Tubulin domains responsible for assembly of dimers and protofilaments.

The protein domains responsible for the dimerization and polymerization of tubulin have been determined using chemical cross-linking and limited proteolysis. The intra-dimer bond is formed by the N-terminal domain of alpha-tubulin and the C-terminal domain of beta-tubulin. Conversely, the inter-dimer bond along protofilaments is formed by the N-terminal domain of beta-tubulin (carrying the exchangeable GTP) and the C-terminal domain of alpha-tubulin. The domains of proteolytically cleaved tubulin remain tightly associated in solution. Apart from the monomer, tubulin shows three levels of assembly: the dimer, oligomer and polymer. Several oligomeric species can be visualized by electron microscopy of rotary shadowed phosphocellulose-tubulin, h.p.l.c. and non-denaturing gel electrophoresis. Tubulin's capacity to form the higher level aggregates is not destroyed by enzymatic nicking.     

Co-assembly properties of higher plant microtubule-associated proteins with purified brain and plant tubulins

The knowledge of higher plant microtubule-associated proteins (MAPs) remains limited to a few examples that illustrate essentially their binding properties to preformed microtubules as described in carrots. Using taxol-stabilized microtubules a putative MAP-enriched fraction has been isolated in maize cultured cell extracts, one of these polypeptides is immunologically related to neural tau. At present, these proteins are being characterized by co-assembly assays that were not possible before. Similar experiments were done also in a heterologous system using brain tubulin. Three polypeptides out of seven that constituted the MAP fraction were found to co-assemble specifically with tubulin subunits of both origins. Their apparent molecular weights are 67, 83 and 125 kDa. A two-dimensional gel immunoblot of the 83 kDa polypeptide with tau antibodies revealed one major spot. Polypeptides were quantiated by scanning the gels. These results shed light on the present debate on higher plant MAPs and their potential activity in the regulation of microtubule assembly and function in the higher plant cell.     

Common antigenic determinants of the tubulin binding domains of the microtubule-associated proteins MAP-2 and tau.

The structural-functional aspects of the tubulin binding domain on the microtubule-associated protein MAP-2, and its relationship with the tubulin binding domain on tau, were studied using anti-idiotypic antibodies that react specifically with the epitope(s) on MAPs involved in their interaction with tubulin in addition to other tau and MAP-2 specific antibodies. Previous studies showed that MAP-2 and tau share common binding sites on tubulin defined by the peptide sequences alpha (430-441) and beta (422-434) of tubulin subunits. Furthermore, binding experiments revealed the existence of multiple sites for the interaction of the alpha- and beta-tubulin peptides with MAP-2 and tau. Most recent studies showed that the synthetic tau peptide Val187-Gly204 (VRSKIGSTENLKHQPGGG) from the repetitive sequence on tau defines a tubulin binding site on tau. Our present immunological studies using anti-idiotypic antibodies which interact with the synthetic tau peptide and antibodies against the Val187-Gly204 tau peptide indicate that MAP-2 and tau share common antigenic determinants at the level of their respective tubulin binding domains. These antigenic determinants appear to be present in the 35 kDa tubulin binding fragment of MAP-2 and in 18-20 kDa chymotryptic fragments containing the tubulin binding site(s) on MAP-2. These findings, along with structural information on these proteins, provide strong evidence in favor of the hypothesis that tubulin binding domains on MAP-2 and tau share similar structural features.     

Tubulin domains for the interaction of microtubule associated protein DMAP-85 from Drosophila melanogaster

The interaction of microtubule associated proteins (MAPs) with the microtubule system has been characterized in depth in neuronal cells from various mammalian species. These proteins interact with well-defined domains within the acidic tubulin carboxyl-terminal regulatory region. However, there is little information on the mechanisms of MAPs-tubulin interactions in nonmammalian systems. Recently, a novel tau-like protein designated as DMAP-85 has been identified in Drosophila melanogaster, and the regulation of its interactions with cytoskeletal elements was analyzed throughout different developmental stages of this organism. In this report, the topographic domains involved in the binding of DMAP-85 with tubulin heterodimer were investigated. Affinity chromatography of DMAP-85 in matrixes of taxol-stabilized microtubules showed the reversible interaction of DMAP-85 with domains on the microtubular surface. Co-sedimentation studies using the subtilisin-treated tubulin (S-tubulin) indicated the lack of association of DMAP-85 to this tubulin moiety. Moreover, studies on affinity chromatography of the purified 4 kDa C-terminal tubulin peptide bound to an affinity column, confirmed that DMAP-85 interacts directly with this regulatory domain on tubulin subunits. Further studies on sequencial affinity chromatography using a calmodulin affinity column followed by the microtubule column confirmed the similarities in the interaction behavior of DMAP-85 with that of tau. DMAP-85 associated to both calmodulin and the microtubular polymer. These studies support the idea that the carboxyl-terminal region on tubulin constitutes a common binding domain for most microtubule-interacting proteins.Abbreviations MAPs microtubule-associated proteins - C-terminal carboxyl-terminal - SDS-PAGE polyacrylamide gel electrophoresis in the presence of SDS - DTT dithiotreitol - BSA bovine serum albumin     

Tubulin binding sites on gamma-tubulin: identification and molecular characterization

gamma-Tubulin is essential to microtubule organization in eukaryotic cells. It is believed that gamma-tubulin interacts with tubulin to accomplish its cellular functions. However, such an interaction has been difficult to demonstrate and to characterize at the molecular level. gamma-Tubulin is a poorly soluble protein, not amenable to biochemical studies in a purified form as yet. Therefore basic questions concerning the existence and properties of tubulin binding sites on gamma-tubulin have been difficult to address. Here we have performed a systematic search for tubulin binding sites on gamma-tubulin using the SPOT peptide technique. We find a specific interaction of tubulin with six distinct domains on gamma-tubulin. These domains are clustered in the central part of the gamma-tubulin primary amino acid sequence. Synthetic peptides corresponding to the tubulin binding domains of gamma-tubulin bind with nanomolar K(d)s to tubulin dimers. These peptides do not interfere measurably with microtubule assembly in vitro and associate with microtubules along the polymer length. On the tertiary structure, the gamma-tubulin peptides cluster to surface regions on both sides of the molecule. Using SPOT analysis, we also find peptides interacting with gamma-tubulin in both the alpha- and beta-tubulin subunits. The tubulin peptides cluster to surface regions on both sides of the alpha- and beta- subunits. These data establish gamma-tubulin as a tubulin ligand with unique tubulin-binding properties and suggests that gamma-tubulin and tubulin dimers associate through lateral interactions.     

Why do tubulin gene families lack diversity in flagellate/ciliate protists?

Summary Considerable amino acid sequence diversity is found among tubulin isotypes encoded by tubulin gene families in animal, higher plant, and fungal systems. In contrast, relatively little diversity is found among the isotypes produced by the gene families in a number of flagellate or ciliate protists. It is possible that proper assembly of the axoneme requires a homogeneous pool of tubulin subunits and that the axoneme thus provides a stringent selection against amino acid replacement substitutions among tubulin genes in these systems.     

Construction of phylogenetic tree of plant tubulins basing on the homology of their protein sequences

We have built phylogenetic tree of alpha-, beta- and gamma-tubulins of plant kingdom, alpha- and beta-tubulins of pig, and all epsilon- and delta-tubulins with known primary sequences and analyzed the levels of homology of tubulin sequences for repersentatives of different groups of organisms. It has been established low heterogeneity of alpha-, gamma-tubulin families and more than two fold higher heterogeneity of beta-tubulins, based on the sequence speciality of alga tubulins. We have showed that sequences of animal tubulins do not fit any cluster formed by on the plant tubulins. Presence in primary tubulin sequences of three major families of insignificant specific differences which are specific for such phylogenetic items of plant kingdom as alga and angiosperms has been also demostrated. The cladogramm shows clear clasterization of investigated sequences according to their belonging to the tubulin families.     

In vitro assembly of microtubules from tubulins of several higher plants

Tubulins were isolated by a combination of affinity (ethyl N-phenylcarbamate-Sepharose 4B) and ion exchange (DEAE-Sephacel) chromatography from several higher plants (mung bean, pea, whole pod bean, zucchini, cucumber seedlings and carrot suspension cultured cells). All these higher plant tubulins readily polymerized to microtubules in a polymerization medium containing GTP, Mg2+, EGTA, leupeptin and DMSO. Tubulins from mung bean, pea and whole pod bean showed identical behaviour on polyacrylamide gel electrophoresis but differed from carrot zucchini and cucumber tubulin. Consequently, tubulin of higher plants seems to have different molecular properties in different plant species.     

