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E Nogales

Publications and source records attributed to E Nogales.

At least 19 recordsLinked to original sources
PubMed2001

Refined structure of alpha beta-tubulin at 3.5 A resolution.

We present a refined model of the alpha beta-tubulin dimer to 3.5 A resolution. An improved experimental density for the zinc-induced tubulin sheets was obtained by adding 114 electron diffraction patterns at 40-60 degrees tilt and increasing the completeness of structure factor amplitudes to 84.7 %. The refined structure was obtained using maximum-likelihood including phase information from experimental images, and simulated annealing Cartesian refinement to an R-factor of 23.2 and free R-factor of 29.7. The current model includes residues alpha:2-34, alpha:61-439, beta:2-437, one molecule of GTP, one of GDP, and one of taxol, as well as one magnesium ion at the non-exchangeable nucleotide site, and one putative zinc ion near the M-loop in the alpha-tubulin subunit. The acidic C-terminal tails could not be traced accurately, neither could the N-terminal loop including residues 35-60 in the alpha-subunit. There are no major changes in the overall fold of tubulin with respect to the previous structure, testifying to the quality of the initial experimental phases. The overall geometry of the model is, however, greatly improved, and the position of side-chains, especially those of exposed polar/charged groups, is much better defined. Three short protein sequence frame shifts were detected with respect to the non-refined structure. In light of the new model we discuss details of the tubulin structure such as nucleotide and taxol binding sites, lateral contacts in zinc-sheets, and the significance of the location of highly conserved residues.

Amino Acid Sequence↗
PubMed2001

The binding conformation of Taxol in beta-tubulin: a model based on electron crystallographic density.

The chemotherapeutic drug Taxol is known to interact within a specific site on beta-tubulin. Although the general location of the site has been defined by photoaffinity labeling and electron crystallography, the original data were insufficient to make an absolute determination of the bound conformation. We have now correlated the crystallographic density with analysis of Taxol conformations and have found the unique solution to be a T-shaped Taxol structure. This T-shaped or butterfly structure is optimized within the beta-tubulin site and exhibits functional similarity to a portion of the B9-B10 loop in the alpha-tubulin subunit. The model provides structural rationalization for a sizeable body of Taxol structure-activity relationship data, including binding affinity, photoaffinity labeling, and acquired mutation in human cancer cells.

Binding Sites↗
PubMed2001

Lipid-layer crystallization and preliminary three-dimensional structural analysis of AcrA, the periplasmic component of a bacterial multidrug efflux pump.

The multidrug efflux complex AcrAB-TolC confers intrinsic drug resistance in Escherichia coli by pumping antibiotics out of the cell. We determined a low-resolution (20 A) structure of AcrA, the periplasmic component, by electron crystallography. Expressed with a His-tag at its carboxyl-terminus, the protein bound to lipid layers containing the nickel-chelating phospholipid DOGS-NTA. Under the lipid layers, AcrA crystallized in layer group P2(1)22, with a unit cell size of 157 by 95 A and a thickness of about 100 A. The four asymmetric units in the unit cell are organized into what appears to be two rings, each with a central opening of 30 A in diameter. Within each ring, the density can be interpreted as following a pseudo-helical path, approximately 210 A long. This length matches that of monomeric AcrA in solution, previously estimated by light scattering and hydrodynamic measurements. On one side the density has a tubular shape, with a thickness of about 25 A, while on the other side the densities of the upper and lower parts of the pseudo-helical path are fused into a shield.

Bacterial Proteins↗
PubMed2001

Alanine-scanning mutagenesis of Aspergillus gamma-tubulin yields diverse and novel phenotypes.

