Structure Molecular Function And Toxicology Of Ricin Chemistry Essay


Ricin is a protein which is isolated from castor bean plant commonly known as Ricinus communis. Ricin is a Lectin which belongs to ribosome-inactivating proteins (Type2) that block protein synthesis in normal eukaryotic cells.

It can be extracted easily from castor beans or from left over waste mash. Ricin is a heterodimer, where one chain contributes in attaching to the cell membrane, while other chain plays a key role in causing toxicity. The special feature of Ricin toxin is retrograde transport to the cytosol (completely reverse to that of normal protein transport to cell membrane, i.e from cell membrane to Golgi complex, then to endoplasmic reticulum and then to cytosol). Since ricin falls in ribosome inactivating proteins group it shares common mechanisms with other group members like Abrin, Shiga toxin. etc.

Toxicity of ricin has been known from long, but it came into light during death of famous Bulgarian novelist Georgi Ivanov Markov. He was poisoned to death by ricin in the form of pellet, which was shot with the help of Umbrella (umbrella gun).Ricin was used as cluster bombs or as a toxic dust during world wars, and now it has been listed as potential bio terror agent.

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Since Structure of ricin is studied extensively, main current research work is going on to find specific vaccine/antidotes against ricin and to potentially use ricin as anti-cancer agent.

Ricin Structure

It consists of 2 glycoprotein chains, RTA (Ricin Toxin A; 32kDa) and RTB (Ricin Toxin B; 34KDa) consisting of 267 and 262 amino acid residues respectively. The two chains are held together by disulphide bond links between 259 residue of RTA and 4 residue of RTB.

Fig 1-Ricin Heterodimer with RTA on upper right and RTB on Bottom Left

RTA is more like a globular protein and is approximately 55Å x 45Å x 35Å thick. RTA consists of 3 structural domains being 30% helical with 7 alpha helices; It also has 15% beta structure which is made up of 5 strands of beta. First domain is made of beta sheet, second domain is composed of residues 118-210 and is dominated by 5 alpha helices, third domain is made of residues 211-267, is like a compact disc like domain which interacts with first two domains and RTB. RTA has a prominent cleft at the interface between all the three domains, which is postulated to be the active site cleft. It is also called as latent active cleft when it is in heterodimer form along with RTB, which partially blocks this active site, which is known to interact with sarcin-ricin loop in ribosome causing depurination and thereby causing ribosome inactivation.

RTB is a product of gene duplication. It consists of 2 domains. Each domain has similar topologies and contain Lactose binding site (shown as pair of circles in fig. 1). By X-ray crystallographic studies it has been seen that galactose of lactose lies in shallow pocket. This pocket/cleft is created by a three residue peptide kink on the bottom, aromatic ring (Try-37 domain 1 & Try 248 in domain 2) at top. At back of cleft in domain 1; C3 and C4 hydroxyl of galactose form hydrogen bond with Asparagine (Asn-46) and Lysine (Lys-40) the key Asparagine residue is held in position by hydrogen bond to Aspartate (Asp-22), Glutamine (Gln-35) forms hydrogen bond with the C4 alcohol of galactose. In domain 2; C3 and C4 form hydrogen bond with Asn-255 and a water molecule bond (analogous to lysine in domain 1) to Asn-255. Asn-255 is stabilised by Asp-234, but there is no analog of glutamine in domain 2. The long arm of around 8-10 residues from amino terminal of B chain interacts with carboxy terminal of A chain (as shown in fig 1).

[Protein Databank Entry of Ricin 2AAI (Structure at 2.5Å)].

Molecular mechanism of Ricin Toxin

Ricin is poisonous if inhaled or injected. In contrary, it is less toxic when taken orally. It leads to toxic effect by the following routes of entry to eukaryotic cells

1) Endocytosis

2) Retrograde Transport

3) Ribosome Inactivation

1) Endocytosis

RTB chain of Ricin usually forms Hydrogen bonds with Carbohydrate moieties present on cell membrane. Ricin is known to bind galactose with its two domains present on RTB, but Ricin also binds to mannose in absence of galactose on cell membrane in some cell types.

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Once bound to membrane, entry into cell is driven by following processes

According to our study we were able to find 3 mechanisms which lead to ricin endocytosis

a) Clathrin Dependent

Clathrin is a protein which enhances the formation of small vesicles (membrane bound) in the cytoplasm; clathrin coated vesicles help in selective transport of molecules to trans-golgi network.

This process dependent on cholesterol.

b) Caveolae

This are small invaginations in plasma membrane (specialised invaginated type of raft), also known as lipid rafts. This flask shaped structures are rich in cholesterol and have known to play major role in endocytosis.

c) Cholestrol independent

There have been postulates that these pathways are dependent on protein kinase A, heterotrimeric G proteins and Cyclo-oxgenase pathway.

Fig 2. Ricin transport route in a Cell.

