Mutational analysis of op18/stathmin-tubulin-interacting surfaces. Binding cooperativity controls tubulin GTP hydrolysis in the ternary complex

Oncoprotein 18 (Op18) is a microtubule regulator that forms a ternary complex with two tubulin heterodimers. Dispersed regions of Op18 are involved in two-site cooperative binding and subsequent modulation of tubulin GTPase activity. Here we have analyzed specific determinants of Op18 that govern both stoichiometry and positive cooperativity in tubulin binding and consequent stimulatory and inhibitory effects on tubulin GTPase activity. The data revealed that the central and C-terminal regions of Op18 contain overlapping binding-motifs contacting both tubulin heterodimers, suggesting that these regions of Op18 are wedged into the previously noted 1-nm gap between the two longitudinally arranged tubulin heterodimers. Both the N- and C-terminal flanks adjacent to the central region are involved in stabilizing the ternary complex, but only the C-terminal flank does so by imposing positive binding cooperativity. Within the C-terminal flank, deletion of a 7-amino acid region attenuated positive binding cooperativity and resulted in a switch from stimulation to inhibition of tubulin GTP hydrolysis. This switch can be explained by attenuated binding cooperativity, because Op18 under these conditions may block longitudinal contact surfaces of single tubulins with consequent interference of tubulin-tubulin interaction-dependent GTP hydrolysis. Together, our results suggest that Op18 links two tubulin heterodimers via longitudinal contact surfaces to form a ternary GTPase productive complex.     

Deciphering the cellular functions of the Op18/Stathmin family of microtubule-regulators by plasma membrane-targeted localization

The Op18/stathmin family of microtubule regulators includes the ubiquitous cytosolic Op18/stathmin (Op18) and the neuronal, primarily Golgi-associated proteins SCG10 and RB3, which all form ternary complexes with two head-to-tail-aligned tubulin heterodimers. To understand the physiological significance of previously observed differences in ternary complex stability, we have fused each of the heterodimer-binding regions of these three proteins with the CD2 cell surface protein to generate confined plasma membrane localization of the resulting CD2 chimeras. Herein, we show that, in contrast to constitutively active CD2-Op18-tetraA, both the CD2-SCG10 and CD2-RB3 chimeras sequestered tubulin at the plasma membrane, which results in >35% reduction of cytosolic tubulin heterodimer levels and consequent delayed formation of mitotic spindles. However, all three CD2 chimeras, including the tubulin sequestration-incompetent CD2-Op18-tetraA, destabilize interphase microtubules. Given that microtubules are in extensive contact with the plasma membrane during interphase, but not during mitosis, these findings indicate that Op18-like proteins have the potential to destabilize microtubules by both sequestration and direct interaction with microtubules. However, the differences in tubulin binding observed in cells also indicate conceptual differences between the functions of low-abundance neural family members, which will accumulate tubulin at specific cellular compartments, and the abundant cytosolic Op18 protein, which will not.     

Stathmin and its phosphoprotein family: general properties, biochemical and functional interaction with tubulin

Stathmin, also referred to as Op18, is a ubiquitous cytosolic phosphoprotein, proposed to be a small regulatory protein and a relay integrating diverse intracellular signaling pathways involved in the control of cell proliferation, differentiation and activities. It interacts with several putative downstream target and/or partner proteins. One major action of stathmin is to interfere with microtubule dynamics, by inhibiting the formation of microtubules and/or favoring their depolymerization. Stathmin (S) interacts directly with soluble tubulin (T), which results in the formation of a T2S complex which sequesters free tubulin and therefore impedes microtubule formation. However, it has been also proposed that stathmin's action on microtubules might result from the direct promotion of catastrophes, which is still controversial. Phosphorylation of stathmin regulates its biological actions: it reduces its affinity for tubulin and hence its action on microtubule dynamics, which allows for example progression of cells through mitosis. Stathmin is also the generic element of a protein family including the neural proteins SCG10, SCLIP and RB3/RB3'/RB3". Interestingly, the stathmin-like domains of these proteins also possess a tubulin binding activity in vitro. In vivo, the transient expression of neural phosphoproteins of the stathmin family leads to their localization at Golgi membranes and, as previously described for stathmin and SCG10, to the depolymerization of interphasic microtubules. Altogether, the same mechanism for microtubule destabilization, that implies tubulin sequestration, is a common feature likely involved in the specific biological roles of each member of the stathmin family.     