Plant mutant tubulin genes as marker selective genes for genetic engineering

The approaches for new marker genes usage in selection of transformed plant cells, which are based on using mutant tubulin genes from natural plant biotypes and, in perspective, from induced plant mutants have been considered. The results of investigations of plant (biotypes, mutants) resistance to herbicides with antimicrotubular mode of action on molecular and cellular levels have been summarized. The reports dealing with study the transferring and the expression of mutant tubulin genes conferring resistance to amiprophosmethyl (phosphorothioamidate herbicide) and to trifluralin (dinitroaniline herbicide) from corresponding N. plumbaginifolia mutants into related and remote plant species by somatic hybridisation methods have been analyzed. The results of experiments on monocotyledonous and dicotyledonous. plant transformation by mutant alpha-tubulin gene conferring resistance to dinitroanilines are described to test the possibility of its using as a marker gene with obtaining, at the same time, a dinitroaniline-resistant plants.     

The Pleiomorphic Plant MTOC： An Evolutionary Perspective

γ-Tubulin is an essential component of the microtubule organizing center （MTOC） responsible for nucleating microtubules in both plants and animals. Whereas γ-tubulin is tightly associated with centrosomes that are inheritable organelles in cells of animals and most algae, it appears at different times and places to organize the myriad specialized microtubule systems that characterize plant cells. We have traced the distribution of γ-tubulin through the cell cycle in representative land plants （embryophytes） and herein present data that have led to a concept of the pleiomorphic and migratory MTOC. The many forms of the plant MTOC at spindle organization constitute pleiomorphism, and stage-specific “migration” is suggested by the consistent pattern of redistribution of γ-tubulin during mitosis. Mitotic spindles may be organized at centriolar centrosomes （only in final divisions of spermatogenesis）, polar organizers （POs）, plastid MTOCs, or nuclear envelope MTOCs （NE-MTOCs）. In all cases, with the possible exception of centrosomes in spermatogenesis, the γ-tubulin migrates to broad polar regions and along the spindle fibers, even when it is initially a discrete polar entity. At anaphase it moves poleward, and subsequently migrates from polar regions （distal nuclear surfaces） into the interzone （proximal nuclear surfaces） where interzonal microtubule arrays and phragmoplasts are organized. Following cytokinesis, γ-tubulin becomes associated with nuclear envelopes and organizes radial microtubule systems （RMSs）. These may exist only briefly, before establishment of hoop-like cortical arrays in vegetative tissues, or they may be characteristic of interphase in syncytial systems where they serve to organize the common cytoplasm into nuclear cytoplasmic domains （NCDs）.     

Folding, stability and polymerization properties of FtsZ chimeras with inserted tubulin loops involved in the interaction with the cytosolic chaperonin CCT and in microtubule formation

To attain its native conformation, the cytoskeletal protein tubulin needs the concourse of several molecular chaperones, among others the cytosolic chaperonin CCT. It has been previously described that denatured tubulin interacts with CCT in a quasi-folded conformation using several loops located throughout its sequence. These loops are also involved in microtubule formation and are absent in its prokaryote homologue FtsZ, which in vitro folds by itself and does not interact with CCT. Several FtsZ/tubulin chimeric proteins were generated by inserting consecutively one, two or three of the CCT-binding domains of tubulin into the corresponding sequence of FtsZ from Methanococccus jannaschii. The insertion of any of the CCT-binding loops generates in the FtsZ/tubulin chimeras the ability to interact with CCT. The accumulation of CCT-binding loops induces in the FtsZ/tubulin chimeras unfolding and refolding properties that are more similar to tubulin than to its prokaryote counterpart. Finally, the insertion of some of these loops generates in the FtsZ/tubulin chimeras more complex polymeric structures than those found for FtsZ. These results reinforce the notion that CCT has coevolved with tubulin to deal with the folding problems encountered by the eukaryotic protein with the appearance of the new sequences involved in microtubule formation.     

