a tracer because of its high affinity for RTA and Stx2A1 by SPR [27,28]. P11 was labeled with the BODIPY™ TMR-X NHS ester dye at the N-terminus and separated from free dye using reverse-phase HPLC. We attached the fluorophore to the N-terminal end of P11 because significant interactions were observed with the C-terminus of the peptide with RTA [20,21] and Stx2A1 [22,27]. BODIPY™ TMR-X NHS was selected as the fluorescent dye because it has a red-shifted absorbance, long emission time and a good separation between absorption and emission. BODIPY-TMR labeled probe gives a Stokes shift of ~10 nm that helps in selection of the bandpass filters. Previous studies have shown very little or no shift in emission maxima between the free dye and BODIPY-TMR labeled peptides, indicating that the fluorophore does not interact with the peptide [34]. The excitation and emission of BODIPY-TMR are 544 nm and 570 nm respectively, with an attenuation coefficient of 60,000 M−1cm−1. The appropriate concentration of the fluorescent probe (labeled P11) was determined by measuring the fluorescence intensity as a function of the concentration [31]. The emission scan of labeled P11 peptide at 0, 0.25, 0.5, 1, 2 and 4 μM was carried out from 535 nm to 700 nm with a step-size of 2 nm keeping the excitation wavelength fixed at 495 nm (Fig. S10). We selected 1 μM as the concentration, which showed fluorescence intensity above 1000 to be used in the FA assay. The affinity of RTA and Stx2A1 for the fluorescent probe was determined by keeping the labeled P11 peptide at a fixed concentration (1 μM) and varying the concentration of each RIP for 30 min at room temperature. As RTA (Fig. 1A) and Stx2A1 (Fig. 1B) concentration increased more complexes formed with labeled P11, leading to an increase of the polarization value until almost all the labeled P11 probe bound to the RIPs. The equilibrium dissociation constant (KD), which represents 50 % of labeled P11 bound to RTA was 1.06 ± 0.2 μM (Fig. 1A) and to Stx2A1 was 0.98 ± 0.2 μM (Fig. 1B).

3.2. Fluorescence anisotropy-based competition assay to determine the affinity of the P-stalk peptides for RTA and Stx2A1

The direct binding results established the conditions to use in the competition assay to determine if unlabeled peptides can competitively displace the labeled P11 peptide from RTA and Stx2A1. Increasing concentrations (0.625, 1.25, 2.5, 5, 10, 20, 31.25, 40, 62.5 and 125 μM) of unlabeled P11 peptide were incubated with 3 μM of RTA or 2.5 μM of Stx2A1 and 1 μM of labeled P11 peptide. As concentration of unlabeled P11 increased, the fluorescent probe was displaced from RTA (Fig. 1C) and Stx2A1 (Fig. 1D) complexes resulting in an increase in the free probe in solution and thus a decrease in the polarization value, reflecting the displacement of the fluorescent probe. At a concentration of 125 μM, the unlabeled P11 completely displaced the fluorescent P11 from both RTA and Stx2A1 (Fig. 1C and D). We tested competition with the PT peptide (RGWGHPSGYS), which binds at the active site of RTA [29]. The PT peptide did not compete with the fluorescent P11 for binding to the P-stalk site of RTA (Fig. 1E) or Stx2A1 (Fig. 1F). A small molecule, BTB13068, which binds RTA away from the P-stalk binding site [29] and SEW06366, which binds Stx2A1 away from the P-stalk binding site, were also tested. BTB13068 or SEW06366 did not compete with P11 for binding to RTA (Fig. 1G) or Stx2A1 (Fig. 1H), respectively. These results established the specificity of the FA assay.

The IC50 value determined by the FA-based competition assay is the concentration of the inhibitor required to replace 50 % of the BODIPY-TMR labeled probe from each toxin. The IC50 values were used to determine the inhibition constant, Ki (Table 1). The Ki values for the unlabeled P11 peptide in the competition assay against RTA (0.5 ± 0.04 μM) (Fig. 1C) and Stx2A1 (1.2 ± 0.04 μM) (Fig. 1D) were similar to that of the KD value of labeled P11 peptide against RTA (Fig. 1A) and Stx2A1 (Fig. 1B), indicating that the interaction between P11 and RTA or Stx2A1 was site-specific.

3.3. The role of the individual amino acids at the C-termini of P-stalk proteins in binding to RTA and Stx2A1

To understand the role of the individual amino acids at the CTD of P-proteins in binding to RTA and Stx2A1, we synthesized peptides of increasing lengths ranging from three to eleven amino acids (P3 to P11) mimicking the P-stalk CTD and evaluated their binding affinity for RTA (Fig. 2A–H) and Stx2A1 (Fig. 3A–H) using the FA assay. The IC50 and the Ki values of each peptide determined against RTA and Stx2A1 are shown in Table 1. The binding affinity of peptides for RTA and Stx2A1 increased with the increasing peptide length. The P3 peptide had an IC50 of 328 μM and a Ki value of 86 μM against RTA (Fig. 2A) but at the same concentration we could not detect competition with Stx2A1 (Fig. 3A), indicating that each toxin interacted with P3 differently. The P4 peptide had an IC50 of 160 μM and Ki value of 42 μM against RTA (Fig. 2B), while it still did not show any competition with Stx2A1 (Fig. 3B).

The ~ 2-fold higher affinity of P4 compared to P3 against RTA might be because P-stalk C-termini forms an α-helix upon binding to RTA [20, 21] and a minimum of 4 residues are required to form an α-helix [24]. The introduction of phenylalanine (Phe7) in P5 did not decrease the IC50 and the Ki for RTA (Fig. 2C), indicating that the peptide sequence and not the length of the peptide directs the binding affinity. In contrast to RTA, introduction of phenylalanine in P5 resulted in a detectable IC50 and Ki for Stx2A1 (Fig. 3C). These results indicated that the N-terminal phenylalanine (Phe7) of the P5 peptide was more important for binding to Stx2A1 than RTA.

The primary distinction observed between Phe7 in the Stx2A1-peptide structure (PDB ID: 8SZ2) and the RTA-peptide structure (PDB ID: 5GU4) seemingly lies in the π-stacking interaction between Phe7 and Arg179 within the Stx2A1-peptide complex [27]. This interaction is not only unique in the Stx2A1-peptide structure but also serves to stabilize the position of Arg179, where it forms two salt-bridges with Asp11 of the P11 peptide, thus providing additional support for the importance of Phe7 in Stx2A1 relative to RTA [20,21].

Introduction of glycine as the sixth residue in P6 lowered the IC50 and Ki values of both RIPs (Fig. 2D and 3D). The greater increase in the binding affinity towards RTA could be because the flexible glycine residue of P6 contributes to an α-helix formation. The X-ray crystal structure of RTA with P11 showed that only the last 6 amino acids, GFGLFD, are inserted into a hydrophobic pocket on RTA [20,21]. The two glycine residues in P6 accommodate the backbone of the peptide to facilitate the insertion of FLF into the hydrophobic pocket of RTA [21]. The X-ray structure of Stx2a with P11 [22] and the cryo-EM structure of Stx2a with P11 [26] also showed that only the GFGLFD motif is involved in direct contacts with the toxin but at a different location compared to that of RTA. The recently determined structure of Stx2A1 with P8 [27] verified these results and showed that only the last 6 residues of P8 bind Stx2A1 in a similar binding mode as the last 6 residues of P11 bind to Stx2a holotoxin [22].