Op18/stathmin mediates multiple region-specific tubulin and microtubule-regulating activities.

Oncoprotein18/stathmin (Op18) is a regulator of microtubule (MT) dynamics that binds tubulin heterodimers and destabilizes MTs by promoting catastrophes (i.e., transitions from growing to shrinking MTs). Here, we have performed a deletion analysis to mechanistically dissect Op18 with respect to (a) modulation of tubulin GTP hydrolysis and exchange, (b) tubulin binding in vitro, and (c) tubulin association and MT-regulating activities in intact cells. The data reveal distinct types of region-specific Op18 modulation of tubulin GTP metabolism, namely inhibition of nucleotide exchange and stimulation or inhibition of GTP hydrolysis. These regulatory activities are mediated via two-site cooperative binding to tubulin by multiple nonessential physically separated regions of Op18. In vitro analysis revealed that NH(2)- and COOH-terminal truncations of Op18 have opposite effects on the rates of tubulin GTP hydrolysis. Transfection of human leukemia cells with these two types of mutants result in similar decrease of MT content, which in both cases appeared independent of a simple tubulin sequestering mechanism. However, the NH(2)- and COOH-terminal-truncated Op18 mutants regulate MTs by distinct mechanisms as evidenced by morphological analysis of microinjected newt lung cells. Hence, mutant analysis shows that Op18 has the potential to regulate tubulin/MTs by more than one specific mechanism.     

Mutations of oncoprotein 18/stathmin identify tubulin-directed regulatory activities distinct from tubulin association

Oncoprotein 18/stathmin (Op18) is a recently identified phosphorylation-responsive regulator of the microtubule (MT) system. It was originally proposed that Op18 specifically regulates dynamic properties of MTs by associating with tubulin, but it has subsequently been proposed that Op18 acts simply by sequestering of tubulin heterodimers. We have dissected the mechanistic action of Op18 by generation of two distinct classes of mutants. One class has interruptions of the heptad repeats of a potential coiled-coil region of Op18, and the other involves substitution at all four phosphorylation sites with negatively charged Glu residues. Both types of mutation result in Op18 proteins with a limited decrease in tubulin complex formation. However, the MT-destabilizing activities of the coiled-coil mutants are more severely reduced in transfected leukemia cells than those of the Glu-substituted Op18 derivative, providing evidence for tubulin-directed regulatory activities distinct from tubulin complex formation. Analysis of Op18-mediated regulation of tubulin GTPase activity and taxol-promoted tubulin polymerization showed that while wild-type and Glu-substituted Op18 derivatives are active, the coiled-coil mutants are essentially inactive. This suggests that Op18-tubulin contact involves structural motifs that deliver a signal of regulatory importance to the MT system.     

Stathmin family proteins display specific molecular and tubulin binding properties

Stathmin family phosphoproteins (stathmin, SCG10, SCLIP, and RB3/RB3'/RB3") are involved in signal transduction and regulation of microtubule dynamics. With the exception of stathmin, they are expressed exclusively in the nervous system, where they display different spatio-temporal and functional regulations and hence play at least partially distinct and possibly complementary roles in relation to the control of development, plasticity, and neuronal activities. At the molecular level, each possesses a specific "stathmin-like domain" and, with the exception of stathmin, various combinations of N-terminal extensions involved in their association with intracellular membrane compartments. We show here that each stathmin-like domain also displays specific biochemical and tubulin interaction properties. They are all able to sequester two alpha/beta tubulin heterodimers as revealed by their inhibitory action on tubulin polymerization and by gel filtration. However, they differ in the stabilities of the complexes formed as well as in their interaction kinetics with tubulin followed by surface plasmon resonance as follows: strong stability and slow kinetics for RB3; medium for SCG10, SCLIP, and stathmin; and weak stability and rapid kinetics for RB3'. These results suggest that the fine-tuning of their stathmin-like domains contributes to the specific functional roles of stathmin family proteins in the regulation of microtubule dynamics within the various cell types and subcellular compartments of the developing or mature nervous system.     

