The Colchicine-Binding and Pyrene-Excimer-Formation Activities of Tubulin Involve a Common Cysteine Residue in the beta Subunit (original) (raw)
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Identification of cysteine 354 of-tubulin as part of the binding site for the A ring of colchicine
1996
The colchicine analog 3-chloroacetyl-3-demethylthiocolchicine (3CTC) is a competitive inhibitor of colchicine binding to tubulin, binds to tubulin at 37°C, but not at 0°C, and covalently reacts with -tubulin at 37°C, but not at 0°C, in a reaction inhibited by colchicine site drugs. The approximate intramolecular distance between the oxygen at position C-3 in 3CTC and the chlorine atom of the 3-chloroacetyl group is 3 Å. Using decylagarose chromatography, we purified -tubulin that had reacted with 3-(chloromethyl-[ 14 C]carbonyl)-3-demethylthiocolchicine ([ 14 C]3CTC). This -tubulin was digested with formic acid, cyanogen bromide, endoproteinase Glu-C, or endoproteinase Lys-C, and the radiolabeled peptide(s) were isolated. The sequences of these peptides indicated that as much as 90% of the covalent reaction between the [ 14 C]3CTC and -tubulin occurred at cysteine 354. This finding indicates that the C-3 oxygen atom of colchicinoids is within 3 Å of the sulfur atom of the Cys-354 residue, suggests that the colchicine A ring lies between Cys-354 and Cys-239, based on the known 9 Å distance between these residues, and may indicate that the tropolone C ring lies between the peptide region containing Cys-239 and the amino-terminal -tubulin sequence, based on the labeling pattern observed following direct photoactivation of tubulinbound colchicine.
Identification of Cysteine 354 of β-Tubulin as Part of the Binding Site for the A Ring of Colchicine
Journal of Biological Chemistry, 1996
The colchicine analog 3-chloroacetyl-3-demethylthiocolchicine (3CTC) is a competitive inhibitor of colchicine binding to tubulin, binds to tubulin at 37°C, but not at 0°C, and covalently reacts with -tubulin at 37°C, but not at 0°C, in a reaction inhibited by colchicine site drugs. The approximate intramolecular distance between the oxygen at position C-3 in 3CTC and the chlorine atom of the 3-chloroacetyl group is 3 Å. Using decylagarose chromatography, we purified -tubulin that had reacted with 3-(chloromethyl-[ 14 C]carbonyl)-3-demethylthiocolchicine ([ 14 C]3CTC). This -tubulin was digested with formic acid, cyanogen bromide, endoproteinase Glu-C, or endoproteinase Lys-C, and the radiolabeled peptide(s) were isolated. The sequences of these peptides indicated that as much as 90% of the covalent reaction between the [ 14 C]3CTC and -tubulin occurred at cysteine 354. This finding indicates that the C-3 oxygen atom of colchicinoids is within 3 Å of the sulfur atom of the Cys-354 residue, suggests that the colchicine A ring lies between Cys-354 and Cys-239, based on the known 9 Å distance between these residues, and may indicate that the tropolone C ring lies between the peptide region containing Cys-239 and the amino-terminal -tubulin sequence, based on the labeling pattern observed following direct photoactivation of tubulinbound colchicine.
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.
Archives of Biochemistry and Biophysics, 1998
such as cell division and trafficking of organelles (1). The posttranslational removal and readdition of ty-Recent studies suggest that at least some diversity rosine at the C-terminus of a-tubulin is associated with of microtubule function is related to the biochemical generation of microtubule populations that differ in heterogeneity of the microtubule subunits, the abintracellular distributions, turnover rates, and sensitubulin heterodimers (2, 3). In addition to the existivities to microtubule-depolymerization agents. Here, tence of a large number of a-and b-tubulin isotypes we compared the in vitro assembly and colchicine (2, 4), a number of posttranslational modifications binding characteristics of tubulin dimer preparations contribute to the biochemical heterogeneity of tucomposed of a-tubulin that had been maximally tybulin. These modifications include the cyclic removal rosinated (Ç40% tyrosinated) by tubulin-tyrosine liand readdition of tyrosine at the C-terminus of agase and maximally detyrosinated (100% detyrositubulin (5, 6), acetylation of the e-amino group of lynated) by carboxypeptidase A. Maximally tyrosinated sine 40 of a-tubulin , phosphorylation of a serine of and detyrosinated tubulins had similar critical conab III -tubulin , the successive addition of glutamyl centrations for polymerization and similar association units to carboxy-terminal glutamic acid residues constants for colchicine binding. Microtubules polyboth of a-and b-tubulin (9), and covalent attachment merized from the two tubulins also had similar steadyof glycyl units to the g-carboxyl group of glutamic state mean lengths and length distributions. The growacid residues both in a-and b-tubulin (10).
Biochemistry, 1998
Linkages between structural components of colchicine (COL) and its biphenyl analogues (allocolchicine, ALLO, and its analogues) in the binding to tubulin and its functional consequences were scrutinized. Three ring ALLO analogues with the carbomethoxyl in position 4′ of ring C′ replaced by a carbomethyl (KAC) and methoxy (MAC) groups were synthesized. The binding properties and consequences of binding (microtubule inhibition, abnormal polymerization, and induction of GTPase activity) were compared within the series of three ring and two ring compounds, as well as between pairs consisting of a two ring and a three ring compound with identical groups in position 4′. Binding measurements showed that the binding of KAC to the COL binding site proceeded with similar chemical characteristics as that of its two ring analogue (TKB), but with the kinetic characteristics of ALLO. The binding constant of KAC was found to be 1.9 × 10 6 M-1 and that of MAC was 4.6 × 10 5 M-1. The binding strength of the three ring analogues in descending order was KAC > ALLO > MAC, with increments similar to the biphenyl compounds, TKB > TCB > TMB. The difference in binding affinities between the pairs of three ring and two ring molecules was invariant (δ∆G°)-1.3 (0.2 kcal/mol-1), showing that in all cases ring B makes only an entropic contribution by suppressing free rotation about the biaryl bond. In the case of microtubule inhibition, all three ring compounds inhibited strongly with similar potencies, even though the spread in inhibition strength between the corresponding two ring molecules was >3.3 kcal mol-1 of free energy. This difference was interpreted in terms of the ability of the various molecules to maintain tubulin in the proper conformation for binding in abnormal geometry to the growth end of a microtubule. This ability attains a maximal plateau value for three ring compounds, independently of the oxygen-containing group in ring C′ (or C) and is maintained for the methyl ketone whether in a two or three ring compound. The induction of the GTPase activity was found to follow in general the binding affinity, with the exception that molecules that contained a methyl ketone were stronger GTPase inducers than expected from their alignment according to binding affinity. The finding that the binding of tropolone methyl ether (ring C of COL) induced a GTPase activity shows that ring C contains the ability to induce both substoichiometric microtubule inhibition and GTPase activity. Rings A and B act only as anchors in the binding, with ring A making an energetic contribution, while the effect of ring B is only entropic. It was concluded that both microtubule assembly inhibition and induction of GTPase activity were modulated by the same postbinding conformational change in tubulin. The difference between the strengths of these activities induced by ligands reflects the difference between a narrow allosteric effect between two well-defined sites in the case of GTPase activity and a broad effect aimed at the multiple sites involved in the incorporation of a tubulin protomer into the microtubule structure. Thus, there seems to be a loose thermodynamic linkage between binding and GTPase activity, while there is none between binding and microtubule inhibition, the two phenomena being linked only kinetically.
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...
European Journal of Biochemistry, 1997
2-Methoxy-5-(2',3',4'-trimethoxy)-2,4,6-cycloheptatrien-l-one (MTC) is a colchicine analogue that lacks the B ring. 2-Methoxy-5-(2',4'-dimethoxypheny1)-2,4,6-cycIoheptatrien-l-one (MD) is an A-ring analogue of MTC, in which one methoxy group is replaced by a hydrogen atom. This paper describes the kinetic features of MDC binding to tubulin, and compares its behaviour with MTC to analyse the effect of the A-ring modification on the recognition process by tubulin. Binding is accompanied by a strong enhancement of MDC fluorescence and quenching of protein fluorescence. The kinetic and thermodynamic parameters were obtained from fluorescence stopped-flow measurements. The kinetics are described by a single exponential, indicating that this drug does not discriminate between the different tubulin isotypes. The observed pseudo-first-order rate constant of the fluorescence increase upon binding increases in a non-linear way, indicating that this ligand binds with a similar overall mechanism as colchicine and MTC, consisting of a fast initial binding of low affinity followed by a slower isomerisation step leading to full affinity. The K , and k, values for MDC at 25°C were 540% 65 M-' and 7 0 % 6 s-I, respectively. From the temperature dependence, a reaction enthalpy change (AH?) of the initial binding of 4 9 5 11 kJ/mol-' and an activation energy for the second step of 2 8 t 9 kJ/mol ' were calculated.