Mechanism of interaction of vinca alkaloids with tubulin: catharanthine and vindoline

The interactions of the vinca alkaloid drugs catharanthine and vindoline with tubulin have been investigated and compared with those of vinblastine and vincristine. Both drugs were found to be less effective in bringing about the inhibition of tubulin self-assembly into microtubules than vincristine and vinblastine, the drug to protein molar ratio required being 3 orders of magnitude greater. An analytical ultracentrifuge study has shown that catharanthine can induce the self-association of tubulin into linear indefinite polymers with an efficacy that is 75% that of vinblastine or vincristine, the intrinsic dimerization constant for the liganded protein being K2 congruent to 1 x 10(5) M-1. The effect of vindoline was marginally detectable. Binding studies of catharanthine using the gel batch and fluorescence perturbation techniques showed a polymerization-linked binding of one catharanthine molecule per tubulin alpha-beta dimer with a binding constant of (2.8 +/- 0.4) x 10(3) M-1. For vindoline, binding to tubulin was marginally detectable by fluorescence spectroscopy, although addition of vindoline to tubulin did generate a difference spectrum. It was concluded that the binding of vinblastine/vincristine to tubulin and its consequences are determined by the interaction of the indole part of catharanthine with tubulin, the role of vindoline being that of an anchor.     

Interaction of vinblastine with calf brain tubulin: multiple equilibria

The binding of the anticancer drug vinblastine to calf brain tubulin was measured by a batch gel filtration method in PG buffer (0.01 M NaPi, 10(-4) M GTP, pH 7.0) at three different protein concentrations. The Scatchard binding isotherms obtained were curvilinear. The binding of the first vinblastine molecule to each tubulin alpha-beta dimer (Mr 110,000) was enhanced by an increase in the protein concentration. Additional binding of vinblastine to the protein was independent of the protein concentration. Theoretical ligand binding isotherms were calculated for a ligand-induced macromolecule self-association involving various ligand stoichiometries and association schemes. Fitting of the experimental data to these isotherms showed that the system can be described best by a one-ligand-induced isodesmic indefinite self-association. The pathway giving the best fit consists of a ligand-mediated plus -facilitated self-association mechanism. The self-association-linked bound vinblastine binds specifically at a site with an intrinsic binding constant K1 = 4 X 10(4) M-1. Additional vinblastine molecules can bind less strongly to tubulin in probably nonspecific fashion, and the previous reports of two specific sites on alpha-beta tubulin for binding vinblastine are incorrect. The self-association constant K2 for liganded tubulin is 1.8 X 10(5) M-1. This analysis is fully consistent with the conclusions derived earlier from the linked function analysis of the vinblastine-induced tubulin self-association [Na, G. C., & Timasheff, S. N. (1980) Biochemistry 19, 1347-1354; Na, G. C., & Timasheff, S. N. (1980) Biochemistry 19, 1355-1365].     

Vincristine-induced self-association of calf brain tubulin

The vincristine-induced self-association of tubulin has been examined in a sedimentation velocity study as a function of free drug concentration in PG buffer (0.01 M NaPi and 10(-4) M GTP, pH 7.0) at 20 degrees C. Analysis of the weight-average sedimentation coefficient (S20,w) as a function of protein concentration showed a good fit with the model of an indefinite, isodesmic self-association mechanism. Analysis of the apparent association constants in terms of the Wyman linkage relations showed a good fit to mediation of the self-association by the binding of one ligand molecule. The intrinsic association constant for dimerization of the vincristine-liganded tubulin was found to be 3.8 X 10(5) M-1, and the intrinsic equilibrium constant for the binding of the self-association-linked vincristine molecule had a value of 3.5 X 10(4) M-1, consistent with that measured by fluorescence in our laboratory [Prakash, V., & Timasheff, S. N. (1983) J. Biol. Chem. 258, 1689-1697]. Both reactions are stronger in the presence of vincristine than of vinblastine, reflecting the oxidation of a -CH3 group to -CHO when going from the latter drug to the former one.     

Aging of tubulin at neutral pH: stabilization by colchicine and its analogues.

