Interaction of tubulin with non-denaturing amphiphiles.

Soluble purified calf brain tubulin contains extensive and easily accessible regions capable of hydrophobic interactions. The binding of non-ionic and mild anionic detergents to this protein has been characterized by difference absorption spectroscopy and equilibrium gel chromatography with labelled ligands. Tubulin bound reversibly and co-operatively 0.42 +/- 0.05 g deoxycholate per g protein and bound octyl glucoside at a minimal stoichiometry of 0.26 g per g protein. Binding of deoxycholate and octyl glucoside perturbed the protein absorption, quenched the fluorescence, and produced a moderate change in the far u.v. circular dichroism of tubulin. These changes have been interpreted as the result of detergent binding near aromatic amino acids and the production of a structural change different from detergent-induced denaturation. Deoxycholate and octyl glucoside inhibited colchicine binding. Octyl glucoside and Triton X-100 inhibited the in vitro self-assembly of tubulin into microtubules, whereas small concentrations of deoxycholate were found to enhance microtubule formation.     

Interaction of tubulin with octyl glucoside and deoxycholate. 1. Binding and hydrodynamic studies

Tubulin purified from calf brain cytoplasm, normally a compact water-soluble dimer, is able to interact with the mild detergents octyl glucoside (a minimum of 60 detergent molecules) and deoxycholate (95 +/- 8 molecules). Binding is cooperative and approaches saturation below the critical micelle concentration of the amphiphiles. Binding is accompanied by a quenching of the intrinsic protein fluorescence, but no spectral shape changes indicating denaturation such as in the case of sodium dodecyl sulfate are observed. Glycerol, which is known to be preferentially excluded from the tubulin domain and to favor the folded and associated forms of this protein, inhibits the binding of the mild detergents. Octyl glucoside induces a rapidly equilibrating tubulin self-association reaction characterized by a bimodal sedimentation velocity profile with boundaries at approximately 5 and 12 S. Full dissociation of this detergent restores the normal sedimentation behavior to 90% of the protein. Binding of deoxycholate slows the sedimentation velocity of tubulin from s(0)20,w = 5.6 +/- 0.2 S to s(0)20,w = 4.8 +/- 0.3 S. Measurements of the molecular weight of the tubulin-deoxycholate complex indicate an increase from 100,000 to 143,000 +/- 5,000. The diffusion rate consistently decreases from (5.3 +/- 0.5) X 10(-7) to (3.8 +/- 0.2) X 10(-7) cm2 S-1. This is most simply interpreted as an expansion of the undissociated tubulin dimer upon detergent binding (a change in the frictional ratio, f/f min, from 1.35 to 1.86). It is concluded that tubulin shows a reversible transition between the water-soluble state and amphipathic detergent-bound forms which constitute a model system of tubulin-membrane interactions.     

Mechanism of colchicine binding to tubulin. Tolerance of substituents in ring C' of biphenyl analogues

The limits of structural variation of the substituent in position 4' of ring C' of biphenyl colchicine analogues (ring C in colchicine) were probed by the synthesis of a number of analogues and the examination of their binding to tubulin and its consequences. Binding was found to require the location in three-dimensional space of the oxygen in the 4'-substituent at a locus not far distant from those of the colchicine ring C oxygens. All those analogues that bind to the colchicine site of tubulin induced the GTPase activity and inhibited microtubule assembly, those containing a carbonyl group substoichiometrically and the others stoichiometrically. A similar relation was found for the induction of the abnormal polymerization of the colchicine analogue-tubulin complex, with methoxy-containing compounds requiring a higher temperature to induce the polymerization. A concerted analysis of the binding thermodynamics of colchicine and its various analogues has shown full consistency with the previously proposed two-step binding pathway that involves two nonidentical binding moieties in the ligand [Andreu, J. M., & Timasheff, S. N. (1982) Biochemistry 21, 534-543]. Comparison of the binding parameters of colchicine, its des(ring B) analogue (MTC), and ring A and C compounds individually with the thermodynamic parameters deduced for the first steps of the bindings of colchicine and MTC [Engelborghs, Y., & Fitzgerald, T. J. (1987) J. Biol. Chem. 262, 5204-5209] have led to the conclusion that binding can occur by two pathways leading to the identical product. In the first pathway, ring A binds first; this is followed by a rate-determining thermodynamically indifferent reaction (protein conformation change), and finally a rapid binding of ring C. In the second pathway, the events are the same except that the order of binding of the rings is reversed. Colchicine, due to the steric hindrance of ring B, can follow only the second pathway. For MTC, both kinetic pathways are open and binding may be initiated by random first contact of either ring A or ring C.     

The solubilization and morphological change of human platelets in various detergents

The solubilization of human gel-filtered platelets by octyl glucoside, Triton X-100, dodecylsulfate, and deoxycholate was compared from the analysis of (1) cell lysis, (2) marker leakiness, and (3) component solubility. These analyses all revealed that the effect of detergent concentration on the solubilization of platelets by these detergents was exerted in three stages, i.e., the prelytic, lytic, and complete platelet-lysis stages. These analyses also indicated several differences among platelets in these detergents. (i) The ratio of the platelet-saturation concentration (PSC) to critical micellar concentration (CMC) was about 1/2 for octyl glucoside. Triton X-100 and dodecylsulfate, while it was close to 1 for deoxycholate. (ii) Platelets in octyl glucoside. Triton X-100, and dodecylsulfate all showed parallel curves in cell lysis, protein solubilization and marker leakiness, while the platelet lysis in deoxycholate was identical to the phospholipid solubilization. (iii) The solubility curves of various components in Triton X-100 and deoxycholate were parallel. However, the solubility of cholesterol in octyl glucoside was lower than that of protein and phospholipid. In dodecylsulfate, the solubility of phospholipid and cholesterol was very low in comparison with that of protein. In addition, morphological studies using scanning electron microscopy (scanning EM) revealed that the solubilization by octyl glucoside or Triton X-100 might occur via membrane area expansion. On the other hand, the solubilization by dodecylsulfate or deoxycholate showed membrane vesiculation prior to cell lysis. Moreover, in the prelytic stage, the morphological change in platelets in octyl glucoside showed only concentration dependence by swelling to an ellipsoid and then to a sphere. However, the morphological change in platelets in the other three detergents was dependent not only on the detergent concentration but also on prolonged incubation. Specifically, in Triton X-100, the cells initially changed to spiculate discs and then reached their final shape as swollen discs with surface invagination. In dodecylsulfate and deoxycholate the morphological changes were almost the same. The cell initially deformed in shape to a spiculate disc and finally to a stretched-out flat form. The results are discussed according to the bilayer couple hypothesis. Also, in the prelytic stage, these detergents caused inhibition of the response of platelets to collagen and ADP-fibrinogen.     

The binding of isocolchicine to tubulin. Mechanisms of ligand association with tubulin

Isocolchicine is a structurally related isomer of colchicine altered in the methoxytropone C ring. In spite of virtual structural homology of colchicine and isocolchicine, isocolchicine is commonly believed to be inactive in binding to tubulin and inhibiting microtubule assembly. We have found that isocolchicine does indeed bind to the colchicine site on tubulin, as demonstrated by its ability to competitively inhibit [3H]colchicine binding to tubulin with a KI approximately 400 microM. Isocolchicine inhibits tubulin assembly into microtubules with an I50 of about 1 mM, but the affinity of isocolchicine for the colchicine receptor site, 5.5 +/- 0.9 x 10(3) M-1 at 23 degrees C, is much less (approximately 500-fold) than that of colchicine. Unlike colchicine, isocolchicine binds rapidly, and the absorption and fluorescence properties of the complex are only modestly altered compared to free ligand. It is proposed that the binding of isocolchicine to tubulin may be rationalized either in terms of conformational states of colchicinoids when liganded to tubulin or by the structural requirements for C-10 substituents for high affinity binding to the colchicine receptor.     

A thermodynamic study of the interaction of tubulin with colchicine site ligands

The bicyclic colchicine analogue 2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-on e (MTC) has been used to study the thermodynamics of specific ligand binding to the colchicine site of tubulin, employing isothermal reaction microcalorimetry. The binding of MTC to purified calf brain tubulin, in 10 mM sodium phosphate buffer, pH 7.0, is characterized by delta H degree = -19 +/- 1 kJ.mol-1, delta G degree = -31.8 +/- 0.6 kJ.mol-1, and delta S degree = 43 +/- 5 J.mol-1.K-1 at 298 K, with a slight variation in the temperature range from 283 to 308 K. The binding thermodynamics of colchicine and allocolchicine are similar to MTC under the conditions examined, suggesting related molecular interactions of the three ligands with the protein binding site. The standard enthalpy changes of binding of colchicine and MTC at 308 K coincide within experimental error. Therefore the more favorable free energy change of binding of colchicine must come from a larger binding entropy change (by about 20 J.mol-1.K-1). This difference could be attributed to the presence of the middle ring of colchicine, which is absent in MTC. Consistently, a similar entropy change is observed by the comparison of allocolchicine to MTC binding at several temperatures. In addition, allocolchicine binding is about 6 kJ.mol-1 less exothermic than MTC binding, which could be attributed to the presence in allocolchicine of a substituted phenyl ring instead of the colchicine-MTC tropolone ring. The present results and analysis are fully compatible with the previously proposed bifunctional binding of colchicine and MTC (through their trimethoxybenzene and tropolone moieties) to a bifocal protein binding site, and also with a partial immobilization of intramolecular rotation of MTC upon binding, which in colchicine is already constrained by its middle ring (Andreu, J. M., Gorbunoff, M. J., Lee, J. C., and Timasheff, S. (1984) Biochemistry 23, 1742-1752).     

Interaction of bacterial F1-ATPase with octyl glucoside and deoxycholate

Purified F₁-ATPase from Micrococcus lysodeikticus (Micrococcus luteus) contains extensive and easily accessible areas capable of hydrophobic interaction. These hydrophobic areas were demonstrated by the binding of a non-ionic and a mild anionic detergent to this protein, evidenced by charge shift electrophoresis and measured by equilibrium gel chromatography with labelled detergents. F₁-ATPase bound 0.06 ± 0.01 g octyl glucoside per g protein and 0.12 ± 0.01 g deoxycholate per g protein, which amount to 81 and 119 amphiphile molecules per protein molecule, respectively. Deoxycholate and octyl glucoside inhibited the Ca²⁺- and Mg²⁺-dependent ATP hydrolytic activity of the enzyme. The inhibition by octyl glucoside was moderately cooperative. Binding of these detergents to the enzyme did not seem to induce any disruption of its quaternary structure, although the spontaneous dissociation of the δ subunit, which is not essential for the enzyme activity, increased during deoxycholate treatment. These results suggest that hydrophobic domains play a role in the enzymatic activity of this coupling factor and / or in its interaction with the membrane.     

Stathmin and Interfacial Microtubule Inhibitors Recognize a Naturally Curved Conformation of Tubulin Dimers

Tubulin is able to switch between a straight microtubule-like structure and a curved structure in complex with the stathmin-like domain of the RB3 protein (T₂RB3). GTP hydrolysis following microtubule assembly induces protofilament curvature and disassembly. The conformation of the labile tubulin heterodimers is unknown. One important question is whether free GDP-tubulin dimers are straightened by GTP binding or if GTP-tubulin is also curved and switches into a straight conformation upon assembly. We have obtained insight into the bending flexibility of tubulin by analyzing the interplay of tubulin-stathmin association with the binding of several small molecule inhibitors to the colchicine domain at the tubulin intradimer interface, combining structural and biochemical approaches. The crystal structures of T₂RB3 complexes with the chiral R and S isomers of ethyl-5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl-carbamate, show that their binding site overlaps with colchicine ring A and that both complexes have the same curvature as unliganded T₂RB3. The binding of these ligands is incompatible with a straight tubulin structure in microtubules. Analytical ultracentrifugation and binding measurements show that tubulin-stathmin associations (T₂RB3, T₂Stath) and binding of ligands (R, S, TN-16, or the colchicine analogue MTC) are thermodynamically independent from one another, irrespective of tubulin being bound to GTP or GDP. The fact that the interfacial ligands bind equally well to tubulin dimers or stathmin complexes supports a bent conformation of the free tubulin dimers. It is tempting to speculate that stathmin evolved to recognize curved structures in unassembled and disassembling tubulin, thus regulating microtubule assembly.     

