Molecular dynamics modeling of tubulin C-terminal tail interactions with the microtubule surface

Tubulin, an α/β heterodimer, has had most of its 3D structure analyzed; however, the carboxy (C)-termini remain elusive. Importantly, the C-termini play critical roles in regulating microtubule structure and function. They are sites of most of the post-translational modifications of tubulin and interaction sites with molecular motors and microtubule-associated proteins. Simulated annealing was used in our molecular dynamics modeling to predict the interactions of the C-terminal tails with the tubulin dimer. We examined differences in their flexibility, interactions with the body of tubulin, and the existence of structural motifs. We found that the α-tubulin tail interacts with the H11 helix of β-tubulin, and the β-tubulin tail interacts with the H11 helix of α-tubulin. Tail domains and H10/B9 loops interact with each other and compete for interactions with positively-charged residues of the H11 helix on the neighboring monomer. In a simulation in which α-tubulin's H10/B9 loop switches on sub-nanosecond intervals between interactions with the C-terminal tail of α-tubulin and the H11 helix of β-tubulin, the intermediate domain of α-tubulin showed more fluctuations compared to those in the other simulations, indicating that tail domains may cause shifts in the position of this domain. This suggests that C-termini may affect the conformation of the tubulin dimer which may explain their essential function in microtubule formation and effects on ligand binding to microtubules. Our modeling also provides evidence for a disordered-helical/helical double-state system of the T3/H3 region of the microtubule, which could be linked to depolymerization following GTP hydrolysis.     

Glycolytic enzyme–tubulin interactions: Role of tubulin carboxy terminals

Tubulin and microtubules were modified with the protease, subtilisin. The modification reduced the length of α-or β-tubulin by cleaving a peptide fragment from the C-terminals. Generation of α′β′-tubulin, which is cleaved at both the α- and β-subunit terminals, and αβ′-tubulin, which is cleaved at the β′-subunit C-terminal, have already been reported. In this work an isotype, α′β-tubulin, was produced. The three modified tubulin isotypes were compared for their ability to interact with glycolytic enzymes. Cleavage of α led to a poorer interaction when tested via affinity chromatography. Tubulin also inhibits the activity of aldolase and glyceraldehyde 3-phosphate dehydrogenase. When the α-subunit C-terminal was intact, inhibition was greatest. These results imply that the C-terminal of the tubulin α-subunit is subunit is responsible for interactions with glycolytic enzymes.     

Bacterial tubulin distinct loop sequences and primitive assembly properties support its origin from a eukaryotic tubulin ancestor

The structure of the unique bacterial tubulin BtubA/B from Prosthecobacter is very similar to eukaryotic αβ-tubulin but, strikingly, BtubA/B fold without eukaryotic chaperones. Our sequence comparisons indicate that BtubA and BtubB do not really correspond to either α- or β-tubulin but have mosaic sequences with intertwining features from both. Their nucleotide-binding loops are more conserved, and their more divergent sequences correspond to discrete surface zones of tubulin involved in microtubule assembly and binding to eukaryotic cytosolic chaperonin, which is absent from the Prosthecobacter dejongeii draft genome. BtubA/B cooperatively assembles over a wider range of conditions than αβ-tubulin, forming pairs of protofilaments that coalesce into bundles instead of microtubules, and it lacks the ability to differentially interact with divalent cations and bind typical tubulin drugs. Assembled BtubA/B contain close to one bound GTP and GDP. Both BtubA and BtubB subunits hydrolyze GTP, leading to disassembly. The mutant BtubA/B-S144G in the tubulin signature motif GGG(T/S)G(S/T)G has strongly inhibited GTPase, but BtubA-T147G/B does not, suggesting that BtubB is a more active GTPase, like β-tubulin. BtubA/B chimera bearing the β-tubulin loops M, H1-S2, and S9-S10 in BtubB fold, assemble, and have reduced GTPase activity. However, introduction of the α-tubulin loop S9-S10 with its unique eight-residue insertion impaired folding. From the sequence analyses, its primitive assembly features, and the properties of the chimeras, we propose that BtubA/B were acquired shortly after duplication of a spontaneously folding α- and β-tubulin ancestor, possibly by horizontal gene transfer from a primitive eukaryotic cell, followed by divergent evolution.     

Comparative immunogold analysis of tubulin isoforms in the mouse sperm flagellum: Unique distribution of glutamylated tubulin

The distribution of different tubulin isoforms in the mouse sperm flagellum was studied using four site-directed antibodies to tubulin: DM1A and DM1B general anti α and β-tubulin, 6-11B-1 anti-acetylated α-tubulin, and GT335 anti-glutamylated α and β-tubulin. Quantitative immunogold analyses were performed on five regions of the flagellum: the middle piece, three successive regions of the principal piece, and the terminal piece. A uniform labeling was observed with DM1A and DM1B along the entire flagellum both for peripheral doublets and the central pair. Similar results were obtained with 6-11B-1 directed to acetylated α-tubulin, an N-terminal-modified tubulin isoform. In contrast, the labeling for glutamylated α and β-tubulin, C-terminal modified isoforms, was not uniform. The highest intensity was found in the middle piece and the terminal piece. The labeling which decreased significantly both for peripheral doublets and central pair along the principal piece was considered as a loss of glutamylated tubulin accessibility. From the middle piece to the end of the principal piece, this labeling was predominant in doublets 1-5-6, corresponding to the plane of the flagellar wave. However, the labeling for doublets 2-3-4-7-8-9 was heterogeneous, showing an increasing asymmetry. These results suggest that in the mammalian sperm cell model, the glutamylated tubulin might be involved in a functional heterogeneity among peripheral doublets of the flagellum. © 1996 Wiley-Liss, Inc.     

In vitro formation of different tubulin polymers from purified tubulin of Ehrlich ascites tumor cells

Preparations of cycled tubulin from Ehrlich ascites tumor cells contain several acessory proteins; once or twice cycled microtubule preparations are usually composed of fibers 10 nm in diameter, but lack vimentin. Highly purified tubulin consists of α- and β-tubulin and a minor component which was identified by peptide mapping as a second β-chain. This pure tubulin is able to form in vitro at low concentrations (1 mg protein/ml) fibers of about 10 nm width, and at higher concentrations (3.5 mg protein/ml) normal microtubules.     

Proteolytic analysis of polymerized maize tubulin: regulation of microtubule stability to low temperature and Ca2+ by the carboxyl terminus of β-tubulin

To obtain information on plant microtubule stability to low temperature and Ca²⁺, the regulatory domain of polymerized tubulin from maize (Zea mays ev. Black Mexican Sweet) was dissected by limited proteolysis with subtilisin. Tubulin in taxol-stabilized microtubules was cleaved in a subtilisin concentration- and time-dependent manner. Immunoblotting of microtubules with antibodies having mapped epitopes on α- and β-tubulins revealed that cleavage initially removed ≤15 residues from the β-tubulin carboxyl terminus to produce αβ_s-microtubules. Subsequent cleavage occurred at an extreme site and an internal site within the α-tubulin carboxyl terminus. Electron microscopy revealed that αβ_s-microtubules were ultra structurally indistinguishable from uncleaved control αβ-micro-tubules. Quantitative polymer sedimentation showed that low temperature treatment (0°C) caused significant depolymerization of αβ-microtubules, but little depolymerization of αβ_s-microtubules. Ca²⁺ enhanced the cold-induced depolymerization of both αβ- and αβ_s-microtubules. However, αβ_s-microtubules were significantly more stable to depolymerization by cold and Ca²⁺ than were αβ-micro-tubules. The results showed that maize microtubules containing shortened β-tubulin carboxyl termini are relatively resistant to the combined depolymerizing effects of cold and Ca²⁺. Thus, the extreme carboxyl terminus of β-tubulin is a crucial element of the plant tubulin regulatory domain and may be involved in the modulation of microtubule stability during the chilling response in plants.     

Molecular cloning and biochemical characterization of α- and β-tubulin from potato plants (Solanum tuberosum L.)

Few studies have investigated microtubules from plants that host pathogenic fungi. Considerable efforts are underway to find an antimitotic agent against plant pathogens like Phytophthora infestans. However, screening the effects of antifungal agents on plant tubulin in vivo or using purified native microtubule in vitro is a time consuming process. A recombinant, correctly folded, microtubule-like structure forming tubulin could accelerate research in this area. In this study, we cloned full length cDNAs isolated from potato leaves using reverse-transcribed polymerase chain reaction (RT-PCR). Solanum tuberosum (Stub) α-tubulin and β-tubulin were predicted to encode 449 and 451 amino acid long proteins with molecular masses of 57 kDa and 60 kDa, respectively. Average yields of α- and β-tubulin were 2.0–3.5 mg l^?1 and 1.3–3.0 mg l^?1 of culture, respectively. The amino acids, His6, Glu198, and Phe170 involved in benomyl sensitivity were conserved in Stub tubulin. The dimerization of tubulin monomers was confirmed by western blot analysis. When combined under appropriate conditions, these recombinant α- and β-tubulins were capable of polymerizing into microtubules. Accessibility of cysteine residues of tubulin revealed that important ligand binding sites were folded correctly. This recombinant tubulin could serve as a control of phytotoxicity of selected antimitotic fungicide compounds during in vitro screening experiments.     