The addition of methionine in P7 caused a slight decrease in the IC50 and Ki values for RTA and Stx2A1 (Fig. 2E and 3E). In contrast, addition of the first Asp in P8 decreased the IC50 and Ki for RTA ~3-fold (Fig. 2F) and decreased the IC50 and Ki for Stx2A1 ~2.5-fold (Fig. 3F). Addition of the second Asp in P9 did not affect the IC50 or Ki for RTA or Stx2A1 (Fig. 2G and 3G). However, addition of the third Asp in P10 decreased the IC50 and Ki for RTA 10- and 14-fold, respectively (Fig. 2H) and caused ~3-fold decrease in the IC50 and Ki for Stx2A1 compared to P7 (Fig. 3H). These results demonstrated that the DDD residues were more important for binding of RTA to the P-stalk CTD compared to Stx2A1. Electron density was not observed for the DDDM motif in the structures of RTA or Stx2A1 in complex with the P-stalk peptides possibly due to the inherent flexibility of these residues. In vitro pull-down assays indicated that the DDDM motif interacts with RTA [21]. Using site-directed mutagenesis we showed that Arg189, Arg191, Arg193, Arg196, Arg197, Arg234 and Arg235 contribute to the ribosome binding and cytotoxicity of RTA, possibly by interacting with the DDD motif [24, 44].

3.4. Comparison of the binding affinity of the P-stalk peptides for RTA and Stx2A1 using the FA assay and the SPR assay

Comparison of the Ki values of peptides obtained by the FA assay with the KD values previously obtained by the SPR assay with Biacore T200 [27,28] indicated that the KD values were larger than the Ki values for both toxins (Table 2). Similarly, the KD values of small molecules against RTA determined by SPR [29] were higher than the values determined by the FA assay [31]. The affinity determined by SPR may be lower due to nonspecific ligand interactions at high concentrations, which can make accurate KD determination challenging. Accurate affinities cannot be determined by SPR when suboptimal concentrations are used for peptides or small molecules with low affinities [29]. Furthermore, SPR values represent total binding, including specific and nonspecific binding to each toxin on the Biacore chip and may be affected by the heterogeneity of the immobilized protein on the chip. In contrast, the FA assay measures the decrease in anisotropy values caused by inhibitors that displace the labeled P11 probe from the P-stalk pocket of RTA or Stx2A1.

Because the FA assay measures site-specific binding in solution, it is more specific than the SPR assay. The Ki values of P6 (28 μM), P9 (4 μM) and P11 (0.5 μM) for RTA obtained by the FA assay were in close agreement with the KD values obtained by isothermal titration calorimetry (ITC) assay for P6 (19 μM), P9 (3 μM) and P11 (2 μM) for RTA [21]. Although the absolute values obtained by the FA and the SPR assay were different, the Ki and the KD values of the peptides were in the same rank order (Table 2). They were also in the same rank order as the IC50 values determined by the qRT-PCR method for inhibition of ribosome depurination in vitro (Table 2) [27,28]. These results demonstrate that peptides that bind at the P-stalk pocket of RTA or Stx2A1 with higher affinity by either the FA or the SPR method, more potently inhibit the depurination activity of each RIP on eukaryotic ribosomes.

We previously showed that P8 had optimal affinity for Stx2A1 by SPR and maximal potency in inhibition of Stx2A1-mediated ribosome depurination in vitro [27]. We obtained similar results for Stx2A1 by the FA assay. Increasing the peptide length from P8 to P11 improved the affinity for Stx2A1 by only 1.6-fold by SPR and ~2-fold by the FA assay (Table 2). In contrast, while the affinity of P11 for RTA improved 1.5-fold by SPR compared to P8, we observed a 12-fold increase in the affinity of P11 for RTA compared to P8 by the FA assay (Table 2). Similarly, the IC50 value of P11 measured by qRT-PCR for RTA improved 5-fold compared to P8 (Table 2). The affinity of P11 for RTA improved 40-fold compared to P7 by the FA assay, while only a 1.5-fold increase was observed in the affinity of P11 for RTA compared to P7 by the SPR assay (Table 2). These results indicate that the FA assay can differentiate between the affinity of P7–P11 peptides against RTA better than the SPR assay. Since increasing the peptide length from P7 to P11 involves addition of the SDDD residues to the N-terminus of the P7 peptide, the negatively charged aspartic acid residues may cause promiscuous binding to RTA outside the P-stalk pocket, leading to similar values by the SPR assay. The KD values of peptides ranging in size from P7 to P11 for RTA may not be distinguishable by SPR due to nonspecific binding, which can make accurate determination of affinity by SPR difficult. In contrast to RTA, binding affinities of P8–P11 peptides for Stx2A1 were similar by both methods. The IC50 values of the P8–P11 peptides for Stx2A1 measured by qRT-PCR were also similar (Table 2). These results indicate that using the FA assay we can more accurately measure the affinity of peptides with varying affinities against RTA compared to the SPR assay.

3.5. The N-terminal aspartic acid residues of the P-stalk peptide are important for the interaction with RTA and Stx2A1

The residues that interact with the P-stalk CTD are not conserved in the different RIPs and the P-stalk binding pockets have different shape, electrostatic charge distribution and differences in the hydrophobic residues, suggesting that the P-stalk CTD interacts with the RIPs using different binding modes [22,45]. Structural analysis showed that although P11 bound to RTA and trichosanthin (TCS), a single chain RIP, at a similar position, P11 adopted a different conformation when interacting with each RIP [21,22]. The negatively charged DDD motif bound to TCS and formed three H-bonds with K173 and R174 and Q169 in TCS [46,47]. Although interaction with the DDD residues was not observed in the structures of RTA-P10 [20], RTA-P11 [21], or in Stx2a-P11 and Stx2A1-P8 complexes [22,27], the FA assay indicates that the DDD residues are important for the affinity of RTA and Stx2A1 for the P-stalk. The addition of these residues decreased the Ki for RTA by 14-fold and the Ki for Stx2A1 by 3-fold (Table 2). While individual Ala substitutions in the DDD residues did not show a significant effect in the affinity of P11 for the A1 subunit of Shiga toxin 1 (Stx1A1), double and triple alanine substitutions at the DDD residues caused at least a 2-fold decrease in the affinity of P11 for Stx1A1 by SPR [48].

The P-stalk CTD adopts an α-helix conformation when bound to RTA [20,21] and has propensity for an α-helix formation when bound to Stx2A1 [26]. A recent study showed that three consecutive aspartic acid residues (DDD) have a propensity for turn-like conformations in intrinsically disordered proteins [49]. The DDD residues may stabilize the helix formation and thus may explain the increased affinity of P11 for RTA and Stx2A1 compared to P7. The α-helical conformation may be responsible for the initiation of a series of conformational changes in RTA and Stx2A1 that orient their active site during approach to the conserved adenine residue of the SRL for productive association and depurination. The intermolecular contacts observed between the last six residues of the P-stalk CTD and RTA or Stx2A1 by X-ray crystallography are not sufficient to explain the binding affinity of the P-stalk peptides. The FA assay measurements suggest that the conformational flexibility of the peptide necessary for α-helix formation may also contribute to the binding affinity.

We previously screened a fragment library by SPR to identify fragments that bind to the P-stalk binding pocket of ricin [29]. Many fragments had low affinities for RTA by SPR, and it was not possible to accurately determine their affinity. It is critically important to accurately determine the affinity of fragments for ranking during hit-to-lead optimization [30]. The FA-based competition assay can accurately measure the binding affinity of peptides that target the P-stalk pocket of two different RIPs even though each RIP interacts with the P-stalk CTD in a unique manner. The assay described here detects only interactions that compete with the labeled P11 peptide and can differentiate between peptides that may be susceptible to nonspecific binding at high concentrations by the SPR assay. The FA assay could be used as an orthogonal assay to validate inhibitor specificity and mechanism of action to accelerate drug discovery. It may also be adapted to examine the interaction of the P-stalk CTD with its diverse binding partners.

4. Conclusions

Ribosome binding sites of ricin or Shiga toxin have not been targeted by small molecules and robust assays are needed to accurately determine the affinity of inhibitors during hit-to-lead optimization in drug discovery. We describe here a novel fluorescence anisotropy-based competition assay for determining the binding affinity of inhibitors that bind the P-stalk binding pocket of ricin A subunit and the A1 subunit of Shiga toxin 2. The FA assay is fast, highly reproducible and exhibits greater specificity than the surface plasmon resonance (SPR) assay because it detects only interactions that compete with the labeled P11 peptide. The FA assay is suitable for validating and ranking inhibitors targeting the P-stalk pocket of RTA and Stx2A1 and could have broad application to mechanism of action studies. It will lay the foundation for screening small molecule inhibitors of RIP-ribosome interactions.

Supplementary Material

Supplement

Acknowledgements

We would like to thank Dr. Jacques Roberge for helpful discussions, Anastasiia Tsymbal with the chemical formulas and Dr. Michael Rudolph for the structural analysis. This work was supported by the National Institutes of Health grant AI178870 to NET and the National Institutes of Health grant R01AI141635 to XPL.

Data availability

Data will be made available on request.

Abbreviations

ACN acetonitrile

A anisotropy

CV column volume

CTD C-terminal domain

Eq equation

FA fluorescence anisotropy

FP fluorescence polarization

HATU hexafluorophosphate azabenzotriazole tetramethyl uronium

IC50 half maximal inhibitory concentration

K D equilibrium dissociation constant

K i the inhibition constant

RIP ribosome inactivating protein

RTA ricin toxin A subunit

SRL sarcin/ricin loop

Stx2A1 Shiga toxin 2 A1 subunit

SPR surface plasmon resonance

TCS trichosanthin

TFA trifluoroacetic acid

Fig. 1. Fluorescence anisotropy assay to examine binding of labeled P11 to RTA and Stx2A1. Varying concentrations of RTA (A) and Stx2A1 (B) were incubated with 1 μM of labeled P11 peptide and scanned using BioTek Synergy 4 microplate reader. The anisotropy values were obtained using excitation and emission bandpass filters of 530/25 nm and 590/35 nm, respectively. Fraction of labeled P11 bound was plotted against RTA (A) and Stx2A1 (B) concentration. Three different measurements are shown. (C & D) Competition of labeled P11 peptide from RTA (C) and Stx2A1 (D) using unlabeled P11 peptide. The mean ± SD of four replicates is shown. Competition of labeled P11 from RTA (E) and Stx2A1 (F) by the PT peptide (sequence: 5’ - RGWGHPSGYS - 3′). Competition of labeled P11 peptide from RTA (G) and Stx2A1 (H) by small molecules BTB13068 (G) and SEW06366 (H). Two different biological replicates are shown.

Fig. 2. Fluorescence anisotropy assay to measure competition of the labeled P11 peptide with P3, P4, P5, P6, P7, P8, P9 and P10 peptides for binding to RTA (A–H). Reactions containing labeled P11 (1 μM), and RTA (3 μM) were incubated with varying peptide concentrations in 40 μl of 1X FA buffer (25 mM Tris-HCl pH 8.0 and 100 mM NaCl) for 30 min in the dark at room temperature followed by centrifugation at 400×g for 2 min and scanned using BioTek Synergy 4 microplate reader. Anisotropy values obtained using 530/25 nm excitation and 590/35 nm emission filters were used to calculate the normalized percentage of binding and were plotted against the peptide concentration. The mean ± SD of four replicates is shown.

Fig. 3. Fluorescence anisotropy to measure competition of the labeled P11 peptide with P3, P4, P5, P6, P7, P8, P9 and P10 peptides for binding to Stx2A1 (A–H). Reactions containing labeled P11 (1 μM) and Stx2A1 (2.5 μM) were incubated with varying peptide concentrations in 40 μl of 1X FA buffer (25 mM Tris-HCl pH 8.0 and 100 mM NaCl) for 30 min in dark at room temperature followed by centrifugation at 400g for 2 min and scanned using BioTek Synergy 4 microplate reader. Anisotropy values obtained using 530/25 nm excitation and 590/35 emission filters were used to calculate the normalized percentage of binding and plotted against the peptide concentration. The mean ± SD of four replicates is shown.

Table 1 The IC50 and Ki values of P-protein peptides against RTA and Stx2A1 determined by the FA-based competition assay.

Peptide	Peptide Sequence	Ricin toxin A subunit (RTA)	Shiga toxin 2 A1 subunit (Stx2A1)	
IC50 (μM)a	Ki (μM)b	IC50 (μM)a	Ki (μM)b	
P3	LFD	327.7 ± 30	85.6 ± 8	>1000	>1000	
P4	GLFD	160.2 ± 21	41.6 ± 7	>1000	>1000	
P5	FGLFD	178.9 ± 21	46.5 ± 7	54.3 ± 6	14.9 ± 2	
P6	GFGLFD	108.4 ± 21	28.0 ± 7	35.4 ± 3	9.6 ± 1	
P7	MGFGLFD	80.5 ± 6	20.6 ± 2	22.9 ± 1	6.0 ± 0.4	
P8	DMGFGLFD	25.0 ± 3	6.0 ± 0.9	10.2 ± 1	2.4 ± 0.4	
P9	DDMGFGLFD	18.6 ± 1	4.3 ± 0.3	9.0 ± 0.2	2.1 ± 0.1	
P10	DDDMGFGLFD	7.9 ± 0.2	1.5 ± 0.1	8.2 ± 0.4	1.8 ± 0.1	
P11	SDDDMGFGLFD	4.0 ± 0.2	0.5 ± 0.04	6.1 ± 0.1	1.2 ± 0.04	
a The IC50 values were determined by FA are shown as mean ± SD of four replicates.

b The Ki values were determined by FA are shown as mean ± SD of four replicates.

Table 2 Comparison of binding affinities of peptides for RTA and Stx2A1 using FA and SPR.

Peptide	Peptide Sequence	Ki (μM) using FA assaya	KD (μM) using SPR assay	IC50 (μM) using qRT-PCRd	
RTA	Stx2A1	RTAb	Stx2A1c	RTA	Stx2A1	
P3	LFD	85.6 ± 8	>1000	>10 mM	>1 M	U.De	U.D.	
P4	GLFD	41.6 ± 7	>1000	451 ± 17	> 2 mM	102 ± 45	U.D.	
P5	FGLFD	46.5 ± 7	14.9 ± 2	497 ± 30	125 ± 27	121 ± 44	U.D.	
P6	GFGLFD	28.0 ± 7	9.6 ± 1	399 ± 20	71 ± 13	63 ± 13	U.D.	
P7	MGFGLFD	20.6 ± 2	6.0 ± 0.4	294 ± 47	66 ± 3	34 ± 10	60 ± 6	
P8	DMGFGLFD	6.0 ± 0.9	2.4 ± 0.4	299 ± 5	36 ± 8	23 ± 4	23 ± 4	
P9	DDMGFGLFD	4.3 ± 0.3	2.1 ± 0.1	309 ± 7	29 ± 5	15 ± 2	26 ± 4	
P10	DDDMGFGLFD	1.5 ± 0.1	1.8 ± 0.1	272 ± 6	20 ± 4	8 ± 2	28 ± 4	
P11	SDDDMGFGLFD	0.5 ± 0.04	1.2 ± 0.04	196 ± 17	22 ± 3	5 ± 1	30 ± 4	
a The Ki values were determined by FA are shown as average ± SD of four replicates.

b The KD values were obtained using Biacore T200 and shown as mean ± SD of three to four replicates [28].

c The KD values were obtained using Biacore T200 and shown as mean ± SD of three replicates [27].

d The IC50 values were determined by qRT-PCR in vitro using yeast ribosomes and shown as mean ± SD of three replicates [38,39].

e U.D. indicates undetermined.

CRediT authorship contribution statement

Arkajyoti Dutta: Writing – original draft, Methodology, Formal analysis, Conceptualization. Zoltan Szekely: Writing – original draft, Investigation, Formal analysis. Hakan Guven: Formal analysis. Xiao-Ping Li: Writing – review & editing, Supervision, Funding acquisition, Formal analysis, Conceptualization. John E. McLaughlin: Formal analysis. Nilgun E. Tumer: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ab.2024.115580.
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