We have created 41 clustered charged-to-alanine scanning mutations of the mipA, gamma-tubulin, gene of Aspergillus nidulans and have created strains carrying these mutations by two-step gene replacement and by a new procedure, heterokaryon gene replacement. Most mutant alleles confer a wild-type phenotype, but others are lethal or conditionally lethal. The conditionally lethal alleles exhibit a variety of phenotypes under restrictive conditions. Most have robust but highly abnormal mitotic spindles and some have abnormal cytoplasmic microtubule arrays. Two alleles appear to have reduced amounts of gamma-tubulin at the spindle pole bodies and nucleation of spindle microtubule assembly may be partially inhibited. One allele inhibits germ tube formation. The cold sensitivity of two alleles is strongly suppressed by the antimicrotubule agents benomyl and nocodazole and a third allele is essentially dependent on these compounds for growth. Together our data indicate that gamma-tubulin probably carries out functions essential to mitosis and organization of cytoplasmic microtubules in addition to its well-documented role in microtubule nucleation. We have also placed our mutations on a model of the structure of gamma-tubulin and these data give a good initial indication of the functionally important regions of the molecule.

Alanine↗
PubMed2001

Structural insight into microtubule function.

Microtubules are polymers that are essential for, among other functions, cell transport and cell division in all eukaryotes. The regulation of the microtubule system includes transcription of different tubulin isotypes, folding of alpha/beta-tubulin heterodimers, post-translation modification of tubulin, and nucleotide-based microtubule dynamics, as well as interaction with numerous microtubule-associated proteins that are themselves regulated. The result is the precise temporal and spatial pattern of microtubules that is observed throughout the cell cycle. The recent high-resolution analysis of the structure of tubulin and the microtubule has brought new insight to the study of microtubule function and regulation, as well as the mode of action of antimitotic drugs that disrupt normal microtubule behavior. The combination of structural, genetic, biochemical, and biophysical data should soon give us a fuller understanding of the exquisite details in the regulation of the microtubule cytoskeleton.

Animals↗
PubMed2001

Structural models for the self-assembly and microtubule interactions of gamma-, delta- and epsilon-tubulin.

alphabeta-tubulin heterodimers self-assemble to form microtubules nucleated by gamma-tubulin in the cell. Gamma-tubulin is believed to recruit the alphabeta-tubulin dimers that form the minus ends of microtubules, but the molecular mechanism of this action remains a matter of heated controversy. Still less is known about the function and molecular interactions of delta-tubulin and epsilon-tubulin. delta-tubulin may seed the formation of the C triplet tubules in the basal bodies of Chlamydomonas and epsilon-tubulin is known to localize to the centrosome in a cell cycle-dependent manner. Using the structure of alphabeta tubulin as a model, we have analyzed the sequences of gamma-, delta- and epsilon-tubulin in regions corresponding to different polymerization interfaces in the tubulin alphabeta dimer. The sequence comparisons sometimes show clear conservation, pointing to similar types of contacts being functionally important for the new tubulin considered. Conversely, certain surfaces show marked differences that rule out equivalent interactions for non-microtubular tubulins. This sequence/structure analysis has led us to structural models of how these special tubulins may be involved in protein-protein contacts that affect microtubule self-assembly. delta-tubulin most likely interacts longitudinally with alpha-tubulin at the minus ends of microtubules, while epsilon-tubulin most likely binds to the plus end of beta-tubulin. Conservation of key residues in gamma-tubulin suggests that it is capable of longitudinal self-assembly. The implications for the protofilament and template models of nucleation are considered.

Amino Acid Sequence↗
PubMed2000

Cold adaptation of microtubule assembly and dynamics. Structural interpretation of primary sequence changes present in the alpha- and beta-tubulins of Antarctic fishes.

The microtubules of Antarctic fishes, unlike those of homeotherms, assemble at very low temperatures (-1.8 degrees C). The adaptations that enhance assembly of these microtubules are intrinsic to the tubulin dimer and reduce its critical concentration for polymerization at 0 degrees C to approximately 0.9 mg/ml (Williams, R. C., Jr., Correia, J. J., and DeVries, A. L. (1985) Biochemistry 24, 2790-2798). Here we demonstrate that microtubules formed by pure brain tubulins of Antarctic fishes exhibit slow dynamics at both low (5 degrees C) and high (25 degrees C) temperatures; the rates of polymer growth and shortening and the frequencies of interconversion between these states are small relative to those observed for mammalian microtubules (37 degrees C). To investigate the contribution of tubulin primary sequence variation to the functional properties of the microtubules of Antarctic fishes, we have sequenced brain cDNAs that encode 9 alpha-tubulins and 4 beta-tubulins from the yellowbelly rockcod Notothenia coriiceps and 4 alpha-tubulins and 2 beta-tubulins from the ocellated icefish Chionodraco rastrospinosus. The tubulins of these fishes were found to contain small sets of unique or rare residue substitutions that mapped to the lateral, interprotofilament surfaces or to the interiors of the alpha- and beta-polypeptides; longitudinal interaction surfaces are not altered in the fish tubulins. Four changes (A278T and S287T in alpha; S280G and A285S in beta) were present in the S7-H9 interprotofilament "M" loops of some monomers and would be expected to increase the flexibility of these regions. A fifth lateral substitution specific to the alpha-chain (M302L or M302F) may increase the hydrophobicity of the interprotofilament interaction. Two hydrophobic substitutions (alpha:S187A in helix H5 and beta:Y202F in sheet S6) may act to stabilize the monomers in conformations favorable to polymerization. We propose that cold adaptation of microtubule assembly in Antarctic fishes has occurred in part by evolutionary restructuring of the lateral surfaces and the cores of the tubulin monomers.

Adaptation, Physiological↗
PubMed2000

A common pharmacophore for epothilone and taxanes: molecular basis for drug resistance conferred by tubulin mutations in human cancer cells.

The epothilones are naturally occurring antimitotic drugs that share with the taxanes a similar mechanism of action without apparent structural similarity. Although photoaffinity labeling and electron crystallographic studies have identified the taxane-binding site on beta-tubulin, similar data are not available for epothilones. To identify tubulin residues important for epothilone binding, we have isolated two epothilone-resistant human ovarian carcinoma sublines derived in a single-step selection with epothilone A or B. These epothilone-resistant sublines exhibit impaired epothilone- and taxane-driven tubulin polymerization caused by acquired beta-tubulin mutations (beta274(Thr-->Ile) and beta282(Arg-->Gln)) located in the atomic model of alphabeta-tubulin near the taxane-binding site. Using molecular modeling, we investigated the conformational behavior of epothilone, which led to the identification of a common pharmacophore shared by taxanes and epothilones. Although two binding modes for the epothilones were predicted, one mode was identified as the preferred epothilone conformation as indicated by the activity of a potent pyridine-epothilone analogue. In addition, the structure-activity relationships of multiple taxanes and epothilones in the tubulin mutant cells can be fully explained by the model presented here, verifying its predictive value. Finally, these pharmacophore and activity data from mutant cells were used to model the tubulin binding of sarcodictyins, a distinct class of microtubule stabilizers, which in contrast to taxanes and the epothilones interact preferentially with the mutant tubulins. The unification of taxane, epothilone, and sarcodictyin chemistries in a single pharmacophore provides a framework to study drug-tubulin interactions that should assist in the rational design of agents targeting tubulin.

Alkaloids↗
PubMed2000

A mutation in gamma-tubulin alters microtubule dynamics and organization and is synthetically lethal with the kinesin-like protein pkl1p.