Trypanosoma cruzi: cell type dependent distribution of a tubulin domain in mammalian stages

The distribution of tubulin domains in the mammalian stages of Trypanosoma cruzi was investigated by using monoclonal antibodies elicited against bovine brain tubulin. Western blotting performed on T. brucei trypomastigotes and T. cruzi epimastigotes showed that the monoclonal antibodies 16D3 and 24E3 reacted only with tubulin in these cell types. Indirect immunofluorescence revealed that, whereas 16D3 stained all microtubules, including subpellicular microtubules, the epitope defined by 24E3 was found in only a part of the tubulin pool of amastigotes and intermediate stages infecting murine fibroblasts and of broad trypomastigotes; the staining was limited to the basal bodies and the distal region of the flagellar adhesion zone in these developmental forms. By contrast, slender trypomastigotes did not exhibit any reaction with 24E3. These results are consistent with a transformation of broad trypomastigotes into slender trypomastigotes during which the tubulin domain recognized by 24E3 would undergo modifications leading to its complete masking in slender forms. The morphogenesis of the mammalian stages of T. cruzi would involve modifications of the tubulin molecule.     

Parkin stabilizes microtubules through strong binding mediated by three independent domains

Mutations of parkin, a protein-ubiquitin isopeptide ligase (E3), appear to be the most frequent cause of familial Parkinson's disease (PD). Our previous studies have demonstrated that parkin binds strongly to alpha/beta tubulin heterodimers and microtubules. Here we show that the strong binding between parkin and tubulin, as well as that between parkin and microtubules, was mediated by three independent domains: linker, RING1, and RING2. These redundant strong interactions made it virtually impossible to separate parkin from microtubules by high concentrations of salt (3.8 m) or urea (0.5 m). Parkin co-purified with tubulin and was found in highly purified tubulin preparation. Expression of either full-length parkin or any of its three microtubule-binding domains significantly attenuated colchicine-induced microtubule depolymerization. The abilities of parkin to bind to and stabilize microtubules were not affected by PD-linked mutations that abrogate its E3 ligase activity. Thus, the tubulin/microtubule-binding activity of parkin and its E3 ligase activity are independent. The strong binding between parkin and tubulin/microtubules through three redundant interaction domains may not only stabilize microtubules but also guarantee the anchorage of this E3 ligase on microtubules. Because many misfolded proteins are transported on microtubules, the localization of parkin on microtubules may provide an important environment for its E3 ligase activity toward misfolded substrates.     

Affinity Purification and Characterization of Functional Tubulin from Cell Suspension Cultures of Arabidopsis and Tobacco