The effect of colchicine and its analogues, allocolchicine, 2,3,4-trimethoxy-4'-carbomethoxy-1,1'biphenyl, 2,3,4,4'-tetramethoxy-1,1'-biphenyl, 2,3,4-trimethoxy-4'-acetyl-1,1'-biphenyl, and tropolone methyl ether, on the aging process of tubulin has been examined. In contrast to the vinca alkaloid drugs which accelerate the formation of the paucidisperse 9 S polymers by a factor of 3.5, the colchicine class of ligands stabilize alpha,beta-tubulin. Less than 10% of the protein is transformed into the aggregates after 50 h of incubation in the presence of 1 x 10(-3) M colchicine, as compared to nearly 70-75% transformation in its absence. These results are supported by fluorescence examination of the retention of colchicine binding ability, as well as circular dichroism spectroscopy. In the presence of colchicine, the rate determining step is a conformational change, just as in its absence. The colchicine analogues which bind to tubulin in a rapidly reversible equilibrium were almost as effective in tubulin stabilization. Addition of vincristine to the system reduced the stability of the tubulin-colchicine complex. Furthermore, vincristine was found to have the same effects on the fresh complex as it does on pure tubulin; i.e., it induced the isodesmic linear polymerization and inhibited assembly into the microtubule-mimicking large polymers. This inhibition, however, was stoichiometric, whereas it is substoichiometric in the case of microtubules.     

Proteome analysis of vinca alkaloid response and resistance in acute lymphoblastic leukemia reveals novel cytoskeletal alterations

Vinca alkaloids are used widely in the treatment of both childhood and adult cancers. Their cellular target is the beta-tubulin subunit of alpha/beta-tubulin heterodimers, and they act to inhibit cell division by disrupting microtubule dynamics. Despite the effectiveness of these agents, drug resistance is a major clinical problem. To identify the underlying mechanisms behind vinca alkaloid resistance, we have performed high resolution differential proteome analysis. Treatment of drug-sensitive human leukemia cells (CCRF-CEM) with vincristine identified numerous proteins involved in the cellular response to vincristine. In addition, differential protein expression was analyzed in leukemia cell lines selected for resistance to vincristine (CEM/VCR R) and vinblastine (CEM/VLB100). This combined proteomic approach identified 10 proteins altered in both vinca alkaloid response and resistance: beta-tubulin, alpha-tubulin, actin, heat shock protein 90beta, 14-3-3tau, 14-3-3epsilon, L-plastin, lamin B1, heterogeneous nuclear ribonuclear protein-F, and heterogeneous nuclear ribonuclear protein-K. Several of these proteins have not previously been associated with drug resistance and are thus novel targets for elucidation of resistance mechanisms. In addition, seven of these proteins are associated with the tubulin and/or actin cytoskeletons. This study provides novel insights into the interrelationship between the microtubule and microfilament systems in vinca alkaloid resistance.     

Linkage between ligand binding and control of tubulin conformation.

The effect of both antimitotic drugs and nucleotide analogues on the magnesium-induced self-association of purified tubulin into 42S double rings has been examined by sedimentation velocity. In the absence of magnesium, all complexes sedimented as the 5.8S species. The binding of colchicine to tubulin led to a small but consistent (-0.1 to -0.2 kcal/mol) enhancement in the self-association of tubulin alpha-beta dimers. In the absence of nucleotide at the exchangeable site, tubulin retained a weak ability (K2 = 7.5 x 10(3) M-1) to self-associate, which was unchanged by the addition of guanosine or GMP. Analogues with altered P-O-P bonds (GMPPCP, GMPPNP) did not support ring formation at the protein concentrations examined, although GMPPCP supported microtubule assembly. When the exchangeable site was occupied by nucleotides altered on the gamma-phosphate (GTP gamma S, GTP gamma F), rings were formed; tubulin-GTP gamma F formed rings to an extent slightly greater than did tubulin-GTP, and tubulin-GTP gamma S to about the same extent as tubulin-GDP. Both of these analogues are inhibitors of microtubule assembly. These results are consistent with a model [Melki, R., Carlier, M.-F., Pantaloni, D., & Timasheff, S. N. (1989) Biochemistry 28, 9143-9152] in which an equilibrium exists between straight (microtubule-forming) and curved (ring-forming) conformations of tubulin. Furthermore, the present results indicate that the "switch" which controls the nature of the final polymeric product via free energy linkages is the occupancy of the gamma-phosphate binding locus of the exchangeable site by a properly coordinated metal-nucleotide complex.     

The mechanism of action of vinblastine. Binding of [acetyl-3H]vinblastine to embryonic chick brain tubulin and tubulin from sea urchin sperm tail outer doublet microtubules.