Sulfhydryl groups of Mimosa pudica tubulin implicated in colchicine binding and polymerization in vitro.

The important characteristic of novel Mimosa pudica tubulin is its ability to bind colchicine only when dithiothreitol is included in the isolation buffer, indicating the involvement of sulfhydryl groups in colchicine binding. Modification of sulfhydryl groups by a sulfhydryl modifying agent also affects the normal assembly of tubulin into microtubules, as revealed by electron microscopic and spectrophotometric studies. The number of free sulfhydryl groups present in tubulin protein responsible for both colchicine binding and polymerization has been found to be 4, distributed in alpha and beta subunits, and is distinctly different from the number reported for animal tubulin.     

Solubilization of DMPC and DPPC vesicles by detergents below their critical micellization concentration: high-sensitivity differential scanning calorimetry, Fourier transform infrared spectroscopy and freeze-fracture electron microscopy reveal two interaction sites of detergents in vesicles

The interaction of sodium deoxycholate, sodium cholate and octyl glucoside with sonicated vesicles of L alpha-dimyristoyl-phosphatidylcholine (DMPC) and L alpha-dipalmitoylphosphatidylcholine (DPPC) at concentrations below the critical micellization concentration (cmc) of the detergents was studied by high-sensitivity DSC (hs-DSC), Fourier transform infrared spectroscopy (FT-IR) and freeze-fracture electron microscopy. The two phospholipids exhibited a striking different thermotropic behaviour in the presence of these detergents. For DPPC vesicles, the detergents were found to interact exclusively in the aqueous interface region of the bilayer below the membrane saturation concentration Rsat while in DMPC vesicles two coexisting interaction sites below this concentration persist. These are detergents which interact at the aqueous interface region (site 1) and in the acyl chain region (site 2) of the DMPC vesicles. The partition coefficients K of the detergents between DPPC vesicles and the water phase were calculated from the hs-DSC results at two detergent/phospholipid molar ratios Rtot less than or equal to Rsat as 0.35, 0.049 and 0.040 mol-1 for sodium deoxycholate, sodium cholate and octyl glucoside, respectively. In contrast, for DMPC the K values for Rtot less than or equal to Rsat were found to be dependent on Rtot due to the occupation of site 2 by the detergents above a certain Rtot. The model is discussed on the basis of the detergents free energies of transfer from the water phase to site 1 and site 2 of the vesicles, respectively. The solubilization behaviour of DPPC vesicles, dependent on whether the total detergent concentration is above or below the cmc at Rsat, differed significantly as revealed by hs-DSC. This suggests that in the latter case an additional hydrophobic effect could facilitate the formation of disc shaped mixed micelles. Moreover, this different behaviour was employed to measure the cmc values of the detergents studied in the presence of the vesicles by hs-DSC.     

Molecular characterization of the human erythrocyte anion transport protein in octyl glucoside

Band 3 protein, the anion transport protein of the human erythrocyte membrane, was purified in the presence of the nonionic detergent octyl glucoside. A molecular characterization was carried out to investigate whether the native structure of the protein was retained in the presence of this detergent. Band 3 bound octyl glucoside below the critical micelle concentration (cmc) of the detergent, approaching saturation above the cmc. At 40 mM octyl glucoside, close to saturating concentrations, 0.64 g of octyl glucoside is bound per gram of band 3 protein, corresponding to 208 molecules of detergent bound per monomer of band 3. Sedimentation velocity and gel filtration studies, performed at 40 mM octyl glucoside, indicated that the band 3-octyl glucoside complex had an average molecular weight of 1.98 X 10(6), which corresponds to a dodecamer. Sedimentation equilibrium experiments confirmed that band 3 in octyl glucoside exists in a heterogeneous and high oligomeric state. This high oligomeric state did not change dramatically over octyl glucoside concentrations ranging from 6 to 60 mM. The circular dichroism spectrum of band 3 changed only slightly over this range of octyl glucoside concentrations. The alpha-helical and beta-sheet contents of band 3 in 2 mM octyl glucoside were calculated to be 40% and 27%, respectively, indicating that no gross alteration in the secondary structure of the protein had occurred in octyl glucoside. The ability of band 3 to bind 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS), a potent inhibitor (Ki = 1 microM) of anion transport, was measured to assess the integrity of the inhibitor binding site of the protein in octyl glucoside.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of tubulin assembly by antileprosy drug dapsone

The effect of dapsone on assembly-disassembly process of bovine brain tubulin was examined. The drug was found to readily bind tubulin dimer and that in its presence colchicine binding to tubulin was enhanced. Although dapsone associated with tubulin at a site other than the colchicine binding site, distinct inhibition of microtubule assembly was detected.     

