Dataset Information

Mechanism of tubulin-colchicine recognition: a kinetic study of the binding of the colchicine analogues colchicide and isocolchicine.

ABSTRACT: Colchicide (IDE) is a colchicine (COL) analogue in which the C-10 methoxy group is replaced by a hydrogen atom. Its binding to tubulin is accompanied by a quenching of the protein fluorescence. The fluorescence decrease shows a monoexponential time dependence. The observed rate constant increases in a non-linear way with the total concentration of IDE, allowing the determination of a binding constant for an initial binding site (K1=5300+/-300 M-1) and the rate constant for the subsequent isomerization (k2=0.071+/-0.002 s-1) at 25 degrees C. The rate constant, k-2, for the reversed isomerization can be determined by displacement experiments. Despite the minor alteration of the C-ring substituent, the kinetic and thermodynamic parameters of binding are substantially different from those of COL itself, for both steps. In isocolchicine (ISO) the carbonyl oxygen atom and the methoxy groups of the C-ring have been interchanged. Its binding to tubulin only results in small fluorescence and absorbance changes. Therefore competition experiments with MTC [2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4, 6-cycloheptatrien-1-one] were performed. ISO competes rapidly and with low affinity with MTC. Fluorimetric titrations of tubulin with MDL (MDL 27048 or trans-1-(2,5 dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2-methyl-2-propen-1 -one) in the presence and absence of ISO give evidence for the existence of a second, slow-reacting low-affinity site for ISO that is not accessible to MTC or MDL. The relevance of these results for the recognition of COL is analysed.

SUBMITTER: Dumortier C

PROVIDER: S-EPMC1218844 | biostudies-other | 1997 Nov

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description: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. Tubulin isotype betaVI, however, is significantly divergent from the other isotypes in sequence, assembly properties, and function. It is the major beta-tubulin isotype of hematopoietic tissue and forms the microtubules of platelet marginal bands. The interaction of the major tubulin isotypes betaI, betaII, betaIII, and betaIotaV with antimicrotubule drugs has been widely studied, but little is known about the drug binding properties of tubulin isotype betaVI. In this investigation, we characterize the activity of various colchicine site ligands with tubulin isolated from Gallus gallus erythrocytes (CeTb), which is approximately 95% betaVI. 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 (K(a) = 0.18 x 10(5) M(-1) vs 5.0 x 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 A of the colchicine binding site compared to betaI, betaII, and betaIV; three of these amino acids are also altered in betaIII-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 isotype betaVI is almost identical to that of its 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.

Dataset Information

Mechanism of tubulin-colchicine recognition: a kinetic study of the binding of the colchicine analogues colchicide and isocolchicine.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets