Fluorescent molecular rotors: a new class of probes for tubulin structure and assembly

9-(Dicyanovinyl)julolidine (DCVJ) is a fluorescent dye whose intramolecular rotational relaxation is solvent dependent. Since its quantum yield increases with decreasing free volume, this molecule has been very useful in monitoring synthetic polymer reactions and measuring local microviscosity changes in phospholipid bilayers [Loutfy, R. O. (1986) Pure Appl. Chem. 58, 1239-1248; Kung, C. E., & Reed, J. K. (1986) Biochemistry 25, 6114-6121]. We have used DCVJ to follow the polymerization of tubulin, a protein that can assemble into a variety of polymorphic microstructures. DCVJ binding to free tubulin is accompanied by an increase in quantum yield, indicating that DCVJ has become partially immobilized. At 4 degrees C, DCVJ binds to a single population of high-affinity hydrophobic sites (Kd = 1.12 +/- 0.26 microM) with a stoichiometry that is protein concentration dependent. n, the number of moles of DCVJ bound per mole of alpha beta dimer, approaches 1 at concentrations less than or equal to 0.5 mg/mL but decreases to a lower limit of approximately 0.3 at concentrations greater than or equal to 2.0 mg/mL. The quantum yield also increases with increasing protein concentration. This trend is unaltered by the presence of microtubule-associated proteins. These results are analyzed in terms of a concentration-dependent oligomerization of tubulin at 4 degrees C. When tubulin is polymerized at 37 degrees C to microtubules or to sheets in the presence of Zn2+, the fluorescence intensity of DCVJ increases although the magnitude of this increase differs significantly. We are able to use the distinct fluorescent and binding characteristics of the bound dye to distinguish between these two polymorphs on a molecular level.     

Anion-induced increases in the affinity of colcemid binding to tubulin

Colcemid binds tubulin rapidly and reversibly in contrast to colchicine which binds tubulin relatively slowly and essentially irreversibly. At 37 degrees C the association rate constant for colcemid binding is 1.88 X 10(6) M-1 h-1, about 10 times higher than that for colchicine; this is reflected in the activation energies for binding which are 51.4 kJ/mol for colcemid and 84.8 kJ/mol for colchicine. Scatchard analysis indicates two binding sites on tubulin having different affinities for colcemid. The high-affinity site (Ka = 0.7 X 10(5) M-1 at 37 degrees C) is sensitive to temperature and binds both colchicine and colcemid and hence they are mutually competitive inhibitors. The low-affinity site (Kb = 1.2 X 10(4) M-1) is rather insensitive to temperature and binds only colcemid. Like colchicine, 0.6 mol of colcemid are bound/mol of tubulin dimer (at the high-affinity site) and the reaction is entropy driven (163 J K-1 mol-1). Similar to colchicine, colcemid binding to tubulin is stimulated by certain anions (viz. sulfate and tartrate) but by a different mechanism. Colcemid binding affinity at the lower-affinity site of tubulin is increased in the presence of ammonium sulfate. Interestingly, the lower-affinity site on tubulin for colcemid, even when converted to higher affinity in presence of ammonium sulfate, is not recognized by colchicine. We conclude that tubulin possesses two binding sites, one of which specifically recognized the groups present on the B-ring of colchicine molecule and is effected by the ammonium sulfate, whereas the higher-affinity site, which could accommodate both colchicine and colcemid, possibly recognized the A and C ring of colchicine.     

The interaction of 1-anilino-8-naphthalene sulfonate with tubulin: A site independent of the colchicine-binding site

Rat brain tubulin binds 1 mole of 1-anilino-8-naphthalene sulfonate (ANS) per dimer (110,000 daltons) with an association constant of 3.2 × 10⁵m^?1. The quantum yield of ANS fluorescence is increased 120-fold over that in water to φ = 0.48 and there is a hypsochromic shift of 56 nm to an emission maximum of 460 nm. There is energy transfer from tryptophan to bound ANS. Vinblastine and Ca²⁺ enhance ANS fluorescence in tubulin by 35%–40%; this can be ascribed to an increased quantum yield, rather than changes in the affinity constant or number of binding sites. The ANS binding site shows minimal decay at 37 °C when colchicine binding has decreased to 50%. It is concluded that the colchicine- and ANS-binding sites occupy different regions of the tubulin molecule.     

The B-ring substituent at C-7 of colchicine and the alpha-C-terminus of tubulin communicate through the "tail-body" interaction

The carboxy terminals of alphabeta-tubulins are flexible regions rich in acidic amino acid residues that play an inhibitory role in the polymerization of tubulin to microtubules. We have shown that the binding of colchicine and its B-ring analogs (with C-7 substituents) to tubulin are pH sensitive and have high activation energies. Under identical conditions, the binding of analogs without C-7 substituents is pH independent and has lower activation energy. Beta-C-terminus-truncated tubulin (alphabeta(s)) shows similar pH sensitivity and activation energy to native tubulin (alphabeta). Removal of the C-termini of both subunits of tubulin (alpha(s)beta(s)) or the binding of a basic peptide P2 to the negatively charged alpha-C-terminus of tubulin causes a colchicine-tubulin interaction independent of pH with a low activation energy. Tubulin dimer structure shows that the C-terminal alpha-tail is too far from the colchicine binding site to interact directly with the bound colchicine. Therefore, it is likely that the interaction of the alpha-C-terminus with the main body of tubulin indirectly affects the colchicine-tubulin interaction via conformational changes in the main body. We therefore conclude that in the presence of tail-body interaction, a B-ring substituent makes contact with the alpha-tubulin and induces significant conformational changes in alpha-tubulin.     

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.     

Spectroscopic and kinetic features of allocolchicine binding to tubulin

Allocolchicine is a structural isomer of colchicine in which colchicine's tropone C ring is replaced with an aromatic ester. In spite of the structural differences between the two ligands, the association parameters for both molecules binding to tubulin are quite similar. The association constant for allocolchicine binding to tubulin was determined by fluorescence titration to be 6.1 x 10(5) M-1 at 37 degrees C, which is about a factor of 5 less than that of the colchicine-tubulin association. In particular, analysis of the kinetics of the association of allocolchicine with tubulin yielded nearly equivalent activation parameters for the two ligands. The activation energy of the allocolchicine binding reaction was found to be 18.4 +/- 1.5 kcal/mol, which is only slightly less than the activation energy for colchicine binding to tubulin. This finding argues against conformational flexibility of the C ring as the structural feature of colchicine responsible for the slow kinetics of colchicinoid-tubulin binding reactions. Tubulin binding promote a dramatic enhancement of allocolchicine fluorescence. Unlike colchicine, the emission energy and intensity of the tubulin-bound allocolchicine fluorescence can be mimicked by solvent, and a general hydrophobic environment for the ligand binding site is indicated. The excitation spectrum of the protein-bound species, however, is shown to possess two bands which center at higher and lower energy than the energy maximum of the spectrum of the ligand in apolar solvents, indicating that properties of the colchicine binding site in addition to a low dielectric constant contribute to the fluorescence of the bound species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Energy-transfer studies of the distance between the high-affinity metal binding site and the colchicine and 8-anilino-1-naphthalenesulfonic acid binding sites on calf brain tubulin

The high-affinity metal divalent cation Mg2+, associated with the exchangeable guanosine 5'-triphosphate (GTP) binding site (E site) on purified tubulin, has been replaced by the transition metal ion Co2+ on tubulin as well as on the tubulin-colchicine, tubulin-allocolchicine and tubulin-8-anilino-1-naphthalenesulfonic acid (tubulin-ANS) complexes. While pure native tubulin readily incorporated 0.8 atom of Co2+ per tubulin alpha-beta dimer, incorporation was reduced to 0.4 atom of Co2+ per mole of tubulin when it was complexed with colchicine, indicating that the conformational change induced in tubulin by the binding of colchicine leads to a reduced accessibility of the divalent cation binding site linked to the E site without necessarily changing the intrinsic binding constant. The fluorescence emission spectra of tubulin-bound colchicine, allocolchicine, and ANS displayed a strong overlap with the Co2+ absorption spectrum, identifying these as adequate donor-acceptor pairs. Fluorescence energy-transfer measurements were carried out between tubulin-bound colchicine (or allocolchicine) and ANS as donors and tubulin-complexed Co2+ as acceptor. It was found that the distance between the ANS and the high-affinity divalent cation binding sites is greater than 28 A, while that between the colchicine and the divalent cation binding sites is greater than 24 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of Vinblastine with Calf Brain Microtubule protein.

The interaction of vinblastine with calf brain tubulin has been studied by velocity sedimentation, gel filtration, and fluorescence. It has been established that vinblastine induces the stable tubulin dimers to dimerize further to tetramers. The sedimentation patterns at low vinblastine concentration were analyzed by the ligand-induced dimerization theory of Cann and Goad ((1972) Arch. Biochem. Biophys. 153, 603-609). The association constant and stoichiometry for the binding of vinblastine to tubulin, determined by gel filtration and spectrofluorometry, were (2.3 +/- 0.1) X 10(4) liters/mol at 25 degrees and two vinblastine binding sites per tubulin dimer of molecular weight 110,000. The binding of vinblastine to tubulin is characterized by an enthalpy change of 5.8 kcal/mol and a positive unitary entropy change. Binding of vinblastine did not induce any significant conformational changes in tubulin as monitored by circular dichroism. However, the vinblastine-tubulin complex displayed an ultraviolet difference spectrum, which appears to reflect mostly the transfer of vinblastine to a less polar environment. Besides binding vinblastine, tubulin was shown to bind vincristine with identical free energy and stoichiometry and to have a single binding site for 8-anilino-1-naphthalene sulfonic acid per tubulin dimer, which is independent of those for vinblastine.     

Interaction of 1-propargyl-5-chloropyrimidin-2-one (a metahalone) with rat brain tubulin

The mitotic inhibitor 1-propargyl-5-chloropyrimidin-2-one (a metahalone) was found to bind to DEAE-cellulose purified rat brain tubulin. A decrease in the fluorescence of 1-propargyl-5-chloropyrimidin-2-one was seen when the drug was incubated in the presence of increasing tubulin concentrations. The decrease in metahalone fluorescence was not affected by the addition of GTP, indicating drug interaction at other portions of the tubulin molecule than the nucleotide binding sites. Scatchard plot analysis following incubation of tubulin with 1-propargyl-5-chloro-[2-14C]pyrimidin-2-one revealed that 1 mol of metahalone bound to 1 mol of tubulin dimer with a measured association constant of 8.0 X 10(3) M-1. Double reciprocal plots of vincristine and colchicine binding to tubulin in the presence of 1-propargyl-5-chloropyrimidin-2-one showed that the metahalone competitively inhibited colchicine binding to tubulin but had no influence on vincristine binding. This conclusion was supported by gel filtration chromatography where an increase in unbound colchicine was measured when 1-propargyl-5-chloropyrimidin-2-one was present in an incubation mixture containing colchicine and tubulin. In the presence of 5 mM 1-propargyl-5-chloropyrimidin-2-one, tubulin self-aggregated into crystalline structures. The binding of 1-propargyl-5-chloropyrimidin-2-one to tubulin at or near the colchicine binding site may be responsible for the metaphase arresting characteristics of this drug.     

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.     

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.     

Podophyllotoxin as a probe for the colchicine binding site of tubulin.

The binding of [3H]podophyllotoxin to tubulin, measured by a DEAE-cellulose filter paper method, occurs with an affinity constant of 1.8 X 10(6) M-1 (37 degrees at pH 6.7). Like colchicine, approximately 0.8 mol of podophyllotixin are bound per mol of tubulin dimer, and the reaction is entropy-driven (43 cal deg-1 mol-1). At 37 degrees the association rate constant for podophyllotoxin binding is 3.8 X 10(6) M-1 h-1, approximtaely 10 times higher than for colchicine; this is reflected in the activation energies for binding which are 14.7 kcal/mol for podophyllotoxin and 20.3 kcal/mol for colchicine. The dissociation rate constant for the tubulin-podophyllotoxin complex is 1.9 h-1, and the affinity constant calculated from the ratio of the rates is close to that obtained by equilibrium measurements. Podophyllotxin and colchicine are mutually competitive inhibitors. This can be ascribed to the fact that both compounds have a trimethoxyphenyl ring and analogues of either compound with bulky substituents in their trimethoxyphenyl moiety are unable to inhibit the the binding of either of the two ligands. Tropolone, which inhibits colchicine binding competitively, has no effect on the podophyllotoxin/tubulin reaction. Conversely, podophyllotoxin does not influence tropolone binding. Moreover, the tropolone binding site of tubulin does not show the temperature and pH lability of the colchicine and podophyllotoxin domains, hence this lability can be ascribed to the trimethoxyphenyl binding region of tubulin. Since podophyllotoxin analogues with a modified B ring do not bind, it is concluded that both podophyllotoxin and colchicine each have at least two points of attachment to tubulin and that they share one of them, the binding region of the trimethoxyphenyl moiety.     

Tubulin photoaffinity labeling study with a plinabulin chemical probe possessing a biotin tag at the oxazole

A new bioactive photoaffinity probe KPU-252-B1 (4) possessing a biotin tag on the oxazole ring of a potent plinabulin derivative KPU-244 (2) was synthesized via the Cu^I-catalyzed Huisgen’s cycloaddition reaction to understand the precise binding mode of the diketopiperazine-based anti-microtubule agent plinabulin on tubulin. Probe 4 showed significant binding affinity toward tubulin and cytotoxicity against an HT-29 cells. A photoaffinity labeling study suggested that probe 4 specifically recognizes tubulin at a binding site that binds plinabulin or colchicine, most likely near or at the colchicine binding site, which is located at the interfacial region formed by ??-and ??-tubulin association. The results also demonstrated that probe 4 may serve as a useful plinabulin chemical probe to investigate the molecular mechanism by which anti-microtubule diketopiperazine derivatives operate.     

Binding to tubulin of the colchicine analog 2-methoxy-5-(2', 3', 4'-trimethoxyphenyl)tropone. Thermodynamic and kinetic aspects

The thermodynamics and kinetics of the binding to tubulin of the colchicine analog 2-methoxy-5-(2', 3', 4'-trimethoxyphenyl) tropone (termed AC because it lacks the B-ring of colchicine) have been characterized by fluorescence techniques. The fluorescence of AC is weak in aqueous solution and is enhanced 250-fold upon binding to tubulin. The following thermodynamic values were obtained for the interaction at 37 degrees C: K = 3.5 X 10(5) M-1; delta G0 = -7.9 kcal/mol; delta H0 = -6.8 kcal/mol; delta S0 = 3.6 entropy units. The AC-tubulin complex is 1-2 kcal/mol less stable than the colchicine-tubulin complex. The change in fluorescence of AC was employed to measure the kinetics of the association process, and quenching of protein fluorescence was used to measure both association and dissociation. The association process, like that of colchicine, could be resolved into a major fast phase and a minor slow phase. The apparent second order rate constant for the fast phase was found to be 5.2 X 10(4) M-1 S-1 at 37 degrees C, and the activation energy was 13 kcal/mol. This activation energy is 7-11 kcal/mol less than that for the binding of colchicine to tubulin. The difference in activation energies can most easily be rationalized by a mechanism involving a tubulin-induced conformational change in the ligand ( Detrich , H. W., III, Williams, R. C., Jr., Macdonald, T. L., Wilson, L., and Puett , D. (1981) Biochemistry 20, 5999-6005). Such a change would be expected to have a small activation energy in AC because it possesses a freely rotating single bond in place of the B-ring of colchicine.     

Distinct and overlapping binding sites for IKP104 and vinblastine on tubulin

IKP104 is one of a group of tubulin-binding drugs whose interaction with tubulin suggests that it may bind to the protein at or close to the region where vinblastine binds. By itself IKP104 is a potent enhancer of tubulin decay as evidenced by the fact that it induces the exposure of the sulfhydryl groups and hydrophobic areas on tubulin. In this respect, IKP104 differs from vinblastine and other drugs such as phomopsin A, dolastatin 10, rhizoxin, and maytansine which are competitive or noncompetitive inhibitors of vinblastine binding. In contrast, however, in the presence of colchicine, IKP104 behaves differently and strongly stabilizes tubulin, to an extent much greater than does colchicine alone. IKP104 appears to have two classes of binding site on tubulin, differing in affinity; the acceleration of decay appears to be mediated by the low-affinity site (Chaudhuriet al., 1998,J. Protein Chem., in press). We investigated the relationship of the binding of IKP104 and vinblastine. We found that the high-affinity site or sites of IKP104 overlap with or interact with the vinblastine-binding sites, but that the low-affinity site is distinctly different.     

The effect of ruthenium red on the assembly and disassembly of microtubules and on rapid axonal transport

The assembly of microtubules was found to decrease in proportion to the amount of added ruthenium red, indicating a high affinity of ruthenium red for the microtubule system. An equimolar amount of ruthenium red per tubulin dimer inhibited the microtubule assembly completely and disassembled existing microtubules. Binding of ruthenium red to tubulin is accompanied by a shift in the absorption maximum from 535 to 538 nm. The binding is very strong, as shown by the finding that ruthenium red could not be displaced from tubulin by gel chromatography on Sephadex, or by the addition of Ca²⁺ or Mg²⁺. The binding of ruthenium red to tubulin did not affect the single colchicine site, nor the Mg²⁺ site(s), as shown by use of Mn²⁺ as an EPR probe. Ruthenium red also interfered with microtubules in an intact cell system, as it inhibited rapid axonal transport in the frog sciatic nerve, measured by the accumulation of [³H]leucine-labelled proteins in front of a ligature.     

Anti-microtubule ‘plinabulin’ chemical probe KPU-244-B3 labeled both α- and β-tubulin

Plinabulin (1, NPI-2358), a potent microtubule-targeting agent derived from the natural diketopiperazine ‘phenylahistin’ with a colchicine-like tubulin depolymerization activity, is an anticancer agent undergoing Phase II clinical trials in four countries including the United States. In order to understand the precise binding mode of plinabulin with tubulin, a new bioactive biotin-tagged photoaffinity probe 4 (KPU-244-B3) was designed and synthesized. Probe 4 showed significant binding affinity to tubulin in a binding assay, and selectively bound to tubulin in an HT-1080 cell lysate without photo-irradiation. In a tubulin photoaffinity labeling study, probe 4 labeled both α- and β-tubulin subunits and these interactions were competitively inhibited by plinabulin during photo-irradiation. These results suggest that plinabulin binds in the boundary region between α- and β-tubulin near the colchicine binding site, and not inside the colchicine binding cavity.     

Immunological detection of microtubule poison-induced conformational changes in tubulin

The interaction of tubulin-microtubule poison complexes with anti-tubulin antisera has been investigated using radioimmunoassay. The binding of the major antiserum used in this study to tubulin does not interfere with the binding of colchicine to the tubulin or affect the decay of the colchicine-binding activity of the tubulin. Conversely, if colchicine is incubated with the tubulin, forming tubulin-colchicine complexes, the tubulin-colchicine complexes are less efficient competitors for antibody-binding sites than tubulin alone. This is the result of the formation of specific colchicine-tubulin complexes, since tubulin, incubated with lumicolchicine or isocolchicine, behaves as if the tubulin were incubated alone in the radioimmunoassay. When tubulin is incubated with other microtubule poisons, podophyllotoxin or vinblastine, the tubulin-drug complexes have diminished ability to compete with tubulin as did the tubulin-colchicine complexes. These changes observed in the binding of tubulin-microtubule poison complexes to anti-tubulin antisera in a tubulin radioimmunoassay suggest that the binding of colchicine, podophyllotoxin, or vinblastine to tubulin induces subtle conformational changes on the surface of the tubulin dimer involving antigenic determinant sites.     

Binding of antimitotic drugs around cysteine residues of tubulin

Two independent approaches provide evidence of cysteine residues in the vicinity of the binding sites of colchicine and vinblastine to tubulin: (1) The reactive bromoacetamide group of the affinity label bromocolchicine covalently binds to cysteine residues of tubulin; (2) vinblastine and colchicine slow down the reaction of DTNB with SH groups of tubulin.     

Anion-induced increases in the rate of colchicine binding to tubulin.

The rate of binding of colchicine to tubulin to tubulin is enhanced by certain anions. Among the inorganic anions tested, only sulfate was effective. The organic anions include mostly dicarboxylic acids, among which tartrate was the most effective. This effect occurs onlt at low concentrations of colchicine (less than 0.6 X 10(-5) M). The rate increase dor sulfate and L-(+)-tartrate is ca. 2.5-fold at 1.0 mM and plateaus at a limiting value of ca. 4-fold at 100mM. The overall dissociation rate of the colchicine from the complex, which includes both the true rate of dissociation and the rate of irreversible denaturation of tubulin, is not influenced by 1.0 mM tartrate. The affinity constants for colchicine determined from the rate constants are 8.7 X 10(6) and 2.1 X 10(7) M-1 in the absence and the presence of 1.0 mM L-(+)-tartrate. The limiting value is 3.2 X 10(7) M-1. The affinity constant calculated from steady-state measurements is 3.2 X 10(6) M-1 with or without anions. The binding of other ligands like podophyllotoxin, vinblastine, and 1 -anilino-8-naphthalenesulfonate to tubulin is not affected by tartrate. No major conformational changes resulting from anion treatment could be detected by circular dichroism or intrinsic fluorescence. However, the ability of tubulin to polymerize is inhibited by L-(+)-tartrate at concentrations that increase the rate of colchicine binding. We conclude that anions must have a local effect at or near the binding site which enhances the binding rate of colchicine and which may be related to inhibition of polymerization.