Microtubules (MT), the main components of the cytoskeleton, are formed of polymers of α and β tubulin expressed as stable heterodimers. They organize to create a stable interphase microtubule network, and the highly dynamic mitotic spindle, during cell division. In eukaryotic cells, microtubules play crucial roles in a number of cellular functions, including cell signaling, cellular transport, morphogenesis, cell motility, and cell division [1
]. Any alteration in tubulin polymerization disrupts the cell homeostasis and leads to mitotic arrest, eventually resulting in cell death [2
]. Several natural compounds of various structures, such as colchicine, vinca alkaloids and taxanes have been found to bind to tubulin and alter MT dynamics. Hence, these compounds have demonstrated an immense potential in clinic as anti-mitotic drugs [3
]. Whereas vinca alkaloids and taxanes turned out to be effective chemotherapeutic agents and have found their way into anti-cancer treatments, colchicine and its analogues and derivatives are still in preclinical stages of development due to their high toxicity and severe side effects.
Colchicine is a plant alkaloid that was first isolated in 1820 from the leaves of Colchicum autumnale
(autumn crocus, also known as meadow saffron). Since then, colchicine has been used—and continues to be used—for the treatment of gout, familial Mediterranean fever, and pericarditis [4
]. Although it has been used as a therapeutic agent for many years, colchicine’s biological action remained unknown until 1968, when Taylor et al. recognized tubulin as its biological target [5
]. In 2004, Ravelli et al. [6
] identified a colchicine-binding site at the interface of the α-β tubulin heterodimer (Figure 1
a). While the binding site for colchicine is located between the α and β tubulin monomers, the principal interaction zone is located on the β subunit. The colchicine-binding pocket was identified within the intermediate domain of β tubulin. Three distinct regions of interaction, which include several amino acids, can be identified within a 6 Å cutoff range of the bound colchicine [7
], namely, residues 235–260, residues 310–320, and residues 350–360. The binding of colchicine to human tubulin induces important structural changes within the tubulin subunits, shifting from a straight to a curved conformation that renders the tubulin dimer assembly incompetent for microtubules. Taking the N-terminal domain of β tubulin as a reference, in going from a straight to a curved conformation, the following conformational changes occur: (I) the H7 helix of the β subunit translates, along with an important rotation of the intermediate domain; (II) the H6–H7 loop and the H6 helix protrude at the longitudinal interface between tubulin subunits [9
] (Figure 1
). The presence of tubulin dimers with curved conformations leads to the blocking of microtubules polymerization, which induces their depolymerization. The MT depolymerization triggered by the tubulin–colchicine complex is the main cause for its cytotoxicity.
In a previous paper [10
], some of the present authors analyzed the structure of tubulin expressed in the yew tree, and compared it to that expressed in Homo Sapiens
. It was concluded that the yew tree tubulin has an overall structural similarity compared to human tubulin, but there is a very significant difference in the paclitaxel-binding site. Consequently, the toxin produced by the yew tree, paclitaxel, has a much lower binding affinity for yew tree tubulin than human tubulin. In humans, paclitaxel is a cytotoxic agent used in cancer chemotherapy, but the yew tree is insensitive to it. In the present paper, we have attempted to analyze a similar situation for colchicine and the plants that produce it. Although colchicine has a high toxic effect on human cells, it does not affect its source plant. Produced as a secondary metabolite of C. autumnale
and Gloriosa superba
, colchicine is involved in these plants’ self-defense mechanisms against pathogens. Interestingly, both plants and humans express the target protein for the action of the toxin. However, only the human cells are susceptible to it. As tubulin is a highly conserved protein in eukaryotic cells, we explored the reasons for the resistance of C. autumnale
to its own toxin given the fact that, at the same time, human cells are extremely sensitive to colchicine. It was suggested that colchicine binding to C. autumnale
tubulin either does not induce conformational changes, or induces less of a conformational change. This is unlike the binding of colchicine to human tubulin, which results in an important conformational shift in the tubulin structure, that is, a shift from a straight conformation to a curved one. In order to test out this hypothesis, molecular models for both human and C. autumnale
tubulins were created. For each model, colchicine-binding affinity was estimated, and possible conformational modifications due to the binding were investigated.