2) Retrograde Transport of Ricin

This transport process is broken down into further 2 processes

Golgi apparatus to ER

Ricin is often transported from early endosomes to golgi complex, and it has been postulated that this pathway is dependent on Rab9 and a protein called Dynamin.

This is assumed to be based on 2 pathways

a) Based on KDEL sequence

It's a C-terminal sequence (Lys-Asp-Glu-Leu), signal for permanent retention of proteins in ER.

The toxins bind to KDEL receptors that cycle between golgi and ER, thus by normal mode toxins are retrieved back to the ER in the same manner as cellular KDEL tagged sequences.

2) Calreticulin

Ricin has been thought to bind protein called calreticulin, a storage protein associated with ER, with the help of RTB. (RTB binds to terminal galactose residue on calreticulin). Catreticulin have been known to carry KDEL sequence.

From the ER Lumen to Cytosol.

This transport is based on Sec61p protein complex, Sec61p is common translocon present on ER, it is known to be involved in retrograde transport of incorrectly folded proteins back in cytosol, where they are degraded by proteosomes (ricin is stable in cytosol, due to less lysine residues, which decrease the activity of proteosomes).

For ricin to act as a potent toxin, RTA must be cleaved from RTB; but exact site and process of this is not understood clearly. But it has been stated that RTA is recognised as a misfolded protein by some chaperones involved in ERAD (Endoplasmic reticulum associated protein degradation pathway), which transport RTA out for degradation.

3) Ribosomes Inactivation

Subunit A also called as Ribosomal RNA N-glycosidase removes a specific adenine from 28S-rRNA. RTA chain is known to cleave glycosidic bonds within rRNA. Ricin targets highly conserved sequences of 12 nucleotides commonly present in almost all eukaryotic ribosomes.

The sequence, 5'-AGUACGAGAGGA-3', also is known as Sarcin-ricin loop plays a very important role in protein synthesis, by providing binding sites to elongation factors. Depurination of Adenine in these sequences inactivates protein synthesis.

Depurination Reaction

Several amino acids are involved in this step

Tyr-80, Tyr-123, Glu-177 and Arg-180.

1) RTA active site binds to Sarcin -Ricin loop, stacking against Tyr80 and Tyr 123.

2) Arginine protonates N-3 adenine, leading to bond break between N-9 of Adenine and C-1 of ribose.

3) Above step leads to formation of oxycarbonium on ribose, this is stabilised by Glu-177 present on ricin

4) Protonation of Arg-180 of ricin and N3 of Adenine leads to deprotonation of nearby water molecule, from where hydroxyl group attacks on ribose carbonium ion.

5) This leads to depurination of Ribose, which is intact in phosphodiester backbone.

Ricin Antidotes

At present there are no antidotes for Ricin, but there are several potential inhibitors which can be used in future as antidotes against ricin.

We can classify Ricin Antidotes into three subclasses where inhibition can be done by using purines or pterin group or by a pyramidine.


Purines are natural inhibitors of Ricin, since ricin acts on depurination in ribosome. So we are not focusing much on this group

Pterin Group

Pterioc Acid

Tautomers of Pterin exist with Ricin, usually it can form 3/6 and 4/2 strong and weak hydrogen bonds respectively.

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Usually Pterin is sandwiched between the rings of Tyr-80 and Tyr-123.

Hydrogen bond formation takes place between

- Carbonyl oxygen atoms of Gly-121 and 2-amino group of Pterin

- N3 of pterioc acid forms two hydrogen bonds with Val-81 and Ser-176

- Arg-180 forms two hydrogen bonds at the 4-oxo and N5 positions.

Due to Benzoate group PTA bends around side-chain of Tyr-80 by making some non-polar interactions, benzoate group is held by hydrogen bonds from carboxylate group and Asn-78 on one side, and one from water molecule which is bound to Arg-258 of RTA.

Fig 3a Fig 3b

Figure 3b-Chemical Structure of Pterin,

3a-Pterin -Ricin complex (Pterin bound to RTA chain of Ricin)

[Protein data bank entry for Ricin-pterin is 1BR6].


The orientation of the pterin ring is similar to that seen for PTA. One difference is the

bonding of Arg180 to the inhibitor. It bonds with the 4-oxo group of neopterin, but does not bond to N5 as occurs in the PTA complex. Instead, a second bond is formed with the proximal hydroxyl of the propane triol moiety; associated with this rearrangement is a 7 rotation of the pterin ring. The other atoms of propane triol moiety of the neopterin does not make any interaction with RTA. Since the propane triol moiety of neopterin is much smaller than the corresponding substituents in PTA, it does not interact with Tyr80 in the same way and appears to lack the van der Waals contribution to binding which might be expected in PTA.

[Protein data bank entry for Ricin-neopterin is 1BR5].


PBA {4-[3-(2-Amino-1,4-dihydro-6-hydroxy-4-oxo-5-pyrimidinyl) propyl]-benzoic acid} binds to Ricin in a very similar way to that of purines and pterins.

