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CLASSIC STRUCTURES
αβ-tubulin

Eva Nogales

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Introduction

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Microtubules are cytoskeletal polymers present in all eukaryotic cells and are required for cellular transport, cell motility, and mitosis. Each microtubule is a cylindrical tube made up of protofilaments, which in turn consist of repeating αβ-tubulin heterodimers bound head to tail. The ability to switch stochastically between growing and shrinking phases is essential to the function of microtubules. This phenomenon, known as dynamic instability, is due to the GTPase activity of the tubulin monomers (see Dynamic instability of microtubules) (3295 ). The structure of the α β-tubulin heterodimer provides insight into how nucleotide hydrolysis affects the polymerization state of microtubules.

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Background

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Tubulin is an αβ dimeric protein that self-assembles into protofilaments. Typically, 13 of these protofilaments associate in parallel to make the microtubule wall. The parallel arrangement of protofilaments gives rise to a structure with a well-defined polarity, such that polymerization occurs more frequently at one end of the microtubule (called the plus end) than at the other (the minus end).

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Tubulin is highly conserved across species, reflecting the sequence constraints imposed by microtubule structure and function. Both α and β subunits exist in numerous isotypic forms and undergo a variety of posttranslational modifications (for a review see 3615). This variability is thought to be important for the interaction of tubulin with a variety of associated proteins and ligands (for reviews on how proteins and ligands affect the properties of microtubules, see 3605 and 3608).

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The α-tubulin and β-tubulin monomers each bind one molecule of GTP, but they differ in their ability to exchange the nucleotide. The nucleotide bound to α-tubulin, at the so-called N-site, cannot be exchanged. The nucleotide bound to β-tubulin, at the E-site, can be exchanged. GTP is required at the E-site in order for tubulin to polymerize. Upon polymerization, however, this nucleotide is hydrolyzed and can no longer be exchanged.

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The effect of GDP on microtubule structure is dramatic. The microtubule loses both its ability to grow and its stability; it quickly falls apart. Microtubules are thought to be stabilized by a cap of GTP-containing tubulin subunits at the microtubule ends. Loss of this "GTP-cap," e.g. through hydrolysis, would result in rapid depolymerization. This model to explain dynamic instability would greatly benefit from structural knowledge of how GTP hydrolysis affects the tubulin components.

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Structure and function

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Figure 1  
Ribbon representation of the αβ tubulin dimer (adapted from 3617). The PDB identifier of this structure is 1TUB.

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>The structure of the tubulin dimer at 3.5 Å resolution was obtained by electron crystallography of tubulin sheets stabilized with the anticancer drug taxol (3617; 3614) (zinc was used to form sheets, in which the protofilaments are arranged in an antiparallel fashion). As shown in Figure 1, the structures of α- and β-tubulin are nearly identical. Each subunit contains one molecule of guanine nucleotide, but only β-tubulin binds taxol.

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Each compact monomer contains three domains. The N-terminal, nucleotide-binding domain is comprised of six parallel β strands (S1-S6) alternating with helices (H1-H6). The loops that connect the β strand with the next helix are directly involved in binding GTP (loops T1 to T6). The core helix H7 connects the nucleotide-binding domain with the smaller, second domain. This domain is formed by three helices (H8-H10) and a mixed β sheet (S7-S10). The C-terminal region is formed by two antiparallel helices (H11-H12) that cross over the previous two domains.

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The structure of tubulin explains the different nucleotide exchangeabilities between α- and β-tubulin and the coupling of polymerization and hydrolysis. The GTP in α-tubulin is not exchangeable because the N-site is buried at the monomer-monomer interface within the dimer. In contrast, the GDP in β-tubulin is exchangeable because the E-site is exposed on the surface of the dimer. Upon polymerization, the E-site nucleotide becomes nonexchangeable because it is buried at the newly formed interface.

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Figure 2  
Loops T1-T6 and helix H7 from β-tubulin constitute the nucleotide binding domain, but loop T7 and residue 254 from α-tubulin are essential for nucleotide hydrolysis. The structure of the tubulin heterodimer contains GDP in the E-site, due to nucleotide hydrolysis during polymerization.

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The interface between the monomers reveals that both subunits contribute residues necessary for GTPase activity. Figure 2 illustrates the interface between dimers. Loop T7, which lies opposite the nucleotide binding site of the next subunit, contains amino acids that are required for nucleotide hydrolysis (GXXNXD). These residues are highly conserved in both tubulin subunits. Residue 254 in the H8 helix completes the interaction across the longitudinal interface. Lys254 in β-tubulin interacts with the γ-phosphate of the N-site nucleotide in α-tubulin. Glu254 in α-tubulin would be close to the γ-phosphate of the E-site nucleotide (in the crystal structure this nucleotide is GDP due to nucleotide hydrolysis during polymerization). The lack of conservation at residue 254 explains why α-tubulin can hydrolyze the GTP in β-tubulin, but β-tubulin cannot hydrolyze the GTP in α-tubulin.

