Crystal structure of ricin toxin A chain complexed with a highly potent pterin-based small-molecular inhibitor

Abstract

Ricin toxin A chain (RTA), from Ricinus communis, is a deadly protein that inactivates ribosomes by degrading an adenine residue at position 4324 in 28S rRNA. Recently, we have demonstrated that pterin-7-carboxamides with peptide pendants were potent RTA inhibitors. Among these, N-(pterin-7-carbonyl)glycyl-L-tyrosine (7PCGY) is the most potent RTA inhibitor as a small organic molecule. However, despite this fascinating inhibitory activity, the mode of interaction of 7PCGY with RTA remains elusive. This study aimed to elucidate the factors responsible for the high RTA inhibitory activity of 7PCGY based on X-ray crystallographic analysis. Herein, we report the successfully resolved X-ray crystal structure of 7PCGY/RTA complexes, revealing that the interaction between the phenolic hydroxy group in 7PCGY and Asn78 of RTA through a hydrogen bonding and the conformational change of Tyr80 and Asn122 are responsible for the high RTA inhibitory activity of 7PCGY.

Keywords:

• Ricin toxin A chain
• ricin toxin A chain inhibitor
• pterin-7-carboxamide
• X-ray crystallography

Introduction

Ricin is a member of ribosome-inactivating proteins (RIPs) and is an N-glycosidase that catalyses the hydrolytic cleavage of an adenine residue at position 4324 (A₄₃₂₄) in 28S ribosomal RNA. Through this degradation, the protein-synthesizing system in the mammalian ribosome is disrupted, leading to cell death^1,2. Ricin is composed of two polypeptide chains, an A-chain and a B-chain, connected via a disulphide bond. The ricin toxin A chain (RTA) is involved in the toxic action of ricin, whereas the ricin toxin B chain (RTB) is a lectin that recognises carbohydrate residues on the cell surface to allow whole ricin to enter a cell. After entry, RTA is detached and subsequently exerts its toxic N-glycosidase activity³. From a public health perspective, the development of antidotes against RTA is a recognised necessity. The active site of RTA consists of two pockets, the primary and secondary pockets with Tyr80 dividing these pockets. In the primary pocket, the A₄₃₂₄ is captured by hydrogen bonds with Val81 and Gly121 and by π-π interactions with Tyr123 and Tyr80, and is cleaved by the catalytic action of Arg180 and Glu177. Whereas, the secondary pocket accommodates a guanine residue adjacent to A₄₃₂₄³. Based on this structural information, extensive efforts have been dedicated to developing RTA inhibitors, but most experiments have yielded unsatisfactory results^4–9. Based on this background, we recently developed several pterin-7-carboxamides (7PCAs) with peptide pendants that exhibited weak to good inhibition of RTA in vitro¹⁰. Among these, N-(pterin-7-carbonyl)glycyl-L-tyrosine (7PCGY, Figure 1) was the most potent, with an IC₅₀ value of 6 µM, followed by N-(pterin-7-carbonyl)glycyl-L-phenylalanyl-L-phenylalanine (7PCGFF, Figure 1) and N-(pterin-7-carbonyl)glycyl-L-phenylalanine (7PCGF, Figure 1), with IC₅₀ values of 15 and 20 µM, respectively. X-ray crystallographic analyses of 7PCGFF/RTA and 7PCGF/RTA cocrystals were performed, and the structural information related to their inhibitory activities was elucidated. However, the crystal structure of RTA incorporating the most potent 7PCGY has not yet been determined; therefore, the details of the interaction between RTA and 7PCGY responsible for such a high inhibitory activity (IC₅₀ = 6 µM) remain elusive. The study aimed to determine the crystal structure of 7PCGY/RTA and the mode of interaction of 7PCGY with RTA.

Figure 1. Structures of the pterin-based RTA inhibitors with peptide pendants.

Materials and methods

Synthesis

The molecule 7PCGY was synthesised by mixing 7-methoxycarbonylpterin (7MCP)¹¹ with glycyl-L-tyrosine in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in methanol at room temperature, as described previously^10,12. Spectral data for this material were in good agreement with those reported previously¹⁰.

Protein production

The pET28a plasmid containing the His-RTA expression sequence was purchased from GenScript (Tokyo, Japan) and transformed into E. coli BL21(DE3) cells following the calcium treatment method. Transformed strains were incubated for 16h at 32 °C on Luria–Bertani (LB) agar culture supplemented with kanamycin. The generated colonies were picked up using a pipet tip and inoculated into 5 L of LB medium, and then incubated at 32 °C with shaking. After approximately 6 h, the bacterial growth was visually confirmed via turbidity. Thereafter, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the medium to induce expression, and the cultivation continued for 16h at 32 °C. The bacteria were collected by centrifugation and were ultrasonically crushed to obtain a crude extract. His-tagged proteins were collected using a TALON column, dialysed against 10 mM sodium acetate buffer (pH 4.0), and concentrated using an ultrafiltration filter to obtain the target proteins. Crystallisation of RTA was subsequently performed by the hanging drop vapour diffusion method using the obtained RTA protein solution, as described below.

Crystallisation, x-ray data collection and processing, structure determination and refinement

RTA crystals were grown at 23 °C following the hanging drop method using 7.8 mg/mL protein, 11% PEG2000, 0.2 M lithium sulphate, and 0.1 M sodium acetate at pH 4.5. Crystals of the RTA complex with synthetic ligands were obtained by soaking RTA crystals in a crystallisation reservoir containing 3.0 mM of the compound.

Diffraction data of the RTA-ligand complex crystals were collected at 95 K using a PILATUS3 S 6 M detector on the beamline BL-5A at the Photon Factory (PF, Tsukuba, Japan). Diffraction images were captured every 0.50° with a total rotation range of 180°. Data with 1.60 Å resolution were integrated using XDS¹³ and subsequently processed with SCALA¹⁴ from the CCP4 suite¹⁵. The crystal belonged to the space group P4₁2₁2, with unit cell parameters of a = b = 67.80 Å, c = 141.38 Å, α = β = γ = 90°. Processed data were phased by the molecular replacement method using MOLREP¹⁶ with the coordinates of RTA with N-(N-(pterin-7-yl)carbonylglycyl)-L-phenylalanine (PDBID: 4HUO)¹⁰. Model building and electron density map inspections were performed using COOT¹⁷. Following model building, refinement was performed using REFMAC5¹⁸. Clear residual electron density was assigned to one 7PCGY, two sulphate anions, and water molecules in the final model. The model was refined to a resolution of 1.60 Å and had an R factor of 21.47% and a R_free of 23.23%. The final model was analysed using PROCHECK¹⁹, which resulted in a Ramachandran plot with 99.2% of the residues in favoured, 0.8% is allowed, and 0% in disallowed regions. The crystallographic statistics are presented in Table 1. The protein structures were prepared using CCP4MG²⁰. The atomic coordinates and structural factors were deposited in the Protein Data Bank as PDB entry 8I7P.

Table 1. Data collection, processing, and refinement statistics^†.

Diffraction source	BL-5A, KEK PF
Wavelength (Å)	1.00000
Temperature (K)	95.000
Detector	Pilatus3 S6M
Crystal-to-detector distance (mm)	287
Rotation range per image (°)	0.50
Total rotation range (°)	180
Exposure time per image (s)	2
Space group	P41212
Unit cell parameters (Å, °)	a = b = 67.80, c = 141.38,
	α = β = γ = 90
Mosaicity (°)	0.17
Resolution range (Å)	48.93–1.60 (1.69–1.60)
Total no. of reflections	571,567
No. of unique reflections	43,942
Completeness (%)	99.2 (98.3)
Multiplicity	13.0 (13.6)
〈I/σ (I) 〉	35.6 (4.1)
Rr.i.m	0.042 (0.801)
Overall B factor from Wilson plot (Å^2)	20.5
Refinement
Resolution (Å)	48.93–1.60
Rfactor (%)	21.47
Rfree (%)	23.23
Root-mean-square deviation
Bond length (Å)	0.0073
Bond angle (°)	1.1417
No. of atoms
Protein	2059
Ligand	41
Water	235
Average B factor (Å^2)
Protein	24.9
Ligand	30.2
Water	37.1
Ramachandran analysis
Favoured (%)	99.2
Allowed (%)	0.8
Outliers (%)	0

^†Values in parentheses are for the outer resolution shells indicated in each section.