Microtubules assemble into several distinct arrays that play important roles in cell division and cell morphogenesis. To decipher the mechanisms that regulate the dynamics and organization of this versatile cytoskeletal component, it is essential to establish in vitro assays that use functional tubulin. Although plant tubulin has been purified previously from protoplasts by reversible taxol-induced polymerization, a simple and efficient purification method has yet to be developed. Here, we used a Tumor Overexpressed Gene (TOG) column, in which the tubulin-binding domains of a yeast (Saccharomyces cerevisiae) TOG homolog are immobilized on resin, to isolate functional plant tubulin. We found that several hundred micrograms of pure tubulin can readily be purified from cell suspension cultures of tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana). The tubulin purified by the TOG column showed high assembly competence, partly because of low levels of polymerization-inhibitory phosphorylation of α-tubulin. Compared with porcine brain tubulin, Arabidopsis tubulin is highly dynamic in vitro at both the plus and minus ends, exhibiting faster shrinkage rates and more frequent catastrophe events, and exhibits frequent spontaneous nucleation. Furthermore, our study shows that an internal histidine tag in α-tubulin can be used to prepare particular isotypes and specifically engineered versions of α-tubulin. In contrast to previous studies of plant tubulin, our mass spectrometry and immunoblot analyses failed to detect posttranslational modification of the isolated Arabidopsis tubulin or detected only low levels of posttranslational modification. This novel technology can be used to prepare assembly-competent, highly dynamic pure tubulin from plant cell cultures.Microtubules (MTs) are important cytoskeletal polymers that are conserved in eukaryotic cells and are assembled from α- and β-tubulin heterodimers (Desai and Mitchison, 1997). In plants, MTs have important functions in essential cellular processes, such as cell division, and in cell morphogenesis. MTs in plant cells adopt several distinct higher order arrays and are remodeled in response to the cell cycle, developmental programs, and environmental cues (Hashimoto, 2015). Genetic, molecular, and cell biological approaches have been used to identify cellular factors that regulate the organization and dynamics of plant MTs. Considerable effort has been devoted to simulating the organization of cortical MT arrays by computational modeling.Cell-free in vitro studies are essential for the biochemical characterization of various MT regulators and for elucidating the mechanistic principles underlying the versatility of this dynamic polymer in cellular functions. The purification of sufficient amounts of assembly-competent tubulin is a prerequisite for these in vitro studies. Tubulin is traditionally purified from mammalian brains, since these tissues contain sufficiently high concentrations of tubulin to allow MT assembly in crude cell extracts. Polymerized MTs and their associated MT-binding proteins are separated from other cellular proteins by sedimentation. Pelleted MTs are then depolymerized upon drug washout under MT-destabilizing conditions, such as high concentrations of salt and calcium and low temperature. A few rounds of assembly-disassembly cycles highly enrich for tubulin and copurify MT-associated proteins, which can subsequently be removed by column chromatography (Borisy et al., 1975). Tubulin has also been purified from several plant sources (Morejohn and Fosket, 1982; Mizuno, 1985; Jiang et al., 1992; Bokros et al., 1993; Moore et al., 1997). However, since tubulin concentrations are low in plant cells, taxol, which stabilizes MTs, is generally included in the polymerization buffer, and cytoplasm-rich miniprotoplasts, which lack vacuoles, are sometimes used as starting material (Hamada et al., 2013). Since it is technically challenging to isolate assembly-competent pure tubulin from nonneural sources (Sackett et al., 2010), general plant science laboratories may hesitate to prepare plant tubulin themselves.Although the primary amino acid sequences of eukaryotic tubulins are fairly well conserved and the biophysical mechanisms of MT assembly and disassembly are thought to be similar for all MTs, the kinetics of MT dynamic instability differ for MTs assembled from animal and plant tubulin (Moore et al., 1997). Interactions with MT-interacting proteins may differ for tubulins isolated from different biological sources, as reported for the MT-dependent activation of kinesin (Alonso et al., 2007). Posttranslational modifications of tubulin, which generate distinct tubulin signatures and may modulate the functions of MT-interacting proteins, such as kinesin (Sirajuddin et al., 2014), are extensive in brain tubulin (Janke, 2014) but may be quantitatively and qualitatively different in plant tubulin. Furthermore, MT nucleation by the γ-tubulin ring complex shows a strong preference for tubulin from the same species (Kollman et al., 2015). Thus, it is important to use plant tubulin, and not brain tubulin, for in vitro studies of plant MTs.Tubulin is folded by a series of molecular chaperones to form an αβ-tubulin heterodimer in which one structural GTP is embedded in the interdimer interface (Lundin et al., 2010). The requirement of these eukaryote-specific chaperones precludes the use of prokaryotic expression systems for synthesizing properly folded and functional tubulin. Bacterially synthesized tubulin can be folded in rabbit reticulocyte lysate to produce functional tubulin, but with moderate yields (Shah et al., 2001). A yeast (Saccharomyces cerevisiae) expression system has been developed to produce modified yeast tubulin (Uchimura et al., 2006; Johnson et al., 2011), but this system is not suitable for the synthesis of animal (Sirajuddin et al., 2014) and plant (our unpublished data) tubulin. A baculovirus-insect cell expression system was recently reported to yield functional human tubulin (Minoura et al., 2013).Tubulin-binding proteins have been used to develop affinity-purification columns. The TOG domains (named after the human MT regulator, colonic and hepatic Tumor Overexpressed Gene [ch-TOG]) are among the best-characterized tubulin-binding domains. ch-TOG and orthologs from other eukaryotes bind to the growing plus ends of MTs and accelerate MT growth (Al-Bassam and Chang, 2011). TOG domains from the yeast ortholog Stu2 were recently used to affinity purify assembly-competent tubulin from fungal and animal sources (Widlund et al., 2012). In this study, we demonstrate that a TOG-based affinity column can be used to purify functional tubulin from tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana). We examined the posttranslational modifications of the isolated tubulins by mass spectrometry and immunoblot analysis and showed that a His-tagged Arabidopsis tubulin isotype could be purified using this column. These results show that wild-type and recombinant functional tubulin from plant sources can be isolated efficiently.     

Submolecular-scale imaging of α-helices and C-terminal domains of tubulins by frequency modulation atomic force microscopy in liquid

In this study, we directly imaged subnanometer-scale structures of tubulins by performing frequency modulation atomic force microscopy (FM-AFM) in liquid. Individual α-helices at the surface of a tubulin protofilament were imaged as periodic corrugations with a spacing of 0.53 nm, which corresponds to the common pitch of an α-helix backbone (0.54 nm). The identification of individual α-helices allowed us to determine the orientation of the deposited tubulin protofilament. As a result, C-terminal domains of tubulins were identified as protrusions with a height of 0.4 nm from the surface of the tubulin. The imaging mechanism for the observed subnanometer-scale contrasts is discussed in relation to the possible structures of the C-terminal domains. Because the C-terminal domains are chemically modified to regulate the interactions between tubulins and other biomolecules (e.g., motor proteins and microtubule-associated proteins), detailed structural information on individual C-terminal domains is valuable for understanding such regulation mechanisms. The results obtained in this study demonstrate that FM-AFM is capable of visualizing the structural variation of tubulins with subnanometer resolution. This is an important first step toward using FM-AFM to analyze the functions of tubulins.     

Characterization of maize microtubule-associated proteins, one of which is immunologically related to tau

Microtubule-associated proteins (MAPs) are identified as proteins that copurify with tubulin, promote tubulin assembly, and bind to microtubules in vitro. Higher plant MAPs remain mostly unknown. One example of non-tubulin carrot proteins, which bind to neural microtubules and induce bundling, has been reported so far [Cyr, R. J., & Palewitz, B. A. (1989) Planta 177, 245-260]. Using taxol, we developed an assay where higher plant microtubules were induced to self-assemble in cytosolic extracts of maize cultured cells and were used as the native matrix to isolate putative plant MAPs. Several polypeptides with an apparent molecular masses between 170 and 32 kDa copolymerized with maize microtubules. These putative maize MAPs also coassembled with pig brain tubulin through two cycles of temperature-dependent assembly-disassembly. They were able to initiate and promote MAP-free tubulin assembly under conditions of nonefficient self-assembly and induced bundling of both plant and neural microtubules. One of these proteins, of about 83 kDa, cross-reacted with affinity-purified antibodies against rat brain tau proteins, suggesting the presence of common epitope(s) between neural tau and maize proteins. This homology might concern the tubulin-binding domain, as plant and neural tubulins are highly conserved and the plant polypeptides coassembled with brain tubulin.     

Direct observation of microtubule treadmilling by electron microscopy

Using an immunoelectron microscopic procedure, we directly observed the concurrent addition and loss of chicken brain tubulin subunits from the opposite ends of microtubules containing erythrocyte tubulin domains. The polarity of growth of the brain tubulin on the ends of erythrocyte microtubules was determined to be similar to growth off the ends of Chlamydomonas axonemes. The flux rate for brain tubulin subunits in vitro was low, approximately 0.9 micron/h. Tubulin subunit flux did not continue through the entire microtubule as expected, but ceased when erythrocyte tubulin domains became exposed, resulting in a metastable configuration that persisted for at least several hours. We attribute this to differences in the critical concentrations of erythrocyte and brain tubulin. The exchange of tubulin subunits into the walls of preformed microtubules other than at their ends was also determined to be insignificant, the exchange rate being less than the sensitivity of the assay, or less than 0.2%/h.     

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.     

Structural insights into FtsZ protofilament formation

The prokaryotic tubulin homolog FtsZ polymerizes into a ring structure essential for bacterial cell division. We have used refolded FtsZ to crystallize a tubulin-like protofilament. The N- and C-terminal domains of two consecutive subunits in the filament assemble to form the GTPase site, with the C-terminal domain providing water-polarizing residues. A domain-swapped structure of FtsZ and biochemical data on purified N- and C-terminal domains show that they are independent. This leads to a model of how FtsZ and tubulin polymerization evolved by fusing two domains. In polymerized tubulin, the nucleotide-binding pocket is occluded, which leads to nucleotide exchange being the rate-limiting step and to dynamic instability. In our FtsZ filament structure the nucleotide is exchangeable, explaining why, in this filament, nucleotide hydrolysis is the rate-limiting step during FtsZ polymerization. Furthermore, crystal structures of FtsZ in different nucleotide states reveal notably few differences.