Tritium-labeled viblastine, specific activity 107 Ci/mol, was prepared by acetylation of desacetylvinblastine with [3H]acetic anhydride, and has been employed in a study of vinblastine binding to tubulin. There are two high affinity vinblastine-binding sites per mole of embryonic chick brain tubulin (KA = 3-5 X 10(5) l./mol). Binding to these sites was rapid, and relatively independent of temperature between 37 and 0degreeC. Vincristin sulfate and desacetylvinblastine sulfate, two other active vinca alkaloid derivatives, competitively inhibited the binding of vinblastine. The inhibition constant for vincristine was 1.7 X 10(-5) M; and for desacetylvinblastine, 2 X 10(-5) M. The vinblastine binding activity of tubulin decayed upon aging, but this property was not studied in detail. Vinblastine did not depolymerize stable sea urchin sperm tail outer doublet microtubules, nor did it bind to these microtubules. However, tubulin solubilized from the B subfiber of the outer doublet microtubules possessed the two high affinity binding sites (KA = 1-3 X 105 l./mol). These data suggest that vinblastine destroys microtubules in cells primarily by inhibition of microtubule polymerization, and does not directly destroy preformed microtubules.     

Binding of dolastatin 10 to tubulin at a distinct site for peptide antimitotic agents near the exchangeable nucleotide and vinca alkaloid sites

Dolastatin 10, a potent antimitotic peptide from a marine animal, strongly inhibits microtubule assembly, tubulin-dependent GTP hydrolysis, and the binding of vinca alkaloids to tubulin. In studies of the binding of [3H]vincristine to the protein, with vinblastine as a control for competitive inhibition (Ki, 6.6 microM), we found that the macrolide antimitotic agents maytansine and rhizoxin were also competitive inhibitors (Ki values, 3.1 and 12 microM). Dolastatin 10 and an unrelated peptide antimitotic, phomopsin A, were more potent but noncompetitive inhibitors (Ki values, 1.4 and 2.8 microM). Since maytansine and, to a much lesser extent, vinblastine interfere with nucleotide exchange on tubulin, all drugs were examined for effects on nucleotide interactions at the exchangeable GTP site. Rhizoxin had effects intermediate between those of vinblastine and maytansine. Both peptides inhibited binding of radiolabeled GTP to tubulin even more strongly than did maytansine, but no drug displaced nucleotide from tubulin. The drugs were evaluated for stabilizing effects on the colchicine binding activity of tubulin. The peptides prevented loss of this activity, and vinblastine provided partial protection, while rhizoxin and maytansine did not stabilize tubulin. A tripeptide segment of dolastatin 10 also effectively inhibits tubulin polymerization and GTP hydrolysis. The tripeptide did not significantly inhibit either vincristine binding or nucleotide exchange, nor did it stabilize colchicine binding. These findings are rationalized in terms of a model with two distinct drug binding sites in close physical proximity to each other and to the exchangeable GTP site on beta-tubulin.     

Photoaffinity labeling of tubulin subunits with a photoactive analogue of vinblastine

A photoactive, radioactive analogue of vinblastine, N-(p-azido[3,5-3H]benzoyl)-N'-(beta-amino-ethyl)vindesine ([ 3H]NABV), was used to localize the Vinca alkaloid binding site(s) on calf brain tubulin after establishing that its in vitro interactions with tubulin were comparable to those of vinblastine. Microtubule assembly was inhibited by 50% with 2 microM NABV or vinblastine. At higher drug concentrations, NABV and vinblastine both induced tubulin aggregation, and both drugs inhibited tubulin-dependent GTP hydrolysis. Vinblastine and NABV inhibited each other's binding to tubulin, but the binding of neither drug was inhibited by colchicine. Two classes of binding sites for NABV and vinblastine were found on calf brain tubulin. High-affinity sites had apparent KD values of 4.2 and 0.54 microM for NABV and vinblastine, respectively, whereas the low-affinity binding sites showed apparent KD values of 26 and 14 microM for NABV and vinblastine, respectively. Mixtures of tubulin and [3H]NABV were irradiated at 302 nm and analyzed for incorporation of radioactivity into protein. Photolabeling of both the alpha- and beta-subunits of tubulin with increasing concentrations of [3H]NABV exhibited a biphasic pattern characteristic of specific and nonspecific reactions. Nonspecific labeling was determined in the presence of excess vinblastine. Saturable specific covalent incorporation into both subunits of tubulin was observed, with an alpha:beta ratio of 3:2 and maximum saturable incorporation of 0.086 and 0.056 mol of [3H]NABV/mol of alpha-tubulin and beta-tubulin, respectively. Such photolabeling of the tubulin subunits will permit precise localization of Vinca alkaloid binding sites, including identification of the amino acid residues involved, an essential requirement for understanding the interactions of these drugs with tubulin.     

