Molecular and structural-biological analysis of <Emphasis Type="Italic">Nicotiana plumbaginifolia</Emphasis> mutants for identification of the site on their β-tubulins of interaction with dinitroanilines and phosphorothioamides

The identification of the location of a point mutation on β-tubulin molecules of amiprophosmethyland trifluralin-resistant Nicotiana plumbaginifolia lines are described in this work. It was shown that in the first case, this mutation is related with the substitution of serine residue for proline in position 248; in the second case, with the substitution of phenylalanine for serine in position 317 of the β-tubulin’s amino acid sequence. Three-dimensional models of the β-tubulin molecule from Chlamydomonas with the well-known location of mutations determining dinitroaniline- and phosphorothioamides resistance (the substitution of lysine residue for methionine in position 350), and β-tubulin from Nicotiana plumbagnifolia have been reconstructed. On the basis of the analysis of the interaction site for dinitroanilines and phosphorothioamides located on the Chlamydomonas β-tubulin’s molecule it was concluded that the revealed mutations on Nicotiana plumbaginifolia β-tubulin are affected by the residues of the amino acids, participating in the formation of this site.     

Molecular dynamics simulation and density functional theory studies on the active pocket for the binding of paclitaxel to tubulin

Paclitaxel (PTX) is used to treat various cancers, but it also causes serious side effects and resistance. To better design similar compounds with less toxicity and more activity against drug-resistant tumors, it is important to clearly understand the PTX-binding pocket formed by the key residues of active sites on β-tubulin. Using a docking method, molecular dynamics (MD) simulation and density functional theory (DFT), we identified some residues (such as Arg278, Asp26, Asp226, Glu22, Glu27, His229, Arg369, Lys218, Ser277 and Thr276) on β-tubulin that are the active sites responsible for interaction with PTX. Another two residues, Leu371 and Gly279, also likely serve as active sites. Most of these sites contact with the “southern hemisphere” of PTX; only one key residue interacts with the “northern hemisphere” of PTX. These key residues can be divided into four groups, which serve as active compositions in the formation of an active pocket for PTX binding to β-tubulin. This active binding pocket enables a very strong interaction (the strength is predicted to be in the range of −327.8 to −365.7 kJ mol⁻¹) between β-tubulin and PTX, with various orientated conformations. This strong interaction means that PTX possesses a high level of activity against cancer cells, a result that is in good agreement with the clinical mechanism of PTX. The described PTX pocket and key active residues will be applied to probe the mechanism of tumor cells resistant to PTX, and to design novel analogs with superior properties.     

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.     

Electrostatic Contributions to Colchicine Binding within Tubulin Isotypes

Tubulin, the structural subunit of microtubules, is the target of some highly successful anti-tumor drugs. Most of these drugs bind to the β-tubulin resulting in the inhibition of microtubule dynamics and eventually cell death. The varied cellular distribution of several human isotypes of β -tubulin provides a platform upon which to construct novel chemotherapeutic agents that are able to differentiate between these types of cells. To test this hypothesis, we have previously created homology models of the nine most frequently observed human β -tubulin isotypes and analyzed them for differences in the colchicine-binding site. Here, we describe the electrostatic properties of the colchicine binding site and how this may affect calculated drug binding affinities between the β -tubulin isotypes.     

h-TBP: an approach based on intron-length polymorphism for the rapid isolation and characterization of the multiple members of the β-tubulin gene family in <Emphasis Type="Italic">Camelina sativa</Emphasis> (L.) Crantz

We have developed a new version of the cTBP (combinatorial tubulin-based polymorphism) method, a previously described approach based on intron-length polymorphism (ILP), to rapidly characterize the β-tubulin gene family of Camelina sativa (L.) Crantz, a plant species of importance for oil production but still largely unexplored at genomic level. The method, named h-TBP, allows the rapid cloning of the β-tubulin genomic sequences that encompass the two introns, invariantly present at fixed positions within the coding region of the vast majority of the plant species. The β-tubulin sequences cloned by h-TBP also comprise part of exon1 and exon3 and the whole sequence of exon2. The h-TBP method has then been used to isolate, clone and characterize the β-tubulin gene family of C. sativa, composed of at least 20 different β-tubulin isotypes, named CsTUB1 through CsTUB20. The relatively high number of β-tubulin genes has been further substantiated by Southern-blot analysis. Comparison of the β-tubulin exon sequences of C. sativa with those of Arabidopsis thaliana, the closest relative among crucifers, defines distinct groups of putative orthologous genes, identified by a UPMGA cluster analysis. Analysis of the C. sativa β-tubulin intron sequences reveals some molecular features that can provide the first hints for the understanding of intron plasticity and evolution. From a more immediate perspective, these data provide the first substantial contribution to the characterization of the largely unexplored genome of C. sativa, and the tools for assisting programmes of breeding and selection of the most productive plants.     