Results and discussion

The inhibitor 7PCGY was synthesised using a previously reported procedure: an ester-amide exchange reaction between 7MCP and unprotected glycyl-L-tyrosine (Gly-Tyr) in the presence of DBU in methanol (Figure 2)^10,12. RTA crystals with 7PCGY were prepared under the same conditions as those for preparing the crystals of 7PCGF/RTA complexes^10,21, and an X-ray diffraction experiment with the crystals was performed. The resulting three-dimensional structure of the complex is shown in Figure 3(A). The active site of RTA consists of two binding pockets: one is the catalytic primary pocket where A₄₃₂₄ is captured and degraded, and the other is a slightly larger secondary pocket which accommodates the guanine residue adjacent to A₄₃₂₄. These two pockets are separated by Tyr80, and the A₄₃₂₄ is captured between Tyr123 and Tyr80 by aromatic interactions in the primary pocket, whereas the guanine residue forms a π-π stacking with Tyr80 to be placed in the secondary pocket³.

Figure 2. Synthesis of N-(pterin-7-carbonyl)glycyl-L-tyrosine (7PCGY).

Figure 3. X-ray crystal structures of ricin toxin A chain (RTA) with or without the pterin-based inhibitors. (A) Omit map for bound N-(pterin-7-carbonyl)glycyl-L-tyrosine (7PCGY). F_o–F_c electron density (shown with black baskets) contoured to 2σ showing evidence for 7PCGY bound to this region. The omit map was generated by removing the ligand from the structure. This image was created using PyMOL²⁷. (B) Surface representation of the 7PCGY/RTA complex. The RTA surface is shown as an atomic charge map. (C) The interactions of 7PCGY in the catalytic primary pocket of RTA observed in the crystal structure of the 7PCGY/RTA complex. Conventional hydrogen bonds are represented as cyan dashed lines, hydrogen bonds between CH and carbonyl oxygen are represented as yellow dashed lines and aromatic (π-π and T-shaped) interactions are represented as magenta dashed lines. (D) Superimposition of protein crystal structures of 7PCGY/RTA (grey) and a ligand-free RTA (PDBID: 1RTC, orange). (E) X-ray crystal structure of the 7PCGF/RTA complex (PDBID: 4HUO). Conventional hydrogen bonds are represented as cyan dashed lines, hydrogen bonds between CH and carbonyl oxygen are represented as yellow dashed lines and aromatic (π-π and T-shaped) interactions are represented as magenta dashed lines. (F) A comparison of crystal structures of 7PCGY/RTA and 7PCGF/RTA. The protein portion of 7PCGY/RTA is shown in grey and that of 7PCGF/RTA in medium blue. All image creations, except (A), and interaction detections were performed using the Discovery Studio Visualiser²⁸.

For 7PCGY, the pterin ring was bound to the catalytic primary pocket (Figure 3(B)) and interacted with Val81, Gly121, Tyr123, and Arg180 via hydrogen bonds, and with Tyr123 via an aromatic (π-π) interaction (Figure 3(C) and Table 2). These interactions were similarly observed in the X-ray crystallography of the cocrystals of all pterin-based RTA inhibitors with RTA, regardless of inhibitory activity^9,10,21–25. The phenyl ring of the tyrosine moiety in 7PCGY interacted with Trp211 in a T-shaped manner (Figure 3(C)), thus, the conformation of Tyr80 was changed from that observed for the inhibitor-free crystal structure (Figure 3(D))²⁶. Together with this movement, the position of Asn122 changed approaching Tyr80 to form a hydrogen bond, as if opening the gate to the secondary pocket. The same conformational changes in Tyr80 and Asn122 were observed with 7PCGF (Figure 3(E)) and 7PCGFF, which exhibited high RTA inhibitory activities¹⁰. Furthermore, the 7PCAs that were capable of interacting with the secondary pocket also induced the same rearrangements of Tyr80 and Asn122 upon binding to RTA^21,25. Based on these facts, it is strongly suggested that the conformational changes of Tyr80 and Asn122 are crucial for achieving high RTA inhibitory activity.