Interaction of tubulin with a new fluorescent analogue of vinblastine

Vinblastine is an antimitotic agent that has been used extensively in cancer chemotherapy. The biological effects of the drug are believed to be the result of its interaction with tubulin, the major component of cellular microtubules. Fluorescence spectroscopy is a powerful and versatile technique for studying drug-tubulin interactions, but it rarely has been applied to studies involving vinca alkaloids. We have prepared a new fluorescent derivative of vinblastine designed to retain high affinity for tubulin while possessing a fluorophore that absorbs and emits visible light. A coumarin derivative of vinblastine, 17-deacetyl-O-(3-carbonylamino-7-diethylaminocoumarin) vinblastine (F-VLB), was prepared by reaction of 17-deacetylvinblastine with 7-diethylaminocoumarin-3-carbonyl azide. F-VLB was a potent inhibitor of in vitro microtubule assembly (IC(50) = 0.5 microM). F-VLB binding to tubulin was inhibited by vinblastine. Tubulin binding induced an increase in the F-VLB emission intensity and shifted the emission maximum to higher energy (from 500 to 480 nm). The Stokes shift of tubulin-bound F-VLB was about the same as the Stokes shift of the molecule in ethanol, indicating that the tubulin-bound fluorophore is probably on the exterior of the vinblastine binding site. Unlike vinblastine, F-VLB failed to induce self-assembly of tubulin that could be detected by light scattering or electron microscopy, although some self-association could be detected by analytical ultracentrifugation. Equilibrium binding parameters were quantitatively determined by monitoring the change in fluorescence anisotropy of F-VLB upon tubulin binding. The apparent equilibrium constant for F-VLB binding to tubulin [K(a)(app) = (7.7 +/- 0.5) x 10(4) M(-1) at 25 degrees C] was identical to the equilibrium constant for vinblastine binding to 2 microM tubulin (K(1)) measured under similar buffer and temperature conditions using ultracentrifugation [Vulevic, B., Lobert, S., and Correia, J. J. (1997) Biochemistry 36, 12828-12835]. Binding allocolchicine to tubulin did not significantly affect F-VLB's affinity for the protein [K(a)(app) = (9.1 +/- 0.4) x 10(4) M(-1) at 25 degrees C]. Analysis of the steady-state emission spectra yielded a distance between the colchicine and vinca binding sites on tubulin of approximately 40 A. F-VLB bound to paclitaxel- and glutaraldehyde-stabilized microtubules, with approximately equal affinity. We conclude that F-VLB can be used to obtain information about the vinblastine binding site on tubulin under equilibrium conditions.     

A study of tubulin dimer conformation by near-UV circular dichroism

The near-UV circular dichroism properties of tubulin dimer have been measured for different preparative methods. Tubulin dimer was obtained from assembly compenent microtubule protein by gel filtration, or phosphocellulose ion-exchange chromatography in the presence of magnesium. Tubulin dimer prepared by the protocol of Weisenberg R.C. and Timasheff, S.N. (1970) Biochemistry 9, 4110–4116, was found to be markedly different due to some apparently irreversible change in conformation. We conclude that the removal of microtubule-associated proteins by phosphocellulose ion-exchange chromatography in the presence of magnesium can be performed without affecting the conformation of native tubulin dimer as judged by near-UV circular dichroism.     

Calmodulin, activated cyclic nucleotide phosphodiesterase, microtubules, and vinca alkaloids