Mitotic segregation of chromosomes requires spindle pole functions for microtubule nucleation, minus end organization, and regulation of dynamics. gamma-Tubulin is essential for nucleation, and we now extend its role to these latter processes. We have characterized a mutation in gamma-tubulin that results in cold-sensitive mitotic arrest with an elongated bipolar spindle but impaired anaphase A. At 30 degrees C cytoplasmic microtubule arrays are abnormal and bundle into single larger arrays. Three-dimensional time-lapse video microscopy reveals that microtubule dynamics are altered. Localization of the mutant gamma-tubulin is like the wild-type protein. Prediction of gamma-tubulin structure indicates that non-alpha/beta-tubulin protein-protein interactions could be affected. The kinesin-like protein (klp) Pkl1p localizes to the spindle poles and spindle and is essential for viability of the gamma-tubulin mutant and in multicopy for normal cell morphology at 30 degrees C. Localization and function of Pkl1p in the mutant appear unaltered, consistent with a redundant function for this protein in wild type. Our data indicate a broader role for gamma-tubulin at spindle poles in regulating aspects of microtubule dynamics and organization. We propose that Pkl1p rescues an impaired function of gamma-tubulin that involves non-tubulin protein-protein interactions, presumably with a second motor, MAP, or MTOC component.

Amino Acid Sequence↗
PubMed2000

Structure-function relationships in yeast tubulins.

A comprehensive set of clustered charged-to-alanine mutations was generated that systematically alter TUB1, the major alpha-tubulin gene of Saccharomyces cerevisiae. A variety of phenotypes were observed, including supersensitivity and resistance to the microtubule-destabilizing drug benomyl, lethality, and cold- and temperature-sensitive lethality. Many of the most benomyl-sensitive tub1 alleles were synthetically lethal in combination with tub3Delta, supporting the idea that benomyl supersensitivity is a rough measure of microtubule instability and/or insufficiency in the amount of alpha-tubulin. The systematic tub1 mutations were placed, along with the comparable set of tub2 mutations previously described, onto a model of the yeast alpha-beta-tubulin dimer based on the three-dimensional structure of bovine tubulin. The modeling revealed a potential site for binding of benomyl in the core of beta-tubulin. Residues whose mutation causes cold sensitivity were concentrated at the lateral and longitudinal interfaces between adjacent subunits. Residues that affect binding of the microtubule-binding protein Bim1p form a large patch across the exterior-facing surface of alpha-tubulin in the model. Finally, the positions of the mutations suggest that proximity to the alpha-beta interface may account for the finding of synthetic lethality of five viable tub1 alleles with the benomyl-resistant but otherwise entirely viable tub2-201 allele.

Animals↗
PubMed2000

Structural insights into microtubule function.

Microtubules are polymers that are essential for, among other functions, cell transport and cell division in all eukaryotes. The regulation of the microtubule system includes transcription of different tubulin isotypes, folding of /¿-tubulin heterodimers, post-translation modification of tubulin, and nucleotide-based microtubule dynamics, as well as interaction with numerous microtubule-associated proteins that are themselves regulated. The result is the precise temporal and spatial pattern of microtubules that is observed throughout the cell cycle. The recent high-resolution analysis of the structure of tubulin and the microtubule has brought new insight to the study of microtubule function and regulation, as well as the mode of action of antimitotic drugs that disrupt normal microtubule behavior. The combination of structural, genetic, biochemical, and biophysical data should soon give us a fuller understanding of the exquisite details in the regulation of the microtubule cytoskeleton.

Animals↗
PubMed2000

Recent structural insights into transcription preinitiation complexes.

Our understanding of the elaborate mechanism of gene transcription initiation in eukaryotes has been widened by recent structural information on some of the key components of the complex preinitiation transcriptional machinery. The high-resolution structures of both bacterial and eukaryotic polymerases are technical landmarks of great biological significance that have given us the first molecular insight into the mechanism of this large enzyme. While new atomic structures of different domains of general transcription factors, such as the double bromodomain of TAF250, have become available by means of X-ray crystallography and NMR studies, more global pictures of multisubunit transcription complexes, such as TFIID, TFIIH or the yeast mediator, have now been obtained by electron microscopy and image-reconstruction techniques. A combination of methodologies may prove essential for a complete structural description of the initial steps in the expression of eukaryotic genes.

DNA↗
PubMed1999

Three-dimensional structure of the human TFIID-IIA-IIB complex.