Op18/Stathmin counteracts the activity of overexpressed tubulin-disrupting proteins in a human leukemia cell line

Op18/stathmin (Op18) is a phosphorylation-regulated and differentially expressed microtubule-destabilizing protein in animal cells. Op18 regulates tubulin monomer-polymer partitioning of the interphase microtubule system and forms complexes with tubulin heterodimers. Recent reports have shown that specific tubulin-folding cofactors and related proteins may disrupt tubulin heterodimers. We therefore investigated whether Op18 protects unpolymerized tubulin from such disruptive activities. Our approach was based on inducible overexpression of two tubulin-disrupting proteins, namely TBCE, which is required for tubulin biogenesis, and E-like, which has been proposed to regulate tubulin turnover and microtubule stability. Expression of either of these proteins was found to cause a rapid degradation of both alpha-tubulin and beta-tubulin subunits of unpolymerized, but not polymeric, tubulin heterodimers. We found that depletion of Op18 by means of RNA interference increased the susceptibility of tubulin to TBCE or E-like mediated disruption, while overexpressed Op18 exerted a tubulin-protective effect. Tubulin protection was shown to depend on Op18 levels, binding affinity, and the partitioning between tubulin monomers and polymers. Hence, the present study reveals that Op18 at physiologically relevant levels functions to preserve the integrity of tubulin heterodimers, which may serve to regulate tubulin turnover rates.     

The oncoprotein 18/stathmin family of microtubule destabilizers.

The past several years have seen major advances in our understanding of the mechanisms of microtubule destabilization by oncoprotein18/stathmin (Op18/stathmin) and related proteins. New structural information has clearly shown how members of the Op18/stathmin protein family bind tubulin dimers and suggests models for how these proteins stimulate catastrophe, the transition from microtubule growth to shortening. Regulation of Op18/stathmin by phosphorylation continues to capture much attention. Studies suggest that phosphorylation occurs in a localized fashion, resulting in decreased microtubule destabilizing activity near chromatin or microtubule polymer. A spatial gradient of inactive Op18/stathmin associated with chromatin or microtubules could contribute significantly to mitotic spindle assembly.     

Thermodynamics of the Op18/stathmin-tubulin interaction

Op18/stathmin (stathmin) is an intrinsically disordered protein involved in the regulation of the microtubule filament system. One function of stathmin is to sequester tubulin dimers into assembly incompetent complexes, and recent studies revealed two tubulin binding sites per stathmin molecule. Using high sensitivity isothermal titration calorimetry, we document that at 10 degrees C and under the conditions of 80 mM PIPES, pH 6.8, 1 mM EGTA, 1 mM MgCl2, 1 mM GTP these two binding sites are of equal affinity with an equilibrium binding constant of K0 = 6.0 x 10(6) m(-1). The obtained large negative molar heat capacity change of deltaCp0 = -860 cal mol(-1) K(-1) (referring to tubulin) for the tubulin-stathmin binding equilibrium suggests that the hydrophobic effect is the major driving force of the binding reaction. Replacing GTP by GDP on beta-tubulin had no significant effect on the thermodynamic parameters of the tubulin-stathmin binding equilibrium. The proposed pH-sensitive dual function of stathmin was further evaluated by circular dichroism spectroscopy and nuclear magnetic resonance. At low temperatures, stathmin was found to be extensively helical but devoid of any stable tertiary structure. However, in complex with two tubulin subunits stathmin adopts a stable conformation. Both the stability and conformation of the individual proteins and complexes were not significantly affected by small changes in pH. A 4-fold decrease in affinity of stathmin for tubulin was revealed at pH 7.5 compared with pH 6.8. This decrease could be attributed to a weaker binding of the C terminus of stathmin. These findings do not support the view that stathmin works as a pH-sensitive protein.     

Op18/stathmin caps a kinked protofilament-like tubulin tetramer

Oncoprotein 18/stathmin (Op18), a regulator of microtubule dynamics, was recombinantly expressed and its structure and function analysed. We report that Op18 by itself can fold into a flexible and extended alpha-helix, which is in equilibrium with a less ordered structure. In complex with tubulin, however, all except the last seven C-terminal residues of Op18 are tightly bound to tubulin. Digital image analysis of Op18:tubulin electron micrographs revealed that the complex consists of two longitudinally aligned alpha/beta-tubulin heterodimers. The appearance of the complex was that of a kinked protofilament-like structure with a flat and a ribbed side. Deletion mapping of Op18 further demonstrated that (i) the function of the N-terminal part of the molecule is to 'cap' tubulin subunits to ensure the specificity of the complex and (ii) the complete C-terminal alpha-helical domain of Op18 is necessary and sufficient for stable Op18:tubulin complex formation. Together, our results suggest that besides sequestering tubulin, the structural features of Op18 enable the protein specifically to recognize microtubule ends to trigger catastrophes.     