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Microtubule structure

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Figure 3  
β-tubulins, with GTP exposed, crown the plus end of the microtubule. Addition of a tubulin dimer induces GTP hydrolysis at the underlying β-tubulin. This illustration includes the microtubule's "seam", where the lateral contacts are not between identical tubulin isoforms.

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The crystal structure of tubulin allowed us to produce a high-resolution model of the microtubule (3619). This was done by docking the structure of the tubulin protofilament into a low-resolution model of the microtubule, which was previously obtained by cryo-electron microscopy and helical image reconstruction (3621). Figure 3 shows the results of our modeling.

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The orientation of the α β-tubulin heterodimer within the microtubule has very important repercussions for the GTP-cap model of microtubule dynamics. The plus end of the microtubule is crowned by β-tubulin subunits exposing their nucleotide surface to the solution, while the minus end is crowned by α-subunits exposing their catalytic end (loop T7 and residue 254). The plus end, then, may be more dynamic because the hydrolyzable GTP is exposed right at the end, where having GTP or GDP has the biggest effect on microtubule stability. In contrast, the minus end consists of α-subunits, which are always bound to GTP.

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Figure 4  
The interactions between protofilaments shed light on the stability of microtubules (adapted from 3619).

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The high-resolution model of the microtubule also showed that the interface between the protofilaments, seen in Figure 4, underlies many properties of the microtubule, including the stabilizing effect of taxol. The M-loop (the loop between S7 and H9) makes close contacts with loop H1-S2 and helix H3 of the next subunit. The M-loop may act as a hinge between subunits, thereby allowing microtubules to be flexible in the number of protofilaments they contain. This explains why reconstituted microtubules do not have the same number of protofilaments. In α-tubulin, the conformation of the M-loop is stabilized by the long S9-S10 loop. In β-tubulin, the S9-S10 loop is not as long, but the M-loop is an essential part of the taxol binding pocket. Taxol and taxol-like compounds may stabilize the M-loop of β-tubulin, thereby affecting microtubule dynamics (3622).

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Interestingly, another element that β-tubulin contributes to the lateral interface, the H3 helix, follows loop T3. Loop T3 is involved in binding the γ-phosphate of the E-site nucleotide. Therefore, the destabilizing effect of nucleotide hydrolysis may be due to a conformational change transmitted to H3 through the γ-phosphate sensing loop.

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Legacy

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More recent structures of tubulin have been used to understand the effect of ligands that interfere with microtubule dynamics. Knossow and coworkers obtained a high-resolution structure of tubulin bound to the C-terminal fragment of RB3, a homolog of stathmin proteins (3606). Their structure suggested that stathmin proteins prevent tubulin polymerization by stabilizing a curved form of protofilaments. This is the conformation protofilaments assume when microtubules depolymerize. More recently, Sackett and coworkers have used the structure of tubulin in an electron microscopy study of tubulin bound to cryptophycin, an antimitotic agent (3623). They found that cryptophycin also induces formation of curved protofilaments.

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The structure of αβ-tubulin has been extremely useful for analyzing other tubulin isoforms. A homology model of γ-tubulin based on the structure of αβ-tubulin has been used to map mutations that affected microtubule dynamics in fission yeast (3620) as well as to map mutants in Aspergillus that give rise to novel phenotypes (3609). Analysis of the sequences of γ-, δ-, and ε-tubulin in regions corresponding to polymerization interfaces in αβ-tubulin allowed us to propose structural models of how different tubulins interact and affect microtubule self-assembly (3607).

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FtsZ, a ubiquitous protein in eubacteria and archebacteria essential for cytokinesis, is the only known structural homolog of tubulin (3612). This was demonstrated when the crystal structure of FtsZ was obtained (3612) and later compared in detail with the structure of tubulin (3617). Structural superposition of tubulin and FtsZ showed that the conserved residues are localized to sites of interaction with the nucleotide as well as T7 on the opposite side of the monomers. This led to a model of FtsZ polymerization in which T7 contributed to the interaction with the nucleotide in the next molecule along the filament. This model has been recently demonstrated by electron microscopy and image analysis of FtsZ filaments formed in vitro (3613).

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The structural homology between tubulin and FtsZ demonstrates another evolutionary link between eukaryotes and prokaryotes. Every single cell, regardless of kingdom, contains either tubulin or a homolog. This universality leads to the intriguing speculation that the original function of tubulin was not moving chromosomes and interacting with motors, but self-assembling into contractile structures.

