Differential colchicine-binding across eukaryotic families: The role of highly conserved Pro268 [beta] and Ala248 [beta] residues in animal tubulin (original) (raw)
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Competitive inhibition of colchicine binding to tubulin by microtubule-associated proteins
Proceedings of the National Academy of Sciences, 1979
Microtubule-associated proteins (MAPs) promote tubulin polymerization, whereas colchicine inhibits this process. In this paper, MAPs have been shown to inhibit colchicine binding to tubulin in a competitive manner. Attempts were made to identify which of the MAPs fraction(s) was responsible; both tau protein (a thermostable molecule with a molecular weight of approximately 70,000) and a high molecular weight fraction (HMW) were able to compete with colchicine. In contrast, Mg2+, which also induces microtubule assembly in vitro, had no effect on colchicine binding to tubulin.
Characterization of the Colchicine Binding Site on Avian Tubulin Isotype βVI
Biochemistry, 2010
Tubulin, the basic component of microtubules, is present in most eukaryotic cells as multiple gene products, called isotypes. The major tubulin isotypes are highly conserved in terms of structure and drug binding capabilities. The tubulin isotype βVI, however, is significantly divergent from the other isotypes in sequence, assembly properties and function. It is the major β-tubulin isotype of hematopoietic tissue and forms the microtubules of platelet marginal bands. The interaction of the major tubulin isotypes βI, βII, βIII and βIV with antimicrotubule drugs has been widely studied, but little is known about the drug binding properties of tubulin isotype βVI. In this investigation, we characterized the activity of various colchicine-site ligands with tubulin isolated from Gallus gallus erythrocytes (CeTb), which is ~95% βVI. Colchicine binding is thought to be a universal property of higher eukaryotic tubulin; however, we were unable to detect colchicine binding to CeTb under any experimental conditions. Podophyllotoxin and nocodazole, other colchicine-site ligands with divergent structures, were able to inhibit paclitaxel-induced CeTb assembly. Surprisingly, the colchicine isomer allocolchicine also inhibited CeTb assembly and displayed measurable, moderate affinity for CeTb (Ka = 0.18 × 10 5 M −1 vs. 5.0 × 10 5 M −1 for bovine brain tubulin). Since allocolchicine and colchicine differ in their C ring structures, the two C-ring colchicine analogues were also tested for CeTb binding. Kinetic experiments indicate that thiocolchicine and chlorocolchicine bind to CeTb, but very slowly and with low affinity. Molecular modeling of CeTb identified five divergent amino acid residues within 6 Å of the colchicine binding site compared to βI, βII, and βIV; three of these amino acids are also altered in βIII-tubulin. Interestingly, the altered amino acids are in the vicinity of the A ring region of the colchicine binding site rather than the C ring region. We propose that the amino acid differences in the binding site constrict the A ring binding domain in CeTb, which interferes with the positioning of the trimethoxyphenyl A ring and prevents C ring binding site interactions from efficiently occurring. Allocolchicine is able to accommodate the altered binding mode because of its smaller ring size and more flexible C ring substituents. The sequence of the colchicine binding domain of CeTb βVI-isotype is almost identical to that of it human hematopoietic counterpart. Thus, through analysis of the interactions of ligands with CeTb, it may be possible to discover colchicine site ligands that specifically target tubulin in human hematopoietic cells. ., sbane@binghamton.edu. SUPPORTING INFORMATION AVAILABLE The inhibition of paclitaxel induced CeTb assembly by allocolchicine, an illustration of three dimensional alignment of tubulin-bound colchicine and podophyllotoxin, and sequence alignment of chicken erythrocyte βVI and human β1 tubulin. This material is available free of charge via the internet at
Variations in the colchicine-binding domain provide insight into the structural switch of tubulin
Proceedings of The National Academy of Sciences, 2009
Structural changes occur in the ␣-tubulin heterodimer during the microtubule assembly/disassembly cycle. Their most prominent feature is a transition from a straight, microtubular structure to a curved structure. There is a broad range of small molecule compounds that disturbs the microtubule cycle, a class of which targets the colchicine-binding site and prevents microtubule assembly. This class includes compounds with very different chemical structures, and it is presently unknown whether they prevent tubulin polymerization by the same mechanism. To address this issue, we have determined the structures of tubulin complexed with a set of such ligands and show that they interfere with several of the movements of tubulin subunits structural elements upon its transition from curved to straight. We also determined the structure of tubulin unliganded at the colchicine site; this reveals that a -tubulin loop (termed T7) flips into this site. As with colchicine site ligands, this prevents a helix which is at the interface with ␣-tubulin from stacking onto a -tubulin  sheet as in straight protofilaments. Whereas in the presence of these ligands the interference with microtubule assembly gets frozen, by flipping in and out the -subunit T7 loop participates in a reversible way in the resistance to straightening that opposes microtubule assembly. Our results suggest that it thereby contributes to microtubule dynamic instability.
The carboxy terminus of the .alpha. subunit of tubulin regulates its interaction with colchicine
Biochemistry, 1990
Controlled proteolysis of goat brain tubulin by subtilisin was carried out to investigate regulatory aspects of the binding of colchicine to tubulin. Tubulin S, obtained by the cleavage of the carboxyl termini of both the CY-and P-subunits of tubulin by subtilisin, exhibited the following differences compared to native tubulin: (a) Reaction with colchicine, which has an optimum pH of 6.8, becomes independent of p H (in the range 5.7-8.0). (b) The colchicine-binding site, which is labile at 37 "C (tl12 = 4-5 h), becomes highly * To whom correspondence should be addressed. Research, Government of India.
Structural Changes, Biological Consequences, and Repurposing of Colchicine Site Ligands
Biomolecules
Microtubule-targeting agents (MTAs) bind to one of several distinct sites in the tubulin dimer, the subunit of microtubules. The binding affinities of MTAs may vary by several orders of magnitude, even for MTAs that specifically bind to a particular site. The first drug binding site discovered in tubulin was the colchicine binding site (CBS), which has been known since the discovery of the tubulin protein. Although highly conserved throughout eukaryotic evolution, tubulins show diversity in their sequences between tubulin orthologs (inter-species sequence differences) and paralogs (intraspecies differences, such as tubulin isotypes). The CBS is promiscuous and binds to a broad range of structurally distinct molecules that can vary in size, shape, and affinity. This site remains a popular target for the development of new drugs to treat human diseases (including cancer) and parasitic infections in plants and animals. Despite the rich knowledge about the diversity of tubulin sequences...
Interactions of long-chain homologues of colchicine with tubulin
European journal of medicinal chemistry, 2016
Several colchicine analogues in which the N-acetyl residue has been replaced by aliphatic, straight-chain acyl moieties, have been synthesized. These compounds show high cytotoxic activity at the nanomolar level against the tumoral cell lines HT-29, MCF-7 and A549. Some of them exhibit activities in the picomolar range against the HT-29 line and are thus two to three orders of magnitude more cytotoxic than colchicine. In this specific cell line, the activities were found to be closely related to the length of the acyl carbon chain, an increase in the latter giving rise to an increase in the cytotoxicity with a maximum in the range of 10-12 carbon atoms, followed by a decrease in activity with still longer chains. Some of the compounds inhibit microtubule assembly and induce the formation of abnormal polymers and present in most cases better apparent affinity constants than colchicine. In addition, at IC50 concentrations the analogues block the cell cycle of A549 cells in the G2/M ph...
Antimitotic activity of colchicine and the structural basis for its interaction with tubulin
Medicinal Research Reviews, 2008
In this review, an attempt has been made to throw light on the mechanism of action of colchicine and its different analogs as anti-cancer agents. Colchicine interacts with tubulin and perturbs the assembly dynamics of microtubules. Though its use has been limited because of its toxicity, colchicine can still be used as a lead compound for the generation of potent anti-cancer drugs. Colchicine binds to tubulin in a poorly reversible manner with high activation energy. The binding interaction is favored entropically. In contrast, binding of its simple analogs AC or DAAC is enthalpically favored and commences with comparatively low activation energy. Colchicine–tubulin interaction, which is normally pH dependent, has been found to be independent of pH in the presence of microtubule-associated proteins, salts or upon cleavage of carboxy termini of tubulin. Biphasic kinetics of colchicines–tubulin interaction has been explained in light of the variation in the residues around the drug-binding site on β-tubulin. Using the crystal structure of the tubulin–DAMAcolchicine complex, a detailed discussion on the pharmacophore concept that explains the variation of affinity for different colchicine site inhibitors (CSI) has been discussed. © 2007 Wiley Periodicals, Inc. Med Res Rev, 28, No. 1, 155–183, 2008
Biochemistry, 2005
Isotypes of vertebrate tubulin have variable amino acid sequences which are clustered at their Cterminal ends. Isotypes bind colchicine at different on-rates and affinity constants. The kinetics of colchicine binding to purified (unfractionated) brain tubulin have been reported to be biphasic under pseudo-first order conditions. Experiments with individual isotypes established that the presence of β III in the purified tubulin is responsible for the biphasic kinetics. Since the isotypes mainly differ at the C-termini, the colchicine binding kinetics of unfractionated tubulin and the β III isotype, cleaved at the C-termini, have been tested under pseudo-first order conditions. Removal of the C-termini made no difference to the nature of the kinetics. Sequence alignment of different β isotypes of tubulin showed that besides the C-terminal region, there are differences in the main body as well. In order to establish whether these differences lie at the colchicine binding site or not, homology modeling of all β tubulin isotypes was done. We found that the isotypes differed from each other in the amino acids located near the A-ring of colchicine at the colchicine-binding site on β-tubulin. While the β III isotype has two hydrophilic residues (Serine 242 and Threonine 317) both β II and β IV have two hydrophobic residues (Leucine 242 and Alanine 317). β II has Isoleucine at position 318, while β III and β IV have Valine at that position. Thus these alterations in the nature of the amino acids surrounding the colchicine site could be responsible for the different colchicine binding kinetics of the different isotypes of tubulin.
Role of B-ring of colchicine in its binding to tubulin
The Journal of biological chemistry, 1981
The chemical specificity of the colchicine-binding site of tubulin is less stringent for the presence of the B-ring than the A- and C-rings of colchicine, Colchicine analogues with modifications in the B-ring bind to tubulin at the same site as colchicine. Analogues with smaller or no substituents in the B-ring bind tubulin remarkably faster than colchicine. Thus, a compound without the B-ring [2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone] binds tubulin even at 4 degrees C and the binding is almost instantaneous at 37 degrees C. Colcemid and 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone bind reversibly to tubulin, whereas colchicine and desacetamidocolchicine bind almost irreversibly, suggesting that the size of the B-ring moiety of colchicine is not related to the reversibility of binding. We conclude that although the presence of the B-ring of colchicine does not appear to be an essential prerequisite for the drug-tubulin interaction, the B-ring substitu...