Microtubule Assembly and Function in Chlamydomonas: Inhibition of Growth and Flagellar Regeneration by Antitubulins and Other Drugs and Isolation of Resistant Mutants

The distribution of microtubules in Chlamydomonas reinhardtii suggests that they are involved in mitosis, cell and nuclear cleavage, and generation of flagella. Vinblastine, colchicine, and podophyllotoxin bind to the protein building block of microtubules (tubulin) and prevent normal assembly. Mutants resistant to these "antitubulin" drugs are candidates to have alterations in tubulin primary structure. We report the ability to inhibit growth, and flagellar regeneration after amputation, of: vinblastine, several colchicine derivatives, two water-soluble derivatives of podophyllotoxin (succinylpodophyllotoxin and epipodophyllotoxin thiuronium bromide), and other substances which may interfere with flagellar assembly or motility (isopropyl N-phenyl carbamate, 2-methoxy-5-nitrotropone, chloral hydrate, caffeine, and nickel acetate). The ability of each drug to inhibit binding of labeled colchicine or podophyllotoxin to mammalian brain tubulin was also determined. The results suggest that only in the cases of colchicine, colcemide, and epipodophyllotoxin thiruonium bromide was the toxicity to Chlamydomonas mediated by inhibition of tubulin assembly. The requirement for high concentrations of colchicine may be due to permeability barriers, since colchicine toxicity was potentiated by deoxycholate. Mutants resistant to antitubulins were isolated after treatment with methyl methanesulfonate. The results with vinblastine were equivocal. Of three mutants resistant to inhibition of growth and flagellar regeneration by colchicine, one was also cross-resistant to epipodophyllotoxin thiuronium bromide.     

Role of B-ring of colchicine in its binding to Zn(II)-induced tubulin-sheets

Colchicine-tubulin dimer comPlex, a Potent inhibitor of normal microtubule assembly undergoes extensive self-assembly in the Presence of 1 X 10^-4 M zinc sulPhate. Polymers assembled from colchicine-tubulin dimer comPlexes are sensitive to cold. Although colchicine can be accomodated within the Polymeric structure, the drug cannot bind to tubulin subunits in the intact Polymers. This is evidenced by the fact that (a) the colchicine binding activity of tubulin is lost when allowed to Polymerize with zinc sulPhate, (b) the loss in colchicine binding could be Prevented by Preincubation of tubulin with 1 X 10^-3 M CaCl₂ or 1 X 10^-5 M vinblastine sulPhate and finally (c) no loss in colchicine binding activity is found when tubulin is kePt at a concentration far below the critical concentration for Polymerization. Unlike colchicine, its B-ring analogues desacetamido colchicine (devoid of the B-ring subtituent) and 2-methoxy-5-(2′, 3′, 4′-trimethoxyPhenyl) troPone (devoid of the B-ring) can bind to tubulin subunits in the intact Polymers. Thus we conclude that the colchicine binding domain on the tubulin molecule is mostly (if not comPletely) exPosed in the Zn(II) -induced Polymers and the B-ring substituent Plays a major role in determining the binding ability of a colchicine analogue to tubulin in the intact Zn(II) -induced sheets.     