Effect of tubulin self-association on GTP hydrolysis and nucleotide exchange reactions

We investigated how the self-association of isolated tubulin dimers affects the rate of GTP hydrolysis and the equilibrium of nucleotide exchange. Both reactions are relevant for microtubule (MT) dynamics. We used HPLC to determine the concentrations of GDP and GTP and thereby the GTPase activity of SEC-eluted tubulin dimers in assembly buffer solution, free of glycerol and tubulin aggregates. When GTP hydrolysis was negligible, the nucleotide exchange mechanism was studied by determining the concentrations of tubulin-free and tubulin-bound GTP and GDP. We observed no GTP hydrolysis below the critical conditions for MT assembly (either below the critical tubulin concentration and/or at low temperature), despite the assembly of tubulin 1D curved oligomers and single-rings, showing that their assembly did not involve GTP hydrolysis. Under conditions enabling spontaneous slow MT assembly, a slow pseudo-first-order GTP hydrolysis kinetics was detected, limited by the rate of MT assembly. Cryo-TEM images showed that GTP-tubulin 1D oligomers were curved also at 36 °C. Nucleotide exchange depended on the total tubulin concentration and the molar ratio between tubulin-free GDP and GTP. We used a thermodynamic model of isodesmic tubulin self-association, terminated by the formation of tubulin single-rings to determine the molar fractions of dimers with exposed and buried nucleotide exchangeable sites (E-sites). Our analysis shows that the GDP to GTP exchange reaction equilibrium constant was an order-of-magnitude larger for tubulin dimers with exposed E-sites than for assembled dimers with buried E-sites. This conclusion may have implications on the dynamics at the tip of the MT plus end.     

Design and characterization of modular scaffolds for tubulin assembly

In cells, microtubule dynamics is regulated by stabilizing and destabilizing factors. Whereas proteins in both categories have been identified, their mechanism of action is rarely understood at the molecular level. This is due in part to the difficulties faced in structural approaches to obtain atomic models when tubulin is involved. Here, we design and characterize new stathmin-like domain (SLD) proteins that sequester tubulins in numbers different from two, the number of tubulins bound by stathmin or by the SLD of RB3, two stathmin family members that have been extensively studied. We established rules for the design of tight tubulin-SLD assemblies and applied them to complexes containing one to four tubulin heterodimers. Biochemical and structural experiments showed that the engineered SLDs behaved as expected. The new SLDs will be tools for structural studies of microtubule regulation. The larger complexes will be useful for cryo-electron microscopy, whereas crystallography or nuclear magnetic resonance will benefit from the 1:1 tubulin-SLD assembly. Finally, our results provide new insight into SLD function, suggesting that a major effect of these phosphorylatable proteins is the programmed release of sequestered tubulin for microtubule assembly at the specific cellular locations of members of the stathmin family.     

The 4 A X-ray structure of a tubulin:stathmin-like domain complex

Phosphoproteins of the stathmin family interact with the alphabeta tubulin heterodimer (tubulin) and hence interfere with microtubule dynamics. The structure of the complex of GDP-tubulin with the stathmin-like domain of the neural protein RB3 reveals a head-to-tail assembly of two tubulins with a 91-residue RB3 alpha helix in which each copy of an internal duplicated sequence interacts with a different tubulin. As a result of the relative orientations adopted by tubulins and by their alpha and beta subunits, the tubulin:RB3 complex forms a curved structure. The RB3 helix thus most likely prevents incorporation of tubulin into microtubules by holding it in an assembly with a curvature very similar to that of the depolymerization products of microtubules.     