PACSIN proteins bind tubulin and promote microtubule assembly

PACSINs are intracellular adapter proteins involved in vesicle transport, membrane dynamics and actin reorganisation. In this study, we report a novel role for PACSIN proteins as components of the centrosome involved in microtubule dynamics. Glutathione S-transferase (GST)-tagged PACSIN proteins interacted with protein complexes containing α- and γ-tubulin in brain homogenate. Analysis of cell lysates showed that all three endogenous PACSINs co-immunoprecipitated dynamin, α-tubulin and γ-tubulin. Furthermore, PACSINs bound only to unpolymerised tubulin, not to microtubules purified from brain. In agreement, the cellular localisation of endogenous PACSIN 2 was not affected by the microtubule depolymerising reagent nocodazole. By light microscopy, endogenous PACSIN 2 localised next to γ-tubulin at purified centrosomes from NIH 3T3 cells. Finally, reduction of PACSIN 2 protein levels with small-interfering RNA (siRNA) resulted in impaired microtubule nucleation from centrosomes, whereas microtubule centrosome splitting was not affected, suggesting a role for PACSIN 2 in the regulation of tubulin polymerisation. These findings suggest a novel function for PACSIN proteins in dynamic microtubuli nucleation.     

Effects of amino-acid substitutions in β tubulin on benomyl sensitivity and microtubule functions inCoprinus cinereus

The sensitivity of the homobasidiomyceteCoprinus cinereus to the benzimidazole fungicide benomyl allowed us to isolate β-tubulin mutants as strains resistant to benomyl. To understand the molecular basis for the interaction between benomyl and β tubulin and for cellular defects in the β-tubulin mutants, we first analyzed the wild-type β1-tubulin gene (benA) ofC. cinereus, revealing thatbenA contains eight introns and encodes a 445 amino-acid protein. We then characterized 16 β1-tubulin mutants. The 16 mutations involved 11 different amino-acid substitutions at 10 different residues in β1 tubulin. The mutated residues were widely distributed along the primary sequence of β1 tubulin, from residue 3 in the N-terminal domain to residue 350 in the intermediate domain, but half of them appeared to be close to the αβ intradimer interface in an atomic model determined by electron crystallography. The benomyl resistant strain BEN 193, which exhibits clear heat sensitivity for hyphal growth and defects in various cellular processes, had a novel mutation, i.e., the Leu to Phe substitution at residue 350. Benomyl resistance and the heat sensitivity in BEN 193 were suppressed by additional amino-acid substitutions at various residues in β1 tubulin, suggesting that conformational changes of β1 tubulin are involved in the alterations. The DDBJ/GeneBank/EMBL accession number for the sequence reported in this paper is AB000116.     

The C-termini of tubulin and the specific geometry of tubulin substrates influence the depolymerization activity of MCAK

MCAK is a Kinesin-13 that depolymerizes microtubules (MTs) and regulates MT dynamics. We used subtilisin-treated MTs (MTs lacking the C-termini of α- and β-tubulin) and alternative tubulin substrates to study which structural and geometrical features of the MT are critical for MCAK activity. We found that removal of the C-termini significantly decreased the efficiency of MCAK-induced depolymerization, which was not due to a reduction of end-specific binding. We also found that depolymerization of SMTs led to an increase in the stabilization of curved oligomeric tubulin products. Using alternative tubulin substrates with different geometries, we found that MCAK depolymerized parallel and anti-parallel tubulin sheets. However, MCAK did not depolymerize tubulin rings regardless of the presence or absence of the tubulin C-termini. We propose that localization of MCAK to the ends of MTs is independent of tubulin C-termini, that MCAK stabilizes a curved conformation at the end of the MT, and that efficient release of this complex is dependent on the presence of the C-termini of tubulin.αβ     