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The author

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Eva Nogales obtained her B.S. degree in Physics by the Universidad Autonoma de Madrid (Spain) in 1988. She did her thesis work at the Synchrotron Radiation Source (U.K.), under the supervision of Dr. Joan Bordas, on the structural dynamics of assembly of tubulin, earning a Ph.D. degree by the Physics Department of the University of Keele in 1992. Her postdoctoral work in Dr. Kenneth Downing's lab at Lawrence Berkeley National Laboratory involved the use of electron crystallography to determine the high-resolution structure of tubulin. In 1998 she became Assistant Professor in the Department of Molecular and Cell Biology at UC Berkeley, and in 2000 Assistant Investigator at HHMI. She is presently a member of the Editorial Board of the Journal of Structural Biology and of the Biophysical Society Council. Her work centers around two main projects: the structural basis of microtubule dynamics and the structural organization of the human transcriptional initiation machinery. She uses electron microscopy, image analysis, homology modeling, and functional biochemical assays to gain new information on these systems and to progress toward models of how they are regulated in the cell.

Contact details

Eva Nogales
Howard Hughes Medical Institute
Molecular and Cell Biology Department
University of California, Berkeley
Berkeley, CA 94720
Phone: 510 642 0557
Fax: 510 642 8806
E-mail: enogales@lbl.gov

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Last Revised on September 23, 2004

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reviews
  • 3605 Desai, A. and Mitchison, T. J. (1997).  Microtubule polymerization dynamics.  Annu. Rev. Cell Dev. Biol. 13, 83-117.  PubMed   Journal
  • 3608 Jordan, A., Hadfield, J. A., Lawrence, N. J., and McGown, A. T. (1998).  Tubulin as a target for anticancer drugs: agents which interact with the mitotic spindle.  Med Res Rev 18, 259-296.  PubMed   Journal
  • 3615 Ludueña, R. F. (1998).  Multiple forms of tubulin: different gene products and covalent modifications.  Int. Rev. Cytol. 178, 207-275.  PubMed  
reviews
  • 3295 Mitchison, T. and Kirschner, M. (1984).  Dynamic instability of microtubule growth.  Nature 312, 237-242.  PubMed  
  • 3606 Gigant, B., Curmi, P. A., Martin-Barbey, C., Charbaut, E., Lachkar, S., Lebeau, L., Siavoshian, S., Sobel, A., and Knossow, M. (2000).  The 4 A X-ray structure of a tubulin:stathmin-like domain complex.  Cell 102, 809-816.  PubMed   Journal
  • 3607 Inclan, Y. F. and Nogales, E. (2001).  Structural models for the self-assembly and microtubule interactions of gamma-, delta- and epsilon-tubulin.  J. Cell Sci. 114, 413-422.  PubMed   Journal
  • 3609 Jung, M. K., Prigozhina, N., Oakley, C. E., Nogales, E., and Oakley, B. R. (2001).  Alanine-scanning mutagenesis of Aspergillus gamma-tubulin yields diverse and novel phenotypes.  Mol. Biol. Cell 12, 2119-2136.  PubMed   Journal
  • 3612 Lowe, J. and Amos, L. A. (1998).  Crystal structure of the bacterial cell-division protein FtsZ.  Nature 391, 203-206.  PubMed   Journal
  • 3613 Lowe, J. and Amos, L. A. (1999).  Tubulin-like protofilaments in Ca2+-induced FtsZ sheets.  EMBO J. 18, 2364-2371.  PubMed   Journal
  • 3614 Lowe, J., Li, H., Downing, K. H., and Nogales, E. (2001).  Refined structure of alpha beta-tubulin at 3.5 A resolution.  J. Mol. Biol. 313, 1045-1057.  PubMed   Journal
  • 3617 Nogales, E., Wolf, S. G., and Downing, K. H. (1998).  Structure of the alpha beta tubulin dimer by electron crystallography.  Nature 391, 199-203.  PubMed   Journal
  • 3619 Nogales, E., Whittaker, M., Milligan, R. A., and Downing, K. H. (1999).  High-resolution model of the microtubule.  Cell 96, 79-88.  PubMed   Journal
  • 3620 Paluh, J. L., Nogales, E., Oakley, B. R., McDonald, K., Pidoux, A. L., and Cande, W. Z. (2000).  A mutation in gamma-tubulin alters microtubule dynamics and organization and is synthetically lethal with the kinesin-like protein pkl1p.  Mol. Biol. Cell 11, 1225-1239.  PubMed   Journal
  • 3621 Sosa, H., Dias, D. P., Hoenger, A., Whittaker, M., Wilson-Kubalek, E., Sablin, E., Fletterick, R. J., Vale, R. D., and Milligan, R. A. (1997).  A model for the microtubule-Ncd motor protein complex obtained by cryo-electron microscopy and image analysis.  Cell 90, 217-224.  PubMed   Journal
  • 3622 Snyder, J. P., Nettles, J. H., Cornett, B., Downing, K. H., and Nogales, E. (2001).  The binding conformation of Taxol in beta-tubulin: a model based on electron crystallographic density.  Proc. Natl. Acad. Sci. USA 98, 5312-5316.  PubMed   Journal
  • 3623 Watts, N. R., Cheng, N., West, W., Steven, A. C., and Sackett, D. L. (2002).  The cryptophycin-tubulin ring structure indicates two points of curvature in the tubulin dimer.  Biochemistry 41, 12662-12669.  PubMed   Journal

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