This study summarizes the interaction of the β tubulin of C. autumnale with colchicine, as assessed by comparative genomics, computational studies, and molecular modeling. Comparison of the molecular model for the C. autumnale tubulin dimer with the human model showed that the resistance to colchicine is related to amino acid substitutions, leading to a weaker interaction with colchicine. Contrary to human tubulin, it is suggested that conformational changes associated with the binding of colchicine to C. autumnale tubulin do not significantly disrupt MT dynamics, thus explaining the high resistance of colchicine for C. autumnale.
Colchicine, a plant alkaloid, has an enormous pharmacological importance due to its ability to bind to tubulin and inhibit microtubule polymerization, which leads to mitotic arrest. Yet, the immense pharmacological potential of colchicine remains unfulfilled due to its severe side effects. Interestingly, while colchicine is extremely toxic for human cells, the plants that endogenously express colchicine, such as C. autumnale and G. superba, seem to be unaffected by its cytotoxicity even though they express tubulin, the biological target of colchicine. This observation suggests that there could be a method of redesigning colchicine analogues to enable differential binding between tubulin isotypes in the human body. This might allow for a reduction of side effects, while maintaining efficacy. In this paper, we performed a computational study in order to explore what might be the cause for the resistance of C. autumnale to colchicine. For this purpose, molecular modeling of C. autumnale tubulin was performed based on the transcriptomic data obtained from the Alberta 1000 Plants Initiative (1KP) database.
Tubulin is a highly conserved protein in all eukaryotic cells, although multiple tubulin isotypes are widespread among eukaryotes. In order to investigate whether the differential colchicine binding to human and C. autumnale tubulin is caused by sequence variations, we performed a sequence alignment. The results revealed a high conservation of the amino acid residues with 86% sequence identity between the C. Autumnale and the human tubulins. In addition, we compared the colchicine-binding domains in both species. Again, high sequence homology was found. The five residues differing in the colchicine-binding site between human and C. autumnale were T238C, A248S, M257L, V258I, and R359I of the β subunit. Interestingly, all of these residues were found to be common among other plants such as Taxus baccata, Podophyllum peltatum, Larrea tridentate and Catharanthus roseus. The high conservation of these residues in other plant species suggests that the differential colchicine binding to human and C. autumnale tubulin cannot be explained only by sequence variations and substitutions in the colchicine-binding site; it also requires an analysis of evolutionary trends in general.
Since sequence variation is not sufficient to explain colchicine resistance within C. autumnale, we investigated whether any difference in the colchicine-binding affinity could be observed between human and C. autumnale tubulins. For that purpose, we performed MM/PBSA and compared the binding free energies of both human and C. autumnale tubulins. It was found that colchicine has a significantly higher affinity for human tubulin than C. autumnale tubulin, even though our results indicated that colchicine is still able to bind to C. autumnale. The energy decomposition per residue revealed eight residues that are critical for colchicine binding to human tubulin, with αAla180, βLeu246, and βLeu253 having the highest energy contribution. αAla180 and αVal181 are both hydrophobic amino acids, which establish van der Waals contacts with the methoxy tropone ring (C ring) of colchicine. βCys239, βLeu246, βAla248, βLeu253, βAla314, βLys350 delimit a hydrophobic pocket in tubulin, and interact with the trimethoxy benzene ring (A ring). The thiol group of βCys239 is involved in a hydrogen bond with the methoxy group of the A ring. On the other hand, in C. autumnale, fewer residues were involved in the binding of colchicine, with a major contribution of βLeu246 and βLys350. Moreover, βLeu257 was found to be involved in the colchicine binding to C. autumnale, but not to human tubulin. Moreover, position 180 in C. autumnale is associated with serine, while it is associated with alanine in human tubulin. However, this substitution does not seem to modify colchicine binding, since both amino acids establish van der Waals contacts with the same strength. Another variation occurs at position 248. In human tubulin, βAla248 turns out to be a key residue for colchicine binding, whereas βSer248 in C. autumnale tubulin does not seem to be involved. These amino acids are located in the hydrophobic pocket of β tubulin. Alanine, being a hydrophobic amino acid, contributes to hydrophobic interactions with the A ring of colchicine. On the contrary, serine, as a polar amino acid with a hydroxyl group, causes local destabilization in the hydrophobic pocket. Surprisingly, βCys239 was not involved in C. autumnale, which suggests a less stable interaction between the A ring of colchicine and β tubulin.
In order to estimate the flexibility of tubulin residues in the presence or absence of colchicine, we looked into the atomic fluctuations of each residue. When analyzing the human tubulin fluctuations, the presence of colchicine induces a higher flexibility of the residues of the β subunit, which are located at its interface within the α subunit. This result confirms the conformational change in human tubulin caused by the binding of colchicine. On the contrary, no significant changes in the residues’ flexibility were observed in C. autumnale
, both in the presence and absence of colchicine. This indicates that C. autumnale
tubulin is more rigid than human tubulin when bound to colchicine, and also undergoes fewer conformational changes. These results were also confirmed by the superposition of the structures bound and unbound to colchicine. The minor shift in the intermediate domain of C. autumnale
β tubulin indicates slight changes in tubulin conformation. Taken together, these findings indicate that colchicine is able to bind to tubulin in C. autumnale
in the identified colchicine-binding domain, but with a weaker affinity when compared to human tubulin. This low affinity is mostly due to weak interactions involving the A ring of colchicine, which is known to be a critical pharmacophore in the binding to tubulin. Analogues and derivatives having the same A ring as colchicine, such as podophyllotoxin and cornigerine, have proven the crucial role of the A ring in this interaction [19
]. Weak interactions of the A ring in C. autumnale
contribute to a decrease in the total binding energy of colchicine, thus resulting in fewer conformational changes in the tubulin structure. These slight conformational changes can be viewed as the result of forming reversible pre-equilibrium complexes without evolving to a stable tubulin–colchicine complex [20
]. Moreover, these conformational changes seem not to alter the microtubules polymerization and their dynamics. In order to further investigate the different conformational changes in tubulin induced by colchicine binding, principal component analysis (PCA) can be performed in the future [21
]. However, this is outside the scope of the present study.
An experimental assay on binding affinity would be necessary to confirm the binding free energy calculations. Since βSer248 seems to cause destabilization in the hydrophobic binding pocket, experiments with βAla248 mutated to βSer248 in human tubulin might give a better understanding of the binding site and its interaction with the A ring. It may also be interesting to investigate whether βSer248 in C. autumnale tubulin includes any post-translational modifications, such as phosphorylation.
In conclusion, this study reported on the features of the C. autumnale tubulin protein and its colchicine-binding site. It provides a better understanding of the interaction of colchicine and its target tubulin in plants endogenously expressing colchicine, and broadens our understanding of this pharmacologically important drug–protein interaction. Since colchicine-binding site agents are examples of tubulin-binding agents, which have not yet reached clinical applications in cancer treatment, it is crucial to understand the biological relevance of the colchicine-binding site. Colchicine has an immense but as yet unrealized potential as an antimitotic drug. The dozens of colchicine derivatives and analogues under development should soon result in clinical trials leading to novel anti-cancer therapeutics. Understanding what types of differences provide resistance against this toxin can aid in the understanding of drug resistance in human cancers as well as contribute to the creation of more specific and more efficacious drugs that bind to the same binding site as colchicine. In a future publication, we intend to examine a similar relation between the tubulin structures expressed by all remaining plants that produce toxins, and the binding affinity for these toxins. We intend this future study to validate the generalization of the hypothesis posed in the present study, that the binding sites are suitably modified to lower the affinity for its own toxic agents. We have seen this in the case of colchicine and C. autumnale and G. superba, as well as in the case of paclitaxel and the yew tree.