Analysis of beta-tubulin cDNAs from taxol-resistant Pestalotiopsis microspora and taxol-sensitive Pythium ultimum and comparison of the taxol-binding properties of their products

The anti-cancer drug taxol binds to β-tubulin in assembled microtubules and causes cell cycle arrest in animal cells; in contrast, in fungi, the effect of taxol varies. For instance, the taxol-producer Pestalotiopsis microspora Ne32, an ascomycete, is resistant to taxol (IC₅₀ greater than 11.7 μM), whereas Pythium ultimum, an oomycete, is sensitive to taxol (IC₅₀ 0.1 μM). In order to understand the differential fungal response to taxol, we isolated cDNAs encoding β-tubulin from both P. microspora and P. ultimum. The deduced amino acid sequence of β-tubulin from P. microspora is very similar to those from other Ascomycetes, many of which are resistant to taxol. The sequence of β-tubulin from P. ultimum is very similar to those from Oomycetes and non-fungal organisms, many of which are sensitive to taxol. To examine the interaction between taxol and fungal microtubules, binding studies were performed with fungal cells, using [³H]taxol. The labeled taxol was found to bind specifically to P. ultimum, but not to P. microspora. In addition, the amount of [³H]taxol specifically bound to P. ultimum was reduced by the microtubule-depolymerizing drug thiabendazole, in a dose-dependent manner. These results suggest efficient binding of taxol to microtubules in P. ultimum, but not in P. microspora, and are consistent with the differential taxol sensitivity of these two organisms. Finally a comparison of previously characterized taxol binding sites in various β-tubulin sequences showed that β-tubulins of taxol-sensitive organisms, including P. ultimum, contain Thr219, but β-tubulins of resistant organisms, including P. microspora, contain Asn or Gln at this position, suggesting an important role for residue 219 in the interaction between taxol and β-tubulin. Received: 16 March 1999 / Accepted: 21 August 1999     

Identification of Phosphate Binding Residues of <Emphasis Type="Italic">Escherichia coli</Emphasis> ATP Synthase

Four positively-charged residues, namely βLys-155, βArg-182, βArg-246, and αArg-376 have been identified as Pi binding residues in Escherichia coli ATP synthase. They form a triangular Pi binding site in catalytic site βE where substrate Pi initially binds for ATP synthesis in oxidative phosphorylation. Positive electrostatic charge in the vicinity of βArg-246 is shown to be one important component of Pi binding.     

Acyl-biotinyl Exchange Chemistry and Mass Spectrometry-Based Analysis of Palmitoylation Sites of In Vitro Palmitoylated Rat Brain Tubulin

Research has shown that the palmitoyl group of α-tubulin mediates the hydrophobic interaction between microtubules and intracellular membranes and that palmitoylated tubulin plays a role in signal transduction. There are 20 cysteine residues per α/β tubulin heterodimer. C376 of α-tubulin was reported to be predominantly palmitoylated and C20, C213 and C305 of α-tubulin were palmitoylated at lower levels. The previous method used for the analysis of the palmitoylation sites on α-tubulin was based on ³H-labeling, enzymolysis, purification and sequencing. This approach, although efficient, is laborious. Mass spectrometry (MS), especially tandem MS, has been shown to be a successful method for identification of various post-translational modifications of proteins. We report here a convenient MS-based method to comprehensively analyze the palmitoylation sites of the α/β tubulin heterodimer. Acyl-biotinyl exchange chemistry and streptavidin agarose affinity purification were applied to enrich palmitoylated peptides from tubulin. After nano-LC-MS/MS analysis, database searching and manual analysis of the spectra revealed that 11 cysteine residues of the α/β tubulin heterodimer were palmitoylated.     

Organization and chromosomal localization ofβ-tubulin genes inLeishmania donovani

The genomic organization and chromosomal location of theβ-tubulin isogenes inLeishmania donovani promastigotes has been studied by nucleic acid hybridization techniques using a cloned β-tubulin gene. We have cloned aβ-tubulin gene fragment, 3.3 kbp long, from genomic DNA ofLeishmania donovani using a heterologousβ-tubulin DNA as probe. Restriction maps of this clone have been prepared. It has been estimated that there are approximately 11–15 copies of theβ-tubulin genes per haploid genome. The majority of these isogenes are arranged in a tandem repeat with a length of 3.5 kbp on a single chromosome. In addition a few dispersed gene copies at different chromosomal loci were detected by pulse field gradient gel electrophoresis. Part of the internal coding region of the gene has been sequenced to confirm the identity of theβ-tubulin clone and is found to be nearly identical to that ofLeishmania mexicana amazonensis.     

