α, β-tubulin (1JFF) from Bos taurus is a protein dimer that polymerizes to form protofilaments, the foundational structure of the microtubule wall present in eukaryotic cell cytoskeletons. Dimers bind head-to-tail to create protofilaments and approximately 13 protofilaments organized parallel to one another comprise the microtubule wall (1). α, β-tubulin provides a foundation that assists in the movement of flagella, organelles, and chromosomes. This foundation consists of microtubules, polymers of tubulin, which direct movement of cellular objects along a fixed path (2). Tubulin has undergone extensive research due to its capability to bind anticancer drugs. Of particular interest is taxol, a member of the taxane family of drugs, which will be discussed later in conjunction with an analogous drug family, epothilones (3). The molecular weight of α, β-tubulin is 101,925.89 Da, and both subunits have an isoelectric point between 5.2 and 5.8 (1).
The α, β-tubulin dimer has two distinct subunits, the α and β monomers, each with two domains (1). Both monomers have a nucleotide binding domain and a C-terminal domain, and in general, both contain a fixed globular region and a more variable C-terminal region (4). Despite the similarities, there is importance in the α and β monomers being somewhat unique for polymerization to occur head-to-tail for the sake of polarity. Indeed, the domain sizes vary by monomer, but sequences are highly conserved across the multiple isotypes. The number of residues contained by the two domains is variable among different species, though in Bos taurus that number is 218 residues and 242 residues for the α and β nucleotide binding domains, respectively, and 194 residues and 184 residues in the respective α and β C-terminal domains, contributing to the distinctiveness of the two subunits (1).
The nucleotide binding domain is located at the N-terminus consisting of six parallel β-strands identified as S1-S6. Between each strand is an α-helix; they are identified as H1-H6. There are loops connecting each β-strand and α-helix, all of which participate in nucleotide binding. Within this domain, each monomer of tubulin can bind one molecule of GTP and one nucleotide. In the α monomer, the nucleotide binds to the N-site and is non-exchangeable due to this position, being buried at the interface between the two monomers. The β subunit binds a nucleotide at the E-site and is exchangeable because it is located on the protein surface. GTP is necessary on the β monomer for polymerization but when GTP binds to the E-site, the nucleotide undergoes hydrolysis and the E-site becomes covered by the interface between monomers, making the nucleotide non-exchangeable. Asn-206 and Asn-228 of the α monomer form hydrogen bonds with GTP molecules in order to form polymers. Residues 11, 15, 16, 69, 71, 99, 101, 140, 145, 168, 179, 206, 224, and 228 form the nucleotide binding pocket, and α:Gly-254 is responsible for the hydrolysis of the nucleotide. For both the α and β subunits, finalization of nucleotide binding occurs by interacting with a core helix (H7) that connects the nucleotide binding domain with the C-terminal domain (1).
The C-terminal domain is smaller than the nucleotide binding domain and consists of two antiparallel α-helices, H11 and H12 (1). It is a negatively charged domain rich in glutamate and tends to be less associated with the rest of the protein structure, projecting outward from the subunit. The C-terminal domain is not only variable in the number of residues, but also in the residues of which it is comprised and the isotypes are interchangeable, having no effect on the function of the region. Because of the exchangeability, different tubulin isotypes can combine to form a single polymer. There are a variety of microtubule-associated proteins and motor proteins that bind to the C-terminal domain important to cellular transport and “dynamic instability,” the alternating growth and shrinking phases characteristic of microtubules. This domain also undergoes glutamylation and polyglycylation in both subunits; detyrosination and deglutamylation in the α subunit only; and phosphorylation in the β subunit only. However, the C-terminal domain has no role in the formation of dimers and does not affect subunit conformations or microtubule formation (4).
The total composition of the secondary structure in α,β-tubulin is 38 α-helices (40.5%), 30 β-sheets (13%), and 46.5% random coil and other secondary structures (1). There is a clear absence of parallel β-sheets, though there are parallel β-strands, as tubulin is primarily composed of antiparallel β-sheets. In the conformation of tubulin, the α-helices are responsible for nucleation and the rate of polymerization, while antiparallel β-sheets serve to regulate these functions as well as attribute to the stability of such a dynamic protein (5).
There are structural and functional similarities between α, β-tubulin and different proteins found in other species. Among these is FtsZ (2R6R), a protein found in prokaryotes with a homologous function to tubulin (6). It has a Z-score of 27.5, an E-value of 0.055, and 23% similarity when compared to tubulin, which can be attributed in part to the evolution of the cell over time (7). FtsZ, like tubulin, uses GTP hydrolysis to drive polymerization in forming protofilaments. In contrast, tubulin produces microtubules that are thousands of subunits in length and FtsZ reaches a polymer maximum around 100 subunