Abstract
Ricin is a potent plant toxin that targets the eukaryotic ribosome by depurinating an adenine from the sarcin-ricin loop (SRL), a highly conserved stem-loop of the rRNA. As a category-B agent for bioterrorism it is a prime target for therapeutic intervention with antibodies and enzyme blocking inhibitors since no effective therapy exists for ricin. Ricin toxin A subunit (RTA) depurinates the SRL by binding to the P-stalk proteins at a remote site. Stimulation of the N-glycosidase activity of RTA by the P-stalk proteins has been studied extensively by biochemical methods and by X-ray crystallography. The current understanding of RTA’s depurination mechanism relies exclusively on X-ray structures of the enzyme in the free state and complexed with transition state analogues. To date we have sparse evidence of conformational dynamics and allosteric regulation of RTA activity that can be exploited in the rational design of inhibitors. Thus, our primary goal here is to apply solution NMR techniques to probe the residue specific structural and dynamic coupling active in RTA as a prerequisite to understand the functional implications of an allosteric network. In this report we present de novo sequence specific amide and sidechain methyl chemical shift assignments of the 267 residue RTA in the free state and in complex with an 11-residue peptide (P11) representing the identical C-terminal sequence of the ribosomal P-stalk proteins. These assignments will facilitate future studies detailing the propagation of binding induced conformational changes in RTA complexed with inhibitors, antibodies, and biologically relevant targets.
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Biological context
Ribosomes play a central role in cellular survival by translating genetic information to direct the biosynthesis of nascent polypeptide chains in a dynamic macromolecular assembly (Steitz 2008; Korostelev et al. 2008; Noeske and Cate 2012). The translational activity is highly susceptible to a family of lethal toxins known collectively as ribosome-inactivating proteins (RIP), which disable ribosome function and trigger apoptosis. These toxins have evolved in diverse organisms such as ricin in plants, alpha-sarcin in fungi and Shiga toxins I/II in pathogenic bacteria (Barbier and Gillet 2018) and are viewed as important targets for developing antibody (Rudolph et al. 2014) and small molecule inhibitors (Tanaka et al. 2001; Li et al. 2021). The domain structure of ricin includes an enzymatically active A subunit (RTA) and a cell binding lectin B subunit (RTB) linked by a disulfide bond (Rutenber and Robertus 1991). The catalytic domain of RTA is an rRNA N-glycosidase which depurinates A4324 in eukaryotic 28 S rRNA or A2660 in E. coli 23 S rRNA in the highly conserved sarcin-ricin loop (SRL) by cleaving an N-glycosidic bond (Endo and Tsurugi 1987). In the ribosome, the SRL rRNA along with the P-stalk proteins form the GTPase associated center involved in regulating mRNA translation (Steitz 2008; Maracci and Rodnina 2016). The highly conserved tetraloop (GAGA) within the SRL rRNA (Szewczak et al. 1993) is selectively depurinated by the RIP toxins (Amukele and Schramm 2004), which in turn inhibit various translation factor interactions within the ribosome debilitating tRNA-mRNA translocation (Voorhees et al. 2010; Shi et al. 2012; Grela et al. 2019).
In the intact ribosome, RTA is tethered to the eukaryotic P-stalk proteins, which facilitate access to the SRL rRNA substrate for steric reasons (Chiou et al. 2008; Li et al. 2010). The association involves interactions between the identical C-terminal 11 residues of the five P-stalk proteins on the ribosome and the P-stalk binding site located in the C-terminal domain of RTA (Li et al. 2018). Blocking the P-stalk binding site with peptide mimics (Li et al. 2018), small molecules (Li et al. 2021) or antibodies (Czajka et al. 2022) suppresses the enzymatic activity of RTA very efficiently despite being physically remote from the active site. This ability to inhibit depurination indirectly underscores the importance of the P-stalk binding site as a novel target for allosteric inhibitors that can overcome the existing discovery challenges of creating inhibitors targeting the active site of RTA (Jasheway et al. 2011; Li et al. 2020).
To date ricin complexed with various inhibitors and antibodies exhibits limited structural variation contrary to evidence from MD simulations, which suggest that dynamics plays an important role in substrate recognition (Olson 1997) and possibly allosteric inhibition by neutralizing antibodies (Dai et al. 2011). More recently the CryoEM structure of the eukaryotic P-stalk pentamer and Shiga toxin 2a (Stx2a), a close homologue of RTA revealed significant conformational heterogeneity in the C-terminal P-stalk binding site (Kulczyk et al. 2023).
An outstanding question in the structural biology of RTA is the nature of the communication between the active site and the P-stalk binding site separated by more than 20Å. Mutational studies concluded that the ~ 200-fold enhancement in the efficiency of depurination observed in the presence of the pentameric P-stalk proteins cannot be attributed solely to the local concentration effect (Li et al. 2013). The prevailing hypothesis is that binding of different modalities at the P-stalk site or at the N-terminal domain (Dai et al. 2011) of RTA may be communicated to the catalytic site of the enzyme by an unknown allosteric mechanism. A distinctive feature of allosteric regulation is coupled conformational and dynamic changes facilitated by a network of intramolecular interactions. Hence mapping such correlated changes is the first step to gain important mechanistic insight into the regulation of the depurination activity of the RIP toxins and to rationalize the high turnover of RTA.
High resolution solution NMR is a versatile technique that has been widely employed to explore the conformational landscape of enzymes with tunable dynamics while demonstrating the effects of structural allostery at the atomic level (Palmer 2015; Alderson and Kay 2021). Our primary objective here is to obtain backbone amide (NH) and methyl group chemical shift assignments (CH3), which will serve as a foundation for future studies comparing RTA interactions with different inhibitors while investigating the role of dynamics in enzyme function.
Methods and experiments
Protein expression
The RTA gene (residues 1-267) from Ricinus communis (castor bean) with two mutations (V76M and Y80A) was subcloned into a modified (original His6) pET His10 Sumo TEV LIC cloning vector (1S) (Addgene plasmid # 29,659). The mutations significantly neutralize the toxin but preserve the basic structure of RTA (Legler et al. 2011). Isotopically labeled protein was expressed in BL21(DE3) pLysS cells. Bacterial cultures were grown in 2 L of LB (OD600 = 0.7) before spinning down the cells (1500g) and transferring the pellet to warm 30 ml M9 media without carbon/nitrogen source and left to shake for 15–30 min at 37 °C. The cells were spun down (1500g) and transferred into warm 500 mL of deuterated (100% D2O) or protonated (100% H2O) M9 minimal media supplemented with 15N-ammonium chloride (1 gm/L), 13C-Glucose (4 gm/L), trace elements and antibiotics (Studier 2005). After 1 h at 37 °C, protein expression was induced by 1 mM IPTG, and the cells grown overnight (16 h) at 20 °C (~ 22 h in D2O). The labeled protein was purified by a previously described protocol involving nickel affinity column and size exclusion chromatography (Czajka et al. 2022). After removing the N-terminal SUMO-His10 tag by TEV cleavage, the purified protein was concentrated and exchanged into NMR buffer with trace protease inhibitors (Roche protease Inhibitor EDTA free tablets), 50 mM Hepes Buffer, 150 mM NaCl, 1 mM TCEP in 95% H2O/5% D2O at pH 7.5.
NMR spectroscopy
The NMR data were acquired on Bruker AVANCE III spectrometers equipped with TCI CryoProbes™ at 18.8T. Two independent samples of RTA with 1H/13C/15N-labeled (190 µM) and ~ 80% deuterated 13C/15N-labeled (250 µM) protein were used for sequence specific backbone and side-chain chemical shift assignments. A standard suite of triple-resonance (HNCO, HNCA, HN(CO)CA, HNCACB, HN(CO)CACB) experiments (Sattler et al. 1999), 3D 15N-edited NOESY-HSQC and 3D (15N,15N)-edited HSQC-NOESY-HSQC were acquired on the deuterated U-13C/15N-labeled RTA (250 µM) at 18.8T. Due to some loss of signal from slow back exchange of amide protons in the beta-sheet of the protein when expressed in D2O, additional assignments were obtained from 3D HNCA, HNCO, CBCA(CO)NH, 15N-edited NOESY-HSQC and 3D (13Caliphatic,15Namide)-edited HSQC-NOESY-HSQC spectra acquired on a uniformly protonated 13C/15N-labeled RTA (190 µM) sample. The side-chain methyl resonances were assigned using a combination of 3D (H)CCH-COSY, (H)CCH-TOCSY, 3D 13C-edited NOESY-HSQC, and 3D (13Caliphatic,13Cmethyl)-edited HMQC-NOESY-HMQC experiments acquired on the protonated sample at 18.8T (Sattler et al. 1999; Cavanagh 2006). The mixing time for the DIPSI2 sequence is set to 16 ms in (H)CCH-TOCSY, and 100 ms in the 1H-1H NOE experiments.
To map the binding site of the P-stalk peptide (P11) (Li et al. 2018), C13/N15-labeled RTA (220 µl and 200 µM) was titrated with 5 µl of 10 mM unlabeled P11 peptide (GenScript) dissolved in NMR buffer. The shifts in the amide and methyl peaks were followed during the titration (8 points) by acquiring 2D 1H-15N HSQC and 2D 1H-13C HMQC spectra respectively at 18.8T after each peptide addition until saturation is reached at ~ 1:9 protein-to-peptide ratio. In the P11 peptide complex the methyl peaks are in fast exchange between the free and bound state. By following the trajectory of the peaks, we were able to assign all the methyl resonances in the complex. Additionally, the backbone assignments in the peptide complex were confirmed by 3D HNCA, HNCO and 3D N15-edited NOESY-HSQC. All the NMR data were processed in Topspin 3.5 from Bruker Biospin and the spectra analyzed in CARA 1.5 (Keller 2004).
Extent of assignment and data deposition
Ricin A subunit is a 30 kDa protein composed of 267 residues which includes 15 prolines in the sequence. The excellent dispersion of resonances in the 2D 1H-15N HSQC spectrum (Fig. 1) allowed us to assign greater than 90% of the amide resonances in the 267-residue protein in the free state and P-stalk peptide complex (Li et al. 2018). Using the backbone walk and 1HN-1HN NOESY correlations we were able to assign all the observed 1H-15N correlations in the 2D N15-edited HSQC to 235 residues (93% of 252) (Fig. 1). Including the prolines, we have assignments for 97% Cα (260/267), 93% Cβ (233/250) and 88% C’ (234/267) carbon atoms, respectively. The missing and unassigned amide resonances generally belong to residues located in flexible loops or are exchange broadened due to adjacent prolines. Notably the amide resonances of nearly all residues at functionally important sites could be assigned except for Glu177 in the active site.
Methyl groups from hydrophobic sidechains are excellent probes of conformational flexibility in large proteins, and useful for monitoring binding induced structural changes. By combining intra-residue carbon-carbon spin correlations from 3D (H)CCH-TOCSY with through space NOE based connectivities, we were able to assign 93% of 125 methyl groups from Ala (26 Cβ), Ile (22/23 Cδ1), Leu (37/44 Cδ1 and Cδ2), Met (4 Cε) and Val (27/28 Cγ1 and Cγ2).
The backbone (N, C’, Cα, Cβ) chemical shifts were analyzed using TALOS-N (Shen and Bax 2015) and the secondary structure predictions compared to the X-ray structure of RTA (Mlsna et al. 1993) (Fig. 2a). The random coil index order parameter (RCI-S2) is a useful parameter to identify relatively dynamic regions of the backbone with values ranging between S2 = 0 (disordered) to S2 = 1 (rigid structure). The TALOS secondary structure predictions are broadly consistent with the αβ-fold present in the N-terminal domain (residues 1-200, NTD) linked to the smaller C-terminal domain (CTD) where the P stalk binding site is located. The opening between the two domains encloses the active site of the enzyme lined by residues important for docking the SRL rRNA sequence (Marsden et al. 2004). Compared to the well-defined core of the protein, the most variable region (0.6 < RCI-S2 < 0.8) is the unstructured loop linking α1 and β12 (residues 33 to 52). The tip of the loop extends across the active site and makes contacts with both α10 at the P-stalk binding site and CTD (Fig. 3c). Notably the P-stalk binding site scaffold is not as well defined as observed in the X-ray structures with increased backbone variation in β31 compared to the more stable β32 strand. The remaining C-terminal backbone is essentially random coil, with the notable exception of residues Ile251 to Leu254 which adopts a strand-like extended conformation (~ 80%) stabilized by interactions with the hinge (Phe181 - Tyr183) flanked by α8 and α9 helices.
To probe the extent of binding induced conformational changes in RTA triggered by the P-stalk peptide, amide (Fig. 3a) and methyl (Fig. 3b) resonances of the protein in complex with the P11 peptide were reassigned. The results from the TALOS analysis of the backbone chemical shifts (N, Cα, C') is plotted in Fig. 2b. The CSP profile (Fig. 2c) is mapped on the three-dimensional structure (Fig. 3c). Some of the largest chemical shift perturbations are clustered in the C-terminal domain which can be rationalized by significant structural rearrangement at the intermolecular contact surface in the complex. These changes are relayed to the N-terminal domain where long-range effects are observed in α1, α3, α8-α10 and loops within the active site (Tyr80, Tyr123, Glu177 and Arg180) (Fig. 3d) and the nucleotide binding site (Marsden et al. 2004).
The remodeling of the CTD is well supported by the TALOS results (Fig. 2a and b), which reflect a shift in the secondary structure preferences at the P-stalk site without concomitant changes in the RCI-S2 profiles. In the NTD, localized changes are detected in the Thr33 - Pro52 loop and β14-β15 strands. A notable dip in RCI-S2 values in the α8-α9 hinge region (Phe181 - Tyr183) can be directly linked to the interaction (Fig. 3e) with the C-terminal residues 251IIAL254 reconfigured in the complex (Fig. 2b). The proximity of the active site (Glu177 and Arg180) to the α8-α9 hinge residues (Fig. 3d), suggests this interaction with the pliable C-terminal domain could be mechanistically important and warrants further investigation.
To summarize the conclusions, we have achieved near complete backbone and side-chain methyl assignments of RTA in the presence and absence of the P-stalk peptide. The secondary structure analysis and CSP mapping presents clear evidence of coupling between the P-stalk site and the active site. The data indicates that the core domain structure is unaffected by the P-stalk interaction, but the selective rearrangement of a subset of residues agrees with the presence of an allosteric network which mediates communication between physically distant sites (Gianni and Jemth 2023). These initial results will be the cornerstone of further studies characterizing the site-specific structural and dynamic response of various ligands with the goal of understanding the functional role of conformational flexibility in allosteric regulation of the enzyme.
Data availability
The plasmids used for producing the protein are available upon reasonable request from the authors and the chemical shifts have been deposited with the BioMagResBank (https://bmrb.io/) under entry number 52271.
References
Alderson TR, Kay LE (2021) NMR spectroscopy captures the essential role of dynamics in regulating biomolecular function. Cell 184:577–595. https://doi.org/10.1016/j.cell.2020.12.034
Amukele TK, Schramm VL (2004) Ricin A-chain substrate specificity in RNA, DNA, and hybrid stem-loop structures. Biochemistry 43:4913–4922. https://doi.org/10.1021/bi0498508
Barbier J, Gillet D (2018) Ribosome inactivating proteins: from Plant Defense to treatments against Human Misuse or diseases. Toxins (Basel) 10:160–163. https://doi.org/10.3390/toxins10040160
Cavanagh J, Skelton N, Fairbrother W, Rance M, Palmer AG (2006) Protein NMR Spectroscopy: Principles and Practice, Second ed., Elsevier
Chiou JC, Li XP, Remacha M, Ballesta JP, Tumer NE (2008) The ribosomal stalk is required for ribosome binding, depurination of the rRNA and cytotoxicity of ricin A chain in Saccharomyces cerevisiae. Mol Microbiol 70:1441–1452. https://doi.org/10.1111/j.1365-2958.2008.06492.x
Curr Opin Chem Biol 12: 674–683. https://doi.org/10.1016/j.cbpa.2008.08.037
Czajka TF, Vance DJ, Davis S, Rudolph MJ, Mantis NJ (2022) Single-domain antibodies neutralize ricin toxin intracellularly by blocking access to ribosomal P-stalk proteins. J Biol Chem 298:101742–101756. https://doi.org/10.1016/j.jbc.2022.101742
Dai J, Zhao L, Yang H, Guo H, Fan K, Wang H, Qian W, Zhang D, Li B, Wang H, Guo Y (2011) Identification of a novel functional domain of ricin responsible for its potent toxicity. J Biol Chem 286:12166–12171. https://doi.org/10.1074/jbc.M110.196584
Endo Y, Tsurugi K (1987) RNA N-glycosidase activity of ricin A-chain. Mechanism of action of the toxic lectin ricin on eukaryotic ribosomes. J Biol Chem 262:8128–8130. https://doi.org/10.1016/S0021-9258(18)47538-2
Evenas J, Tugarinov V, Skrynnikov NR, Goto NK, Muhandiram R, Kay LE (2001) Ligand-induced structural changes to maltodextrin-binding protein as studied by solution NMR spectroscopy. J Mol Biol 309:961–974. https://doi.org/10.1006/jmbi.2001.4695
Gianni S, Jemth P (2023) Allostery frustrates the experimentalist. J Mol Biol 435:167934–167941. https://doi.org/10.1016/j.jmb.2022.167934
Grela P, Szajwaj M, Horbowicz-Drozdzal P, Tchorzewski M (2019) How Ricin damages the ribosome. Toxins (Basel) 11:241–256. https://doi.org/10.3390/toxins11050241
Jasheway K, Pruet J, Anslyn EV, Robertus JD (2011) Structure-based design of ricin inhibitors. Toxins (Basel) 3:1233–1248. https://doi.org/10.3390/toxins3101233
Keller RLJ (2004) Optimizing the process of nuclear magnetic resonance spectrum analysis and computer aided resonance assignment. ETH Zurich Thesis No. 15947, Switzerland
Korostelev A, Ermolenko DN, Noller HF (2008) Structural dynamics of the ribosome
Kulczyk AW, Sorzano COS, Grela P, Tchorzewski M, Tumer NE, Li XP (2023) Cryo-EM structure of Shiga toxin 2 in complex with the native ribosomal P-stalk reveals residues involved in the binding interaction. J Biol Chem 299:102795–102807. https://doi.org/10.1016/j.jbc.2022.102795
Legler PM, Brey RN, Smallshaw JE, Vitetta ES, Millard CB (2011) Structure of RiVax: a recombinant ricin vaccine. Acta Crystallogr D Biol Crystallogr 67:826–830. https://doi.org/10.1107/S0907444911026771
Li XP, Grela P, Krokowski D, Tchorzewski M, Tumer NE (2010) Pentameric organization of the ribosomal stalk accelerates recruitment of ricin a chain to the ribosome for depurination. J Biol Chem 285:41463–41471. https://doi.org/10.1074/jbc.M110.171793
Li XP, Kahn PC, Kahn JN, Grela P, Tumer NE (2013) Arginine residues on the opposite side of the active site stimulate the catalysis of ribosome depurination by ricin A chain by interacting with the P-protein stalk. J Biol Chem 288:30270–30284. https://doi.org/10.1074/jbc.M113.510966
Li XP, Kahn JN, Tumer NE (2018) Peptide mimics of the ribosomal P stalk inhibit the activity of Ricin A Chain by preventing ribosome binding. Toxins (Basel) 10:371–383. https://doi.org/10.3390/toxins10090371
Li XP, Harijan RK, Kahn JN, Schramm VL, Tumer NE (2020) Small molecule inhibitors targeting the Interaction of Ricin Toxin A Subunit with ribosomes. ACS Infect Dis 6:1894–1905. https://doi.org/10.1021/acsinfecdis.0c00127
Li XP, Harijan RK, Cao B, Kahn JN, Pierce M, Tsymbal AM, Roberge JY, Augeri D, Tumer NE (2021) Synthesis and structural characterization of ricin inhibitors targeting ribosome binding using fragment-based methods and structure-based design. J Med Chem 64:15334–15348. https://doi.org/10.1021/acs.jmedchem.1c01370
Maracci C, Rodnina MV (2016) Review: translational GTPases. Biopolymers 105:463–475. https://doi.org/10.1002/bip.22832
Marsden CJ, Fulop V, Day PJ, Lord JM (2004) The effect of mutations surrounding and within the active site on the catalytic activity of ricin A chain. Eur J Biochem 271:153–162. https://doi.org/10.1046/j.1432-1033.2003.03914.x
Mlsna D, Monzingo AF, Katzin BJ, Ernst S, Robertus JD (1993) Structure of recombinant ricin a chain at 2.3 A. Protein Sci 2:429–435. https://doi.org/10.1002/pro.5560020315
Noeske J, Cate JH (2012) Structural basis for protein synthesis: snapshots of the ribosome in motion. Curr Opin Struct Biol 22:743–749. https://doi.org/10.1016/j.sbi.2012.07.011
Olson MA (1997) Ricin A-chain structural determinant for binding substrate analogues: a molecular dynamics simulation analysis. Proteins 27:80–95. https://doi.org/10.1002/(sici)1097-0134(199701)27:1<80::aid-prot9>3.0.co;2-r
Palmer AG 3rd (2015) Enzyme dynamics from NMR spectroscopy. Acc Chem Res 48:457–465. https://doi.org/10.1021/ar500340a
Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. 25:1605–1612. https://doi.org/10.1002/jcc.20084
Rudolph MJ, Vance DJ, Cheung J, Franklin MC, Burshteyn F, Cassidy MS, Gary EN, Herrera C, Shoemaker CB, Mantis NJ (2014) Crystal structures of ricin toxin’s enzymatic subunit (RTA) in complex with neutralizing and non-neutralizing single-chain antibodies. J Mol Biol 426:3057–3068. https://doi.org/10.1016/j.jmb.2014.05.026
Rutenber E, Robertus JD (1991) Structure of ricin B-chain at 2.5 a resolution. Proteins 10:260–269. https://doi.org/10.1002/prot.340100310
Sattler M, Schleucher J, Greisinger C (1999) Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulse field gradients. Prog NMR Spectrosc 34:93–158. https://doi.org/10.1016/S0079-6565(98)00025-9
Shen Y, Bax A (2015) Protein structural information derived from NMR chemical shift with the neural network program TALOS-N. Methods Mol Biol 1260:17–32. https://doi.org/10.1007/978-1-4939-2239-0_2
Shi X, Khade PK, Sanbonmatsu KY, Joseph S (2012) Functional role of the sarcin-ricin loop of the 23S rRNA in the elongation cycle of protein synthesis. J Mol Biol 419:125–138. https://doi.org/10.1016/j.jmb.2012.03.016
Steitz TA (2008) A structural understanding of the dynamic ribosome machine Nat R Mol Cell Biol 9: 242–253. https://doi.org/10.1038/nrm2352
Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41:207–234. https://doi.org/10.1016/j.pep.2005.01.016
Szewczak AA, Moore PB, Chang YL, Wool IG (1993) The conformation of the sarcin/ricin loop from 28S ribosomal RNA. Proc Natl Acad Sci U S A 90:9581–9585. https://doi.org/10.1073/pnas.90.20.9581
Tanaka KS, Chen XY, Ichikawa Y, Tyler PC, Furneaux RH, Schramm VL (2001) Ricin A-chain inhibitors resembling the oxacarbenium ion transition state. Biochemistry 40:6845–6851. https://doi.org/10.1021/bi010499p
Voorhees RM, Schmeing TM, Kelley AC, Ramakrishnan V (2010) The mechanism for activation of GTP hydrolysis on the ribosome. Science 330:835–838. https://doi.org/10.1126/science.1194460
Acknowledgements
Data collected at 800MHz AVANCE III spectrometer is supported by NIH grant S10OD016432. Some of the work presented here was conducted at the Center on Macromolecular Dynamics by NMR Spectroscopy located at the NYSBC, supported by NIH NIGMS grants P41 GM118302 and RM1 GM145397. The plasmids used for sub-cloning RTA were kindly provided by COMPAÅ funded by NIH P41 GM116799. This work was supported by NIH grants R01AI072425 and R01AI178870 to N. Tumer.
Funding
NIH grant S10OD016432.
NIH NIGMS P41 GM118302.
NIH NIGMS RM1 GM145397.
NIH NIGMS P41 GM116799.
NIH NIAID R01 AI072425.
NIH NIAID R01 AI178870.
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SB acquired and analyzed the data, prepared the first draft of the manuscript and the figures. TD and MJR provided the samples. SB, MJG, MJR and NET conceived the project. All authors reviewed and approved the manuscript.
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Bhattacharya, S., Dahmane, T., Goger, M.J. et al. 1H, 13C, and 15N backbone and methyl group resonance assignments of ricin toxin A subunit. Biomol NMR Assign (2024). https://doi.org/10.1007/s12104-024-10172-8
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DOI: https://doi.org/10.1007/s12104-024-10172-8