The multisubunit transcription factor IID (TFIID) is an essential component of the eukaryotic RNA polymerase II machinery that works in concert with TFIIA (IIA) and TFIIB (IIB) to assemble initiation complexes at core eukaryotic promoters. Here the structures of human TFIID and the TFIID-IIA-IIB complex that were obtained by electron microscopy and image analysis to 35 angstrom resolution are presented. TFIID is a trilobed, horseshoe-shaped structure, with TFIIA and TFIIB bound on opposite lobes and flanking a central cavity. Antibody studies locate the TATA-binding protein (TBP) between TFIIA and TFIIB at the top of the cavity that most likely encompasses the TATA DNA binding region of the supramolecular complex.

Binding Sites↗
PubMed1999

Visualizing a new binding site of ncd-motor domain on tubulin.

Ncd is a microtubule minus-end directed motor of the kinesin superfamily. Previously it has been shown that ncd and kinesin motor domains share the same major binding site on microtubules. Here we report a three-dimensional EM reconstruction of negatively stained two-dimensional Zn-induced tubulin crystal sheets (Zn-sheets) decorated with the ncd motor domain at a resolution of 16 A. This work has revealed a second specific binding site for the ncd motor domain. The motor binding site on the tubulin Zn-sheets spans both alpha and beta tubulin subunits. This binding site is located at a position different from the previously identified ncd binding site on microtubules and may play a role in motor function.

Animals↗
PubMed1999

Editorial

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Journal Article↗
PubMed1999

A structural view of microtubule dynamics.

The essential microtubule property of dynamic instability is based on the binding, hydrolysis and exchange of GTP in each tubulin dimer. The recent high-resolution structures of tubulin and the microtubule have given us the first view at atomic level of properties such as nucleotide exchangeability, the linkage between polymerization and nucleotide hydrolysis. and the origin of microtubule destabilization, as well as the mode of action of antimitotic agents such as taxol.

Amino Acid Sequence↗
PubMed1999

Alf1p, a CLIP-170 domain-containing protein, is functionally and physically associated with alpha-tubulin.

Tubulin is a heterodimer of alpha- and beta-tubulin polypeptides. Assembly of the tubulin heterodimer in vitro requires the CCT chaperonin complex, and a set of five proteins referred to as the tubulin cofactors (Tian, F., Y. Huang, H. Rommelaere, J. Vandekerckhove, C. Ampe, and N.J. Cowan. 1996. Cell. 86:287-296; Tian, G., S.A. Lewis, B. Feierbach, T. Stearns, H. Rommelaere, C. Ampe, and N.J. Cowan. 1997. J. Cell Biol. 138:821-832). We report the characterization of Alf1p, the yeast ortholog of mammalian cofactor B. Alf1p interacts with alpha-tubulin in both two-hybrid and immunoprecipitation assays. Alf1p and cofactor B contain a single CLIP-170 domain, which is found in several microtubule-associated proteins. Mutation of the CLIP-170 domain in Alf1p disrupts the interaction with alpha-tubulin. Mutations in alpha-tubulin that disrupt the interaction with Alf1p map to a domain on the cytoplasmic face of alpha-tubulin; this domain is distinct from the region of interaction between alpha-tubulin and beta-tubulin. Alf1p-green fluorescent protein (GFP) is able to associate with microtubules in vivo, and this localization is abolished either by mutation of the CLIP-170 domain in Alf1p, or by mutation of the Alf1p-binding domain in alpha-tubulin. Analysis of double mutants constructed between null alleles of ALF1 and PAC2, which encodes the other yeast alpha-tubulin cofactor, suggests that Alf1p and Pac2p act in the same pathway leading to functional alpha-tubulin. The phenotype of overexpression of ALF1 suggests that Alf1p can act to sequester alpha-tubulin from interaction with beta-tubulin, raising the possibility that it plays a regulatory role in the formation of the tubulin heterodimer.

Amino Acid Sequence↗