Aromatic ring of PBA is stacked between side chains of Try-80.

Hydrogen bonds are formed between :

Exocyclic amino group of PBA with carbonyl oxygen of Val-81 and Gly-121,

N1 (one ring) and Amido group of valine

N3 (second ring) and Carbonyl oxygen of Gly-121

The pyrimidine ring of PBA usually overlaps half pterin ring, (when both structures are superimposed) but corresponding polar atoms play the same role in RTA binding.

The only difference between pterin and PBA is the binding between Arg-180and oxyanion at position 6 of PBA, which is considered stronger than the ketone at same position of of PTA.

Fig 5. PBA-Ricin complex

Comparison with Other Toxins.


It is a potent toxin also belonging to ribosome inactivating protein (RIP 2), which is obtained from seeds of Abrus precatorius (or Rosary pea). Abrin is a hetrodimer consisting of chains A and chain B with a disulfide bond between cys247 of A chain and Cys8 of B chain.

A chain is composed of 251 aminoacids and divided into 3 folding domains, B chain is composed of 268 amino acids. A chain depurinates adenine from position 4 and 324 of 28s rRNA and inhibits protein synthesis.

Abrin shares carbohydrate binding site with Ricin, nearly 60% of residues are similar to that of RICIN. B chain a lectin which binds to galactose present on the cell membrane. Abrin is regarded as more toxic then ricin (70 times).

Fig 6. Crystallographic Structure of Abrin Fig7. 3-D structure of Shiga Toxin

Shiga Toxin

Shiga toxins are AB5-toxins consisting of a pentameric binding moeity (stxB) and enzymatically active A-moeity (stxA). B-chain is a pentamer consisting of 5 small chains of 7.7kDa each, which interact non-covalently. Each B chain contains a binding site of glycosphingolipid gb3.

A chain is of 32.2kDa and is attached to B chain with help of Disulfide bond between cysteines 242-261.

Shiga Toxin and Ricin share very common structure, as in, both have Lectin as one of there subunits which specifically binds to carbohydrate moiety on cell membrane, during internationalisation both the subunits are detached from themselves so as to release N-glycosidase enzyme which will specially act on Ribosome. Both toxins have been known to follow the same retrograde transport to reach cytosol and depurinate ribosome.

Ricin as an Anti-Cancer Drug and other Medicinal uses.

Work is being carried out to use ricin as an anti-cancer drug. Ricin can induce apoptosis in Human Cervical Cancer cells (HeLa) but the exact mechanism is very poorly understood and studies are going on with the use of RTA/RTB labeled antibodies.

The foreign molecule has been usually fused to the N-terminal domain of RTB to avoid steric hindrance by the antigen with RTB galactose receptor binding sites. Research has demonstrated that ricin toxin B subunit is an excellent candidate to enhance immunogenicity at the i.p. and i.n. routes of HIV, and probably other, antigens, and potentially to boost cytotoxic T-lymphocyte responses in the context of mucosal protection, a major requirement for a potential HIV vaccine candidate.


Our study shows that Structure of Ricin has been studied extensively; postulates supporting the mode of transport and toxicity have also been established well.

During comparison of Ricin with other toxins (Abrin and Shiga toxin) from same family or RIP (type2), we found out that there is high resemblance in there structural topology, mode of entry into cell and mechanism of inactivating ribsomes(by depurination of adenine in sarcin-ricin loop of ribosome).

At present, we did not find any antidotes for ricin, but we have presented some potential antidotes like PBA and Pterins, among the two we did find out that PBA binds more efficiently to Ricin (blocking completely its active site)

Ricin protein came to light as toxin, but studies have shown that it can induce apoptosis in highly dividing cells (Cancer cells) but the exact mechanism and reason for this is yet to be explored but we suggest some way where ricin can be used in countering cancer cells:

There are some instances where RTB has been used successfully as a carrier fused to with other molecules; the use of affibody technology and nano sized carrier system increases the selectivity of ricin. Affibody molecules are small in size, highly specific, highly stable, and easily engineered and have a high degree of folding and they are used in identifying tumors due to their high selectivity. Therefore, toxin conjugated affibody can be used (ricin conjugated affibody) to increase the efficiency of drug delivery system in targeting cancer cells. Also, targeting ricin can be enhanced by nano sized carrier system through passive targeting by binding ricin on carrier complexes where they are attracted to tumor receptors according to a certain property like pH taking advantage from the acidic medium of the micro-environment of cancer unlike the pH normal cells, solving the internalization problem and increasing selectivity.


In our study we have found that Structure, mode of action of Ricin has been established, but many aspects like regulation of endocytic pathway, retrograde transport and transport intermediates need to be researched with the special focus. Ricin antidotes and other possible medicinal applications of ricin like in the cancer and all should be concentrated.