Ultra performance liquid chromatography-mass spectrometric determination of the site specificity of modification of β-casein by glucose and methylglyoxal

Modification of protein by carbonyl compounds under in vitro physiological conditions is site-directed. There are few reports of the site specificity of glycation of proteins using heating conditions of relevance to food processing. The aim of this study was to determine the site specificity of modification of β-casein (βCN) by glucose and methylglyoxal (MGO). βCN (1.33 M, 3.2%) was heated with either glucose (1.345 M, 4.6%) or MGO (1 mM) at 95°C for up to 4 h. Tryptic digests were prepared and analysed by ultra performance liquid chromatography electrospray ionisation mass spectrometry (UPLC-ES/MS). The sites of formation of the Amadori product, N ^ε -(fructosyl)lysine (FL), and the advanced glycation end-products, N ^ε -(carboxymethyl)lysine (CML), MGO-derived dihydroxyimidazolidine (MG-DH) and MGO-derived hydroimidazolone (MG-HI), were located. FL and CML were detected at K107 and K176 residues in βCN/glucose incubations. Indigenous N ^ε -(lactulosyl)lysine was detected at K107 only. MG-DH and MG-HI were detected at R202 and possibly R183 residues in both βCN/glucose and βCN/MGO incubations. Glycation of βCN by glucose and MGO resulted in similar site specificity for MG-DH and MG-HI formation.     

Physicochemical aspects of carbohydrate binding to some plant lectins with binding preference forN-acetylgalactosamine and galactose

This contribution illustrates the advantages of some chromophoric and fluorophoric carbohydrate derivatives such asp-nitrophenyl (pNO₂Phe) or 4-methylumbelliferyl (MeUmb) glycosides andN-dansylgalactosamine in studies of the binding equilibrium and kinetics with some plant lectins. The methods used involve continuous titrations of changes in ligand or protein absorption and ligand fluorescence, including substitution titrations as well as stopped-flow, temperature-jump or pressure-jump relaxation kinetics. When monitored by temperature-jump relaxation, binding of MeUmbαGal to the bloodgroup A specific lectin GSAI-A₄ fromGriffonia simplicifolia is a simple bimolecular association with parametersk ₊ = 9.4 × 10⁴ M^-1 s^-1 andk _-1 = 5.3 s^-1 at 23°C, but binding to the GSAI-B₄ lectin is biphasic. The complementarity of the peanut agglutinin binding site with Galβ1 → 3GalNAc that occurs in manyO-glycoproteins follows from enthalpic considerations and also from the value of the dissociation-rate parameterk _-1 = 0.24 s^-1 of the MeUmbβGalβl → 3GalNAc.lectin complex. This value, obtained by stopped-flow kinetics is 100 times smaller than for other mono-and disaccharides investigated. The binding mechanism is simple and the derivatisation of Galβ1 → 3GalNAc does not affect the affinity to a considerable degree. The binding preference of tetravalentsoybean agglutinin for MeαGalNAc over MeαGal by a factor of 25 is mainly of enthalpic origin with an additional 7 kJ mol^-1; the NAc group causes perturbation of a tryptophanyl residue, evidenced by protein difference absorption spectrometry. In the glycosides, a large aglycon likeβpNO₂ Phe orβMeUmb hardly affects the affinity of SBA but a largeN-dansyl group increases the affinity by a factor 20 as compared to GalNAc. The 10-fold increase in carbohydrate-specificN-dansylgalactosamine fluorescence, together with a very favourable entropic contribution point at the presence of a hydrophobic region in the vicinity of the carbohydrate-binding site. The dissociation-rate parameter of the MeUmbβGalNAc SBA complex is slower than for any reported monosaccharide-lectin complex: 0.4 s^-1. The divalent lectin fromErythrina cristagalli preferentially binds the Galβ1 → 4GlcNAc structure that occurs in manyN-glycoproteins. The combining site was mapped thermodynamically with carbohydrates ranging from mono-to pentasaccharides as derived fromN-glycoproteins. Here, N-dansylgalactosamine was used as a fluorescent indicator ligand in substitution titrations. When Galβ1 → 4GlcNAc was linkedα1 → 2 orα1 → 6 to Man, the binding enthalpy and entropy remained practically constant. Application of stopped flow kinetics and pressure-jump relaxation withN-dansylgalactosamine gave mono-exponential signal changes with a concentration dependence corresponding tok ₊ = 4.8 x 10⁴ M^-1 s^-1 k _- = 0.4 to 0.66 s^-1 and a change in reaction volume of+7ml/mol.     

The β-tubulin gene as a molecular phylogenetic marker for classification and discrimination of the <Emphasis Type="Italic">Saccharomyces</Emphasis> <Emphasis Type="Italic">sensu stricto</Emphasis> complex

The Saccharomyces sensu stricto complex comprises seven very closely related species. In this study, we compared the use of two different phylogenetic markers, the 26S rDNA and β-tubulin genes, for discriminating phylogenetic relationships among Saccharomyces sensu stricto strains using sequencing as well as RFLP methods. The average sequence similarity for the β-tubulin gene (90.0%) among seven strains was significantly less than that for 26S rDNA (98.6%). This result demonstrates that β-tubulin gene sequences provided higher resolution than 26S rDNA sequences. Species-specific restriction profiles of the Saccharomyces strains were obtained by cutting them with the Tsp509I enzyme. Our data indicate that phylogenetic relationships between these strains are best resolved using sequencing or RFLP analysis of the β-tubulin gene. The β-tubulin gene sequence data reported in this paper appear in the GenBank nucleotide sequence database with the following accession numbers: FJ238316–FJ238341.     

Distinct localization of a beta-tubulin epitope in the Tetrahymena thermophila and Paramecium caudatum cortex

Summary. Many of the highly organized microtubular arrangements in ciliates are located in the cortical area containing membrane vesicles and vacuoles. In Tetrahymena thermophila and Paramecium caudatum, immunofluorescence microscopy with the monoclonal antibody TU-06, directed against β-tubulin, revealed distinct staining of this cortical region alone, while the cilia and other microtubular structures were unstained. The specificity of the antibody was confirmed by immunoblotting and by preabsorption of the antibody with purified tubulin. Double-label immunofluorescence with antibodies against γ-tubulin, detyrosinated α-tubulin, and centrin showed that the TU-06 epitope is localized outside the basal body region. This was also confirmed by immunogold electron microscopy of thin sections. Proteolytic digestion of porcine brain β-tubulin combined with a peptide scan of immobilized, overlapping peptides disclosed that the epitope was in the β-tubulin region β81–95, a region which is phylogenetically highly conserved. As known posttranslational modifications of β-tubulin are located outside this area, the observed staining pattern cannot be interpreted as evidence of subcellular sequestration of modified tubulin. The limited distribution of the epitope could rather reflect the dependence of TU-06 epitope exposition on conformations of tubulin molecules in microtubule arrangements or on differential masking by interacting proteins. Correspondence and reprints: Institute of Molecular Genetics, Czech Academy of Sciences, Vídeňská 1083, 14220 Prague, Czech Republic.     

Alteration of β-tubulin in Nicotiana plumbaginifolia confers resistance to amiprophos-methyl

 A Nicotiana plumbaginifolia plant (apm5^r) resistant to amiprophos-methyl (APM), a phosphoro-amide herbicide, was isolated from protoplasts prepared from leaves of haploid plants. Genetic analysis revealed that the resistance is coded for by a dominant nuclear mutation and is associated with the increased stability of cortical microtubules. Two-dimensional polyacrylamide-gel electrophoresis, combined with immunoblotting using anti-tubulin monoclonal antibodies, showed that part of the β-tubulin in the resistant plant possessed lower isoelectric points than the β-tubulin of susceptible wild-type plants. These results provide evidence that the resistance to APM is associated with a mutation in a β-tubulin gene. The APM-resistant line showed cross-resistance to trifluralin, a dinitroaniline herbicide, suggesting a common mechanism of resistance between these two classes of herbicides. Received: 26 January 1997 / Accepted: 17 February 1998     

Dioxygenases Without Requirement for Cofactors: Identification of Amino Acid Residues Involved in Substrate Binding and Catalysis, and Testing for Rate-Limiting Steps in the Reaction of 1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase

1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod), catalyzing cleavage of its heteroaromatic substrate to form carbon monoxide and N-acetylanthranilate, belongs to the α/β hydrolase fold family of enzymes. Analysis of protein variants suggested that Hod has adapted active-site residues of the α/β hydrolase fold for the dioxygenolytic reaction. H251 was recently shown to act as a general base to abstract a proton from the organic substrate. Residue S101, which corresponds to the nucleophile of the catalytic triad of α/β-hydrolases, presumably participates in binding the heteroaromatic substrate. H102 and residues located in the topological region of the triad’s acidic residue appear to influence O₂ binding and reactivity. A tyrosine residue might be involved in the turnover of the ternary complex [HodH⁺–3,4-dioxyquinaldine dianion–O₂]. Absence of viscosity effects and kinetic solvent isotope effects suggests that turnover of the ternary complex, rather than substrate binding, product release, or proton movements, involves the rate-determining step in the reaction catalyzed by Hod.     

Acrylamide Alters Cytoskeletal Protein Level in Rat Sciatic Nerves

Occupational exposure and experimental intoxication with acrylamide (ACR) produce neuropathy characterized by nerve degeneration. To investigate the mechanism of ACR-induced neuropathy, male adult Wistar rats were given ACR (20, 40 mg/kg i.p. 3 days/week) for 8 weeks. Sciatic nerves were Triton-extracted and centrifuged at a high speed (100,000 × g) to yield pellet and supernatant fractions. The contents of six cytoskeletal proteins (NF-L, NF-M, NF-H, α-tubulin, β-tubulin, and β-actin) in both fractions were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Results showed that the three neurofilament (NF) subunits (NF-L, NF-M, NF-H) in both the pellet and the supernatant fraction decreased significantly (P < 0.01) in the high-dosing group, except for NF-M in the pellet. α-tubulin, β-tubulin, and β-actin increased significantly in the supernatant (P < 0.01), whereas both α-tubulin and β-tubulin decreased significantly in the pellet (P < 0.01). However, β-actin was not altered significantly in the sciatic nerves pellet. These findings suggest that ACR altered the cytoskeletal protein level in sciatic nerve, which may be one of the molecular mechanisms of ACR-induced peripheral neuropathy.     

Mechanism of action of N-phenyl-N′-(2-chloroethyl)ureas in the colchicine-binding site at the interface between α- and β-tubulin

Computational tools such as CoMSIA and CoMFA models reported in a recent study revealed the structure–activity relationships ruling the interactions occurring between hydrophobic N-phenyl-N′-(2-chloroethyl)ureas (CEU) and the colchicine-binding site (C-BS) on β_ΙΙ-tubulin. Here, we describe the mechanisms involved in the covalent binding of three subsets of CEU derivatives to the C-BS. The FlexiDock experiments confirmed that the interaction of non-covalent portions of the CEU auxophore moiety of CEU is involved in the binding of the drug to the C-BS facilitate the nucleophilic attack of Glu-β198 rather than Cys-β239. In addition, these studies suggest that Cys-β239 together with Asn-α99, Ser-α176, Thr-α177, Leu-β246, Asn-β247, Ala-β248, Lys-β252 and Asn-β256 are implicated in the stabilization of a C-BS–CEU complex prior to the acylation of Glu-β198 by CEU. Our molecular models propose the formation of a stabilized C-BS–CEU complex before the completion of the Glu-β198 acylation; acylation triggering conformational changes of β-tubulin, microtubule depolymerization and anoikis. The computational models presented here might be useful to the design of selective and more potent C-BS inhibitors. Of interest, in vivo acylation of acidic amino acid residues by xenobiotics is an unusual reaction and may open new approaches for the design of irreversible protein inhibitors such as tubulin.     

Functional Significance May Underlie the Taxonomic Utility of Single Amino Acid Substitutions in Conserved Proteins

We hypothesized that some amino acid substitutions in conserved proteins that are strongly fixed by critical functional roles would show lineage-specific distributions. As an example of an archetypal conserved eukaryotic protein we considered the active site of β-tubulin. Our analysis identified one amino acid substitution—β-tubulin F224—which was highly lineage specific. Investigation of β-tubulin for other phylogenetically restricted amino acids identified several with apparent specificity for well-defined phylogenetic groups. Intriguingly, none showed specificity for “supergroups” other than the unikonts. To understand why, we analysed the β-tubulin Neighbor-Net and demonstrated a fundamental division between core β-tubulins (plant-like) and divergent β-tubulins (animal and fungal). F224 was almost completely restricted to the core β-tubulins, while divergent β-tubulins possessed Y224. Thus, our specific example offers insight into the restrictions associated with the co-evolution of β-tubulin during the radiation of eukaryotes, underlining a fundamental dichotomy between F-type, core β-tubulins and Y-type, divergent β-tubulins. More broadly our study provides proof of principle for the taxonomic utility of critical amino acids in the active sites of conserved proteins.