Calmodulin 1.8 and 10.6 microM, inhibited the polymerization of bovine brain microtubules by 30 and 50%, respectively. Two 55,000- to 68,000-dalton calmodulin-binding protein as well as calmodulin-dependent and -independent phosphodiesterases (PDE) were found associated with microtubule proteins. Among the antimicrotubule drugs, such as colchicine, podophyllotoxin, griseofulvin, and vinca alkaloids (vinblastine, desacetylvinblastine amide, and vincristine), the vinca alkaloids were selective inhibitors of calmodulin-activated PDE activity. This action of vinca alkaloids resides in the catharanthine moiety of vinblastine molecule. An alpha 2 inhibitor, yohimbine, affects the microtubules, and a series of alpha-adrenoceptor blocking agents were examined for their effects on calmodulin-dependent PDE. The relative order of the potency is phenoxybenzamine = dibenamine greater than phentolamine greater than yohimbine greater than prazosin greater than tolazoline, and the first four drugs in this series were selective inhibitors of calmodulin action. Inasmuch as phenoxybenzamine and dibenamine are alkylating agents, the effects of antineoplastic alkylating agents on the calmodulin action were also examined. Busulphan, melphalan, 1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea, and streptozotocin (up to 4 mM) were not selective inhibitors of calmodulin action. Maytansine, a vinca alkaloid-type antimicrotubule agents as well as an alkylating agent, selectively inhibited the calmodulin with a potency similar to vincristine. In addition, phenoxybenzamine affected Ca2+-dependent fluorescence induced by the interaction between calmodulin and hydrophobic fluorescent probes, whereas vinblastine was ineffective. However, the binding of vinblastine to calmodulin is calcium dependent. Studies such as these suggest the importance of physical and structural considerations in drugs binding to calmodulin as well as at least two different binding sites for drugs on calmodulin.     

Interaction of tubulin with bifunctional colchicine analogues: an equilibrium study

The interaction of tubulin with simple analogues of colchicine that contain both its tropolone and trimethoxyphenyl rings has been characterized, and the results were analyzed in terms of the simple bifunctional ligand model developed for the binding of colchicine [ Andreu , J. M., & Timasheff , S. N. (1982) Biochemistry 21, 534-543] on the basis of interactions of tubulin with single-ring analogues. The compound 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6- cycloheptatrien -1-one has been found to bind reversibly to 0.86 +/- 0.06 site of purified calf brain tubulin with an equilibrium constant of (4.9 +/- 0.3) X 10(5) M-1 (25 degrees C), delta H degrees app = -1.6 +/- 0.7 kcal mol-1, and delta S degrees app = 20.5 +/- 2.5 eu. The binding appears specific for the colchicine site. The closely related compound 2-methoxy-5-[[3-(3,4,5-trimethoxyphenyl)-propionyl]amino] -2,4,6- cycloheptatrien -1-one interacts weakly with tubulin. Binding of the first analogue is accompanied by ligand fluorescence appearance, quenching of protein fluorescence, perturbation of the far-ultraviolet circular dichroism of tubulin, and induction of the tubulin GTPase activity, similarly to colchicine binding. Substoichiometric concentrations of the analogue inhibit microtubule assembly in vitro. Excess analogue concentration under microtubule-promoting conditions induces an abnormal cooperative polymerization of tubulin, similar to that of the tubulin-colchicine complex.     

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.     

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.     

GDP state of tubulin: stabilization of double rings

Purified tubulin, with GDP occupying the exchangeable nucleotide binding site, has been examined conformationally and for its ability to self-associate into double rings. The circular dichroism spectrum increased by ca. 10% in negative amplitude between 205 and 225 nm over the spectrum of tubulin in the GTP state, but there were no significant shape changes. This indicates that replacement of GTP by GDP induces tubulin to adopt a more ordered conformation. The sedimentation coefficients of tubulin alpha-beta dimers in the GDP and GTP states were identical, with s20,w = 5.8 S. A sedimentation velocity study of tubulin in the GDP state showed that, in the presence of magnesium ions, this protein undergoes a reversible Gilbert-type self-association. The end product of this reaction was found to be 26 subunit double rings identical with those described by Frigon and Timasheff [(1975) Biochemistry 14, 4567-4599] for a similar polymerization of tubulin in the GTP state. Analysis of the data showed that Tu-GDP has a much stronger propensity for the formation of double rings than Tu-GTP, the corresponding equilibrium with constants for the 26Tu in equilibrium Tu26 being 4.2 X 10(119) M-25 and 2.27 X 10(109) M-25 for Tu-GDP and Tu-GTP, respectively. This leads to Tu-GTP being predominantly in the form of alpha-beta dimers and Tu-GDP in the form of double rings under normal experimental conditions used in the study of microtubule assembly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural insight into the inhibition of tubulin by vinca domain peptide ligands

The tubulin vinca domain is the target of widely different microtubule inhibitors that interfere with the binding of vinblastine. Although all these ligands inhibit the hydrolysis of GTP, they affect nucleotide exchange to variable extents. The structures of two vinca domain antimitotic peptides--phomopsin A and soblidotin (a dolastatin 10 analogue)--bound to tubulin in a complex with a stathmin-like domain show that their sites partly overlap with that of vinblastine and extend the definition of the vinca domain. The structural data, together with the biochemical results from the ligands we studied, highlight two main contributors in nucleotide exchange: the flexibility of the tubulin subunits' arrangement at their interfaces and the residues in the carboxy-terminal part of the beta-tubulin H6-H7 loop. The structures also highlight common features of the mechanisms by which vinca domain ligands favour curved tubulin assemblies and destabilize microtubules.     

Heat capacity microcalorimetry of the in vitro reconstitution of calf brain microtubules.

The self-assembly of calf brain tubulin, purified by the modified Weisenberg procedure, was examined in an adiabatic differential heat capacity microcalorimeter. Tubulin solutions at concentrations between 6 and 17 mg/mL were heated from 8 to 40 degrees C at heating rates between 0.1 and 1.0 deg/min in a pH 7.0 phosphate buffer containing 1 X 10(-3) M GTP, 1.6 X 10(-2) M MgCl2, and 3.4 M glycerol. The heat capacity change, deltaCp of the microtubule growth reaction was found to be -1600 +/- 500 cal/(deg mol) per 110 000 molecular weight tubulin dimer incorporated into microtubules, in agreement with the reported van't Hoff deltaCp value of -1500 cal/(deg mol) [Lee, J.C., & Timasheff, S.N. (1977) Biochemistry 16, 1754-1765]. The assembly reaction is characterized by a complex heat uptake pattern comprising both endothermic and exothermic processes.     

Steric exclusion is the principal source of the preferential hydration of proteins in the presence of polyethylene glycols.

The preferential interactions of bovine serum albumin, lysozyme, chymotrypsinogen, ribonuclease A, and beta-lactoglobulin with polyethylene glycols (PEGs) of molecular weight 200-6,000 have been measured by dialysis equilibrium coupled with high precision densimetry. All the proteins were found to be preferentially hydrated in all the PEGs, and the magnitude of the preferential hydration increased with increasing PEG size for each protein. The change in the chemical potentials of the proteins with the addition of the PEGs had highly positive values, indicating a strong thermodynamic destabilization of the system by the PEGs. A viscosity study of the PEGs showed them to be randomly coiled polymers, as their radii of gyration were related to the molecular weight by Rg = aM0.55. The thickness of the effective shell impenetrable to PEG around protein molecules, calculated from the preferential hydration, was found to vary with PEG molecular weight in similar fashion as the PEG radius of gyration, supporting the proposal (Arakawa, T. & Timasheff, S.N., 1985a, Biochemistry 24, 6756-6762) that the preferential exclusion of PEGs from proteins is due principally to the steric exclusion of PEG from the protein domain, although favorable interactions with protein surface residues, in particular nonpolar ones, may compete with the exclusion. These thermodynamically unfavorable preferential exclusion interactions lead to the action of PEGs as precipitants, although they may destabilize protein structure at higher temperatures.     

The thermodynamics of vinca alkaloid-induced tubulin spirals formation

Vinca alkaloids are antimitotic, anticancer agents that induce tubulin to form spiral polymers at physiological protein concentrations. We used sedimentation velocity to investigate the effects of six vinca alkaloids on tubulin spiraling. Fitting to a Wyman linkage model reveals a drug dependent change of over two orders of magnitude in spiraling potential, K(1)K(2). Thermodynamic analysis of LnK(1)K(2) data demonstrates large and positive DeltaS values, indicating that tubulin spiral formation is entropically-driven. From the curvature in van't Hoff plots of vinblastine data, we estimate DeltaC(p) for GTP and GDP conditions to be -439 and -396 cal/mol K. Partitioning of DeltaS into the hydrophobic effect, DeltaS(HE), change in rotational/translational freedom, DeltaS(RT) and change in protein conformation, DeltaS(other), demonstrates that the major driving force for tubulin spiral formation is burial of hydrophobic surfaces and that protein conformational changes do not make a significant contribution. Spiraling potential is an indicator of antimitotic activity in vivo, although turbidity studies indicate that there is no correlation between spiraling potential and microtubule inhibition in vitro. Mechanisms that explain this discrepancy are discussed.