Reversible inhibition of microtubules and cell growth by the bicyclic colchicine analogue MTC

2-methoxy-5-(2,3,4-trimethoxyphenyl) 2,4,6-cycloheptatrien-1-one (MTC) is a synthetic colchicine analogue, lacking the B ring of the alkaloid (Fitzgerald: Biochem. Pharmacol. 25:1381-1387, 1976). MTC has been shown to bind reversibly to the colchicine binding site of tubulin and to inhibit microtubule assembly in vitro (Andreu et al: Biochemistry 23:1742-1752, 1984; Bane et al: J. Biol. Chem. 259:7391-7398, 1984). Its action on different cultured cell lines (PtK2, Pk15, and SV-3T3) has now been studied. 0.2 X 10(-6) M MTC stopped Pk15 and SV-3T3 cell growth, inducing an accumulation of mitoses in a few hours. Removal of MTC from the culture medium rapidly restored normal mitotic index and growth rates. Partial depolymerization of the cytoplasmic microtubules of PtK2 cells was observed at concentrations ranging from 2 to 5 X 10(-7) M. Maximal microtubule network depolymerization was obtained after 4 h of treatment with 2 to 5 X 10(-6) M MTC or at a higher MTC concentration (2 X 10(-5) M) for less than 2 h. Removal of 2 X 10(-5) M MTC (the highest MTC concentration used) from the culture medium resulted in almost complete microtubule polymerization after 10 min of drug recovery and a normal microtubule network in 20-30 min. MTC constitutes an antimitotic drug directed to the colchicine site. It is water-soluble, shows a fast and reversible action, and may therefore be employed as a convenient tool to study cellular microtubule-dependent functions.     

Colchicine-binding sites of brain tubulins from an antarctic fish and from a mammal are functionally similar, but not identical: implications for microtubule assembly at low temperature.

The tubulins of Antarctic fishes possess adaptations that favor microtubule formation at low body temperatures (Detrich et al.: Biochemistry 28:10085-10093, 1989). To determine whether some of these adaptations may be present in a domain of tubulin that participates directly or indirectly in lateral contact between microtubule protofilaments, we have examined the energetics of the binding of colchicine, a drug thought to bind to such a site, to pure brain tubulins from an Antarctic fish (Notothenia gibberifrons) and from a mammal (the cow, Bos taurus). At temperatures between 0 and 20 degrees C, the affinity constants for colchicine binding to the fish tubulin were slightly smaller (1.5-2.6-fold) than those for bovine tubulin. van't Hoff analysis showed that the standard enthalpy changes for colchicine binding to the two tubulins were comparable (delta H degrees = +10.6 and +7.4 kcal mol-1 for piscine and bovine tubulins, respectively), as were the standard entropy changes (delta S degrees = +61.3 eu for N. gibberifrons tubulin, +51.2 eu for bovine tubulin). At saturating concentrations of the ligand, the maximal binding stoichiometry for each tubulin was approximately 1 mol colchicine/mol tubulin dimer. The data indicate that the colchicine-binding sites of the two tubulins are similar, but probably not identical, in structure. The apparent absence of major structural modifications at the colchicine site suggests that this region of tubulin is not involved in functional adaptation for low-temperature polymerization. Rather, the colchicine site of tubulin may have been conserved evolutionarily to serve in vivo as a receptor for endogenous molecules (i.e., "colchicine-like" molecules or MAPs) that regulate microtubule assembly.     