Cytosolic carboxypeptidase 1 is involved in processing α- and β-tubulin

The Purkinje cell degeneration (pcd) mouse has a disruption in the gene encoding cytosolic carboxypeptidase 1 (CCP1). This study tested two proposed functions of CCP1: degradation of intracellular peptides and processing of tubulin. Overexpression (2-3-fold) or knockdown (80-90%) of CCP1 in human embryonic kidney 293T cells (HEK293T) did not affect the levels of most intracellular peptides but altered the levels of α-tubulin lacking two C-terminal amino acids (delta2-tubulin) ≥ 5-fold, suggesting that tubulin processing is the primary function of CCP1, not peptide degradation. Purified CCP1 produced delta2-tubulin from purified porcine brain α-tubulin or polymerized HEK293T microtubules. In addition, CCP1 removed Glu residues from the polyglutamyl side chains of porcine brain α- and β-tubulin and also generated a form of α-tubulin with two C-terminal Glu residues removed (delta3-tubulin). Consistent with this, pcd mouse brain showed hyperglutamylation of both α- and β-tubulin. The hyperglutamylation of α- and β-tubulin and subsequent death of Purkinje cells in pcd mice was counteracted by the knock-out of the gene encoding tubulin tyrosine ligase-like-1, indicating that this enzyme hyperglutamylates α- and β-tubulin. Taken together, these results demonstrate a role for CCP1 in the processing of Glu residues from β- as well as α-tubulin in vitro and in vivo.     

Increased levels of mRNAs for tubulin and other flagellar proteins after amputation or shortening of Chlamydomonas Flagella

A dramatic stimulation of synthesis of flagellar proteins occurs in Chlamydomonas following flagellar removal or experimentally induced resorption of the flagella into the cell. In this report we show that this stimulation involves an increase in the levels of mRNAs for tubulin and many other flagellar proteins. Total RNA and poly(A) RNA were isolated from cells after deflagellation or flagellar resorption, and were then translated in the reticulocyte lysate system. Two-dimensional gel analysis of the translation products demonstrates that the RNA-directed in vitro synthesis of α and β tubulins, and a number of other flagellar proteins, increases after deflagellation or flagellar resorption. Surprisingly, the α-tubulin synthesized in vitro does not co-migrate on two-dimensional gels with mature flagellar α-tubulin. Moreover, in vivo labeling experiments show that the major α-tubulin synthesized in the cell after deflagellation co-migrates with the major α-tubulin made in vitro, not with the major α-tubulin present in the flagella. These results suggest that flagellar α-tubulin is synthesized as a precursor, and undergoes post-translational modification before assembly into the flagella. In addition, we report that the synthesis of tubulin and other flagellar proteins can be specifically inhibited, as well as stimulated. Treatment of cells with IBMX, which induces flagellar resorption, causes a marked decrease in the levels of translatable mRNAs for tubulin and other flagellar proteins, without affecting levels of mRNAs for nonflagellar proteins.     

Overexpression,purification, and functional analysis of recombinant human tubulin dimer

Microtubules consisting of tubulin dimers play essential roles in various cellular functions. Investigating the structure–function relationship of tubulin dimers requires a method to prepare sufficient quantities of recombinant tubulin. To this end, we simultaneously expressed human α1- and β3-tubulin using a baculovirus-insect cell expression system that enabled the purification of 5 mg recombinant tubulin per litre of cell culture. The purified recombinant human tubulin could be polymerized into microtubules that glide on a kinesin-coated glass surface. The method provides a powerful tool for in vitro functional analyses of microtubules.     

TTLL10 can perform tubulin glycylation when co-expressed with TTLL8

Tubulin can undergo unusual post-translational modifications, glycylation and glutamylation. We previously failed to find glycylase (glycine ligase) for tubulin while identifying TTLL10 as a polyglycylase for nucleosome assembly protein 1. We here examine whether TTLL10 performs tubulin glycylation. We used a polyclonal antibody (R-polygly) raised against a poly(glycine) chain, which does not recognize monoglycylated protein. R-polygly strongly reacted with mouse tracheal cilia and axonemal tubulins. R-polygly detected many proteins in cell lysates co-expressing TTLL10 with TTLL8. Two-dimensional electrophoresis revealed that the R-polygly-reactive proteins included α- and β-tubulin. R-polygly labeling signals overlapped with microtubules. These results indicate that TTLL10 can strongly glycylate tubulin in a TTLL8-dependent manner. Furthermore, these two TTLL proteins can glycylate unidentified 170-, 110-, 75-, 40-, 35-, and 30-kDa acidic proteins.     

Structural mechanisms of interaction of cyanolcrylates with plant tubulin

The structural mechanisms underlying the specific binding of cyanoacrylate compounds with tubulin of higher plants have been studied by the example of the interaction of ethyl-(2Z)-3-amino-2-cyano-4-ethylhex-2-enoate (CA1) and isopropyl-(2Z)-3-amino-2-cyano-4-ethylhex-2-enoate (CA2) with Arabidopsis thaliana α-tubulin. It was revealed that the cyano group of cyanoacrylates is a functional analogue of the nitrile group, which determines the processes of specific interaction with plant tubulin for dinitroaniline compounds. Based on data on spatial structure fluctuations, the dynamics of hydrogen bonds and the interaction energy of CA1 and CA2 (the most probable binding mode for these compounds with plant α-tubulin) was identified and the appropriate site of interaction was characterized. Seven out of ten residues composing this site (Gln-133, Asn-249, Val-250, Asp-251, Val-252, Asn-253, and Glu-254) are obligatory components of dinitroanilines’ binding site on the plant α-tubulin surface. Thus, the binding site on the α-tubulin surface characterized by us is able to recognize and specifically bind substances, which are cardinally different by their chemical nature and have no common pharmacophore groups, under the condition of a certain similarity of their electrostatic topology.     

Calmodulin-dependent protein kinase (kinase II) which is involved in the activation of tryptophan 5-monooxygenase catalyzes phosphorylation of tubulin

Tubulin was shown to be an endogenous substrate of the calmodulin-dependent protein kinase (kinase II), which is involved in the activation of tryptophan 5-monooxygenase [T. Yamauchi and H. Fujisawa (1983) Eur. J. Biochem.132, 15–21]. Serine and threonine were identified as the phosphate acceptor amino acids of tubulin. The V_max of the phosphorylation of tubulin and the apparent K_m value for tubulin of calmodulin-dependent protein kinase II were 89 nmol phosphate transferred min^?1 mg kinase II^?1 and 1.7 μm, respectively. The maximum ³²P incorporation into tubulin was 0.18 mol P_i/mol α-tubulin and 0.13 mol P_i/mol β-tubulin. The phosphorylation of tubulin was decreased by the denaturation of tubulin. The phosphorylation of tubulin by kinase II did not affect the assembly of microtubules.     

Isolation of separate mRNAs for α- and β-Tubulin and characterization of the corresponding in vitro translation products

The messenger RNAs coding for α- and β-tubulin have been isolated from embryonic chick brain. Although the mRNAs for the two tubulin subunits have been resolved on native gels, they are very similar in molecular weight (650,000 daltons) as judged by mobility on denaturing gels containing methyl mercury. The mRNAs for β- and γ-actin have also been resolved on native gels, but migrate as an unresolved peak (molecular weight 650,000–700,000 daltons) under denaturing conditions. Since the nonmuscle actins are substantially smaller proteins than α- and β-tubulin, the large size of chick nonmuscle actin mRNAs suggests an unusually long untranslated region.Since tubulin and actin polypeptides are internal structural proteins, one would expect them to be synthesized only on free polysomes. Translation of mRNA derived directly from a purified membrane fraction or by puromycin release from that fraction, however, showed the synthesis of a small proportion of these proteins on polysomes that are membrane-associated. Peptide mapping has in all cases confirmed the identity of the products of cell-free synthesis with authentic α-tubulin, β-tubulin and actin. Approximately 67% of the α- and 13% of the β-tubulin chains produced by in vitro translation are competent for co-assembly into microtubules with added carrier microtubule protein.     

ZNRF1 interacts with tubulin and regulates cell morphogenesis

The ubiquitin-proteasome system has been implicated in neuronal degeneration and regeneration. We demonstrated that overexpression of ZNRF1, which has been identified as a crucial molecule in nerve regeneration, causes morphological changes such as neurite-like elongation. Molecular dissections showed that both the RING finger domain and zinc finger domain are required for morphological changes. Furthermore, we identified β-tubulin type 2 (Tubb2) as a ZNRF1-binding protein by yeast two-hybrid screening. In vivo binding assay showed that ZNRF1 interacts with Tubb2 and immunofluorescent staining suggests that ZNRF1 is colocalized with Tubb2. These results suggest that ZNRF1 mediates regulation of neuritogenesis via interaction with tubulin.