Table 2. Interactions between pterin-based inhibitors and RTA.

Residues in RTA	7PCGY (PDBID: 8I7P)		7PCGF (PDBID: 4HUO)
	Interaction type	Distance /Å	Interaction type	Distance /Å
Asn78	H-bond	3.06	–	–
Tyr80	aromatic (π-π)	3.59	aromatic (π-π)	3.50
Val81	H-bond	2.88	H-bond	2.96
Gly121	H-bond	2.75	H-bond	2.68
Asn122	^αCH-n	2.62	^αCH-n	2.66
Tyr123	aromatic (π-π) H-bond	4.24 2.82	aromatic (π-π) H-bond	4.15 2.92
Arg180	H-bond (N4 of 7PC) H-bond (5-CO of 7PC)	3.12 2.84	H-bond (N4 of 7PC) H-bond (5-CO of 7PC)	3.07 2.86
Asn209	^αCH-π (ligand’s CO) CH-π (chain CO)	2.54 2.48	^αCH-π (ligand’s CO) –	2.60 –
Trp211	aromatic (T-shaped)	4.86	aromatic (T-shaped)	4.89

Although 7PCGY and 7PCGF differ only in the presence or absence of a hydroxy group on the phenyl group, the RTA inhibitory activity of 7PCGY is approximately three times higher than that of 7PCGF. When the RTA structure of 7PCGY/RTA was superimposed on that of 7PCGF/RTA, the two structures were perfectly comparable (Figure 3(F)). The positions of the pterin ring in 7PCGY and 7PCGF were completely overlapped, whereas the remaining dipeptide moieties in 7PCGY and 7PCGF were slightly offset from each other. A closer examination of the crystal structure of the 7PCGY/RTA complex revealed that the phenolic hydroxy group in 7PCGY interacted with Asn78 of RTA (Figure 3(C) and Table 2). This interaction anchored the tyrosine moiety of 7PCGY, which consequently anchored the arrangement of the other portions of 7PCGY, thereby forming an additional interaction with Asn209 (Figure 3(C) and Table 2). These extra interactions were not observed in 7PCGF (Figure 3(E) and Table 2) and probably led to the higher RTA inhibitory activity of 7PCGY than that of 7PCGF. These results indicated the conformational changes of Tyr80 and Asn122, and that further interactions between an inhibitor and RTA are required to obtain a high inhibitory activity against RTA. Thus, the factors responsible for the high RTA inhibitory activity of 7PCGY have been elucidated, achieving the objective of this study. These findings will facilitate the development of compounds with even higher RTA inhibitory activity.

Conclusion

In the present study, the previously unknown structure of 7PCGY-RTA complex was unambiguously determined using X-ray crystallography. Comparison of the crystal structure of the previously reported 7PCGF-RTA complex with that of the present 7PCGY-RTA complex reveals that the hydrogen bonding between the phenolic hydroxy group in 7PCGY and Asn78 of RTA and the conformational change of Tyr80 and Asn122 are responsible for the high RTA inhibitory activity of 7PCGY. The findings of this study will guide future optimisation of pterin-based RTA inhibitors, with the aim of increasing the inhibitory activity.

Ethical statement

This study was performed with the approval of the Photon Factory Program Advisory Committee (proposal No. 2018G520 and 2022G603).

Acknowledgement

We would like to thank the staff of the Photon Factory for their assistance with the data collection. The authors would also like to thank Editage (www.editage.jp) for the English language editing.

Disclosure statement

The authors report no conflicts of interest.

Additional information

Funding

This research was supported by JSPS KAKENHI Grant Number 19K05699 and Japan Society for the Promotion of Science.