Roles of colchicine rings B and C in the binding process to tubulin

The interactions of tubulin with colchicine analogues in which the tropolone methyl ether ring had been transformed into a p-carbomethoxybenzene have been characterized. The analogues were allocolchicine (ALLO) and 2,3,4-trimethoxy-4'-carbomethoxy-1,1'-biphenyl (TCB), the first being transformed colchicine and the second transformed colchicine with ring B eliminated. The binding of both analogues has been shown to be specific for the colchicine binding site on tubulin by competition with colchicine and podophyllotoxin. Both analogues bind reversibly to tubulin with the generation of ligand fluorescence. The binding of ALLO is slow, the fluorescence reaching a steady state in the same time span as colchicine; that of TCB is rapid. The displacement of ALLO by podophyllotoxin proceeds with a half-life of ca. 40 min. Binding isotherms generated from gel filtration and fluorescence measurements have shown that both analogues bind to tubulin with a stoichiometry of 1 mol of analogue/mol of alpha-beta tubulin. The equilibrium binding constants at 25 degrees C have been found to be (9.2 +/- 2.5) x 10(5) M-1 for ALLO and (1.0 +/- 0.2) X 10(5) M-1 for TCB. Binding of both analogues was accompanied by quenching of protein fluorescence, perturbation of the far-ultraviolet circular dichroism of tubulin, and induction of the tubulin GTPase activity, similarly to colchicine binding. Both inhibited microtubule assembly in vitro, ALLO substoichiometrically, and both induced the abnormal cooperative polymerization of tubulin, which is characteristic of the tubulin-colchicine complex. Analysis in terms of the simple bifunctional ligand binding mechanism developed for colchicine [Andreu, J.M., & Timasheff, S.N. (1982) Biochemistry 21, 534-543] and comparison with the binding of the colchicine two-ring analogue, 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one [Andreu, J. M., Gorbunoff, M. J., Lee, J. C., & Timasheff, S. N. (1984) Biochemistry 23, 1742-1752], have shown that transformation of the tropolone methyl ether part of colchicine into p-carbomethoxybenzene weakens the standard free energy of binding to tubulin by 1.4 +/- 0.1 kcal/mol, while elimination of ring B weakens it by 1.0 +/- 0.1 kcal/mol. The roles of rings C and B of colchicine in the thermodynamic and kinetic mechanisms of binding to tubulin were analyzed in terms of these findings.     

The effect of TN-16 on the alkylation of tubulin

The synthetic anti-tumor drug 3-(1-anilinoethylidene)-5-benzylpyrrolidine-2,4-dione (TN-16) is known to block microtubule assembly and colchicine binding to tubulin, although its structure does not resemble those of either colchicine, podophyllotoxin, or nocodazole (Arai, FEBS Lett. 155:273-276 (1983]. We have found that TN-16 affects the intra-chain cross-linking of beta-tubulin by N,N'-ethylene-bis(iodoacetamide) in a manner identical to that of colchicine, podophyllotoxin, and nocodazole, but different from that of vinblastine or maytansine. TN-16 also inhibits alkylation of tubulin by iodo[14C]acetamide, as do colchicine and its congeners. TN-16 appears to bind to tubulin at the colchicine binding site and one of its phenyl groups is likely to bind at the site on tubulin where colchicine's A ring binds.     

Structure of a benzylidene derivative of 9(10H)-anthracenone in complex with tubulin provides a rationale for drug design

Microtubules are composed of αβ-tubulin heterodimers and have been treated as highly attractive targets for antitumor drugs. A broad range of agents bind to tubulin and interfere with microtubule assembly, including colchicine binding site inhibitors (CBSIs). Tubulin Polymerization Inhibitor I (TPI1), a benzylidene derivative of 9(10H)-anthracenone, is a CBSI that inhibits the assembly of microtubules. However, for a long time, the design and development of anthracenone family drugs have been hindered by the lack of structural information of the tubulin-agent complex. Here we report a 2.3 Å crystal structure of tubulin complexed with TPI1, the first structure of anthracenone family agents. This complex structure reveals the interactions between TPI1 and tubulin, and thus provides insights into the development of new anthracenone derivatives targeting the colchicine binding site.     

4',6-Diamidino-2-phenylindole, a fluorescent probe for tubulin and microtubules

A new fluorophor for tubulin which has permitted the monitoring of microtubule assembly in vitro is reported. DAPI (4',6-diamidino-2-phenylindole), a fluorophor already known as a DNA intercalator, was shown to bind specifically to a unique tubulin site as a dimer (KD(app) = 43 +/- 5 microM at 37 degrees C) or to tubulin associated in microtubules (KD(app) = 6 +/- 2 microM at 37 degrees C) with the same maximum enhancement in fluorescence. When tubulin polymerization was induced with GTP, the change in DAPI affinity for tubulin resulted in an enhancement of DAPI binding and, consequently, of fluorescence intensity. DAPI, whose binding site is different from that of colchicine, vinblastine, or taxol, did not interfere greatly with microtubule polymerization. It induced a slight diminution of the critical concentration for tubulin assembly due to a decrease in the depolymerizing rate constant. Moreover, DAPI did not interfere with GTP hydrolysis correlated with tubulin polymerization, but it decreased the GTPase activity at the steady state of tubulin assembly. Even at substoichiometric levels DAPI can be used to follow the kinetics of microtubule assembly.