Tubulin exchanges divalent cations at both guanine nucleotide-binding sites

The tubulin heterodimer binds a molecule of GTP at the nonexchangeable nucleotide-binding site (N-site) and either GDP or GTP at the exchangeable nucleotide-binding site (E-site). Mg2+ is known to be tightly linked to the binding of GTP at the E-site (Correia, J. J., Baty, L. T., and Williams, R. C., Jr. (1987) J. Biol. Chem. 262, 17278-17284). Measurements of the exchange of Mn2+ for bound Mg2+ (as monitored by atomic absorption and EPR) demonstrate that tubulin which has GDP at the E-site possesses one high affinity metal-binding site and that tubulin which has GTP at the E-site possesses two such sites. The apparent association constants are 0.7-1.1 x 10(6) M-1 for Mg2+ and approximately 4.1-4.9 x 10(7) M-1 for Mn2+. Divalent cations do bind to GDP at the E-site, but with much lower affinity (2.0-2.3 x 10(3) M-1 for Mg2+ and 3.9-6.6 x 10(3) M-1 for Mn2+). These data suggest that divalent cations are involved in GTP binding to both the N- and E-sites of tubulin. The N-site metal exchanges slowly (kapp = 0.020 min-1), suggesting a mechanism involving protein "breathing" or heterodimer dissociation. The N-site metal exchange rate is independent of the concentration of protein and metal, an observation consistent with the possibility that a dynamic breathing process is the rate-limiting step. The exchange of Mn2+ for Mg2+ has no effect on the secondary structure of tubulin at 4 degrees C or on the ability of tubulin to form microtubules. These results have important consequences for the interpretation of distance measurements within the tubulin dimer using paramagnetic ions. They are also relevant to the detailed mechanism of divalent cation release from microtubules after GTP hydrolysis.     

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.     

Mechanism of the microtubule GTPase reaction

The rate of GTP hydrolysis by microtubules has been measured at tubulin subunit concentrations where microtubules undergo net disassembly. This was made possible by using microtubules stabilized against disassembly by reaction with ethylene glycol bis-(succinimidylsuccinate) (EGS) as sites for the addition of tubulin-GTP subunits. The tubulin subunit concentration was varied from 25 to 90% of the steady state concentration, and there was no net elongation of stabilized microtubule seeds. The GTPase rate with EGS microtubules was linearly proportional to the tubulin-GTP subunit concentration when this concentration was varied by dilution and by using GDP to compete with GTP for the tubulin E-site. The linear dependence of the rate is consistent with a GTP mechanism in which hydrolysis is coupled to the tubulin-GTP subunit addition to microtubule ends. It is inconsistent with reaction schemes in which: microtubules are capped by a single tubulin-GTP subunit, which hydrolyzes GTP when a tubulin-GTP subunit adds to the end; hydrolysis occurs primarily in subunits at the interface of a tubulin-GTP cap and the tubulin-GDP microtubule core; hydrolysis is not coupled to subunit addition and occurs randomly in subunits in a tubulin-GTP cap. It was also found that GDP inhibition of the microtubule GTPase rate results from GDP competition for GTP at the tubulin subunit E-site. There is no additional effect of GDP on the GTPase rate resulting from exchange into tubulin subunits at microtubule ends.     

Dissociation of the Tubulin-sequestering and Microtubule Catastrophe-promoting Activities of Oncoprotein 18/Stathmin

Oncoprotein 18/stathmin (Op18) has been identified recently as a protein that destabilizes microtubules, but the mechanism of destabilization is currently controversial. Based on in vitro microtubule assembly assays, evidence has been presented supporting conflicting destabilization models of either tubulin sequestration or promotion of microtubule catastrophes. We found that Op18 can destabilize microtubules by both of these mechanisms and that these activities can be dissociated by changing pH. At pH 6.8, Op18 slowed microtubule elongation and increased catastrophes at both plus and minus ends, consistent with a tubulin-sequestering activity. In contrast, at pH 7.5, Op18 promoted microtubule catastrophes, particularly at plus ends, with little effect on elongation rates at either microtubule end. Dissociation of tubulin-sequestering and catastrophe-promoting activities of Op18 was further demonstrated by analysis of truncated Op18 derivatives. Lack of a C-terminal region of Op18 (aa 100–147) resulted in a truncated protein that lost sequestering activity at pH 6.8 but retained catastrophe-promoting activity. In contrast, lack of an N-terminal region of Op18 (aa 5–25) resulted in a truncated protein that still sequestered tubulin at pH 6.8 but was unable to promote catastrophes at pH 7.5. At pH 6.8, both the full length and the N-terminal–truncated Op18 bound tubulin, whereas truncation at the C-terminus resulted in a pronounced decrease in tubulin binding. Based on these results, and a previous study documenting a pH-dependent change in binding affinity between Op18 and tubulin, it is likely that tubulin sequestering observed at lower pH resulted from the relatively tight interaction between Op18 and tubulin and that this tight binding requires the C-terminus of Op18; however, under conditions in which Op18 binds weakly to tubulin (pH 7.5), Op18 stimulated catastrophes without altering tubulin subunit association or dissociation rates, and Op18 did not depolymerize microtubules capped with guanylyl (α, β)-methylene diphosphonate–tubulin subunits. We hypothesize that weak binding between Op18 and tubulin results in free Op18, which is available to interact with microtubule ends and thereby promote catastrophes by a mechanism that likely involves GTP hydrolysis.     

JNK1 phosphorylation of SCG10 determines microtubule dynamics and axodendritic length

c-Jun NH(2)-terminal kinases (JNKs) are essential during brain development, when they regulate morphogenic changes involving cell movement and migration. In the adult, JNK determines neuronal cytoarchitecture. To help uncover the molecular effectors for JNKs in these events, we affinity purified JNK-interacting proteins from brain. This revealed that the stathmin family microtubule-destabilizing proteins SCG10, SCLIP, RB3, and RB3' interact tightly with JNK. Furthermore, SCG10 is also phosphorylated by JNK in vivo on sites that regulate its microtubule depolymerizing activity, serines 62 and 73. SCG10-S73 phosphorylation is significantly decreased in JNK1-/- cortex, indicating that JNK1 phosphorylates SCG10 in developing forebrain. JNK phosphorylation of SCG10 determines axodendritic length in cerebrocortical cultures, and JNK site-phosphorylated SCG10 colocalizes with active JNK in embryonic brain regions undergoing neurite elongation and migration. We demonstrate that inhibition of cytoplasmic JNK and expression of SCG10-62A/73A both inhibited fluorescent tubulin recovery after photobleaching. These data suggest that JNK1 is responsible for regulation of SCG10 depolymerizing activity and neurite elongation during brain development.     

Insight into the GTPase activity of tubulin from complexes with stathmin-like domains

Microtubules are dynamically unstable tubulin polymers that interconvert stochastically between growing and shrinking states, a property central to their cellular functions. Following its incorporation in microtubules, tubulin hydrolyzes one GTP molecule. Microtubule dynamic instability depends on GTP hydrolysis so that this activity is crucial to the regulation of microtubule assembly. Tubulin also has a much lower GTPase activity in solution. We have used ternary complexes made of two tubulin molecules and one stathmin-like domain to investigate the mechanism of the tubulin GTPase activity in solution. We show that whereas stathmin-like domains and colchicine enhance this activity, it is inhibited by vinblastine and by the N-terminal part of stathmin-like domains. Taken together with the structures of the tubulin-colchicine-stathmin-like domain-vinblastine complex and of microtubules, our results lead to the conclusions that the tubulin-colchicine GTPase activity in solution is caused by tubulin-tubulin associations and that the residues involved in catalysis comprise the beta tubulin GTP binding site and alpha tubulin residues that participate in intermolecular interactions in protofilaments. This site resembles the one that has been proposed to give rise to GTP hydrolysis in microtubules. The widely different hydrolysis rates in these two sites result at least in part from the curved and straight tubulin assemblies in solution and in microtubules, respectively.     

Inhibition of tubulin self-assembly and tubulin-colchicine GTPase activity by guanosine 5'-(gamma-fluorotriphosphate)

The inhibitory effects of guanosine 5'-(gamma-fluorotriphosphate) [GTP(gamma F)] on both the polymerization and the colchicine-dependent GTPase activity of calf brain tubulin have been studied. The results demonstrate that this analogue of GTP, with a fluorine atom on the gamma-phosphate, is a reversible competitive dead-end inhibitor of the colchicine-induced GTPase activity with a K1 value of (1.8 +/- 0.6) X 10(-4) M. GTP(gamma F) did not promote assembly of tubulin from which the E-site guanine nucleotide had been removed. It binds to the exchangeable nucleotide site competitively with respect to GTP, diminishing both the rate and extent of tubulin polymerization. Treatment in terms of the Oosawa-Kasai model of the inhibitory effect of GTP(gamma F) on the assembly led to a value of Kdis = 1.1 X 10(-6) M for the complex GTP(gamma F)-tubulin. This analogue does not bind to the postulated third site. The growing of tubulin polymers at 37 degrees C was arrested by GTP(gamma F), and only limited depolymerization was induced by the addition of this analogue after assembly in the presence of GTP. This result confirms that the E-site is blocked in the polymer and that this analogue can bind only to the ends of the polymers. Sedimentation velocity and circular dichroism studies showed that the conformation of the tubulin-GTP(gamma F) complex is not identical with that of tubulin-GTP. This is caused by the replacement of the hydroxyl group in the gamma-phosphate by the fluorine group, which have 2.20- and 1.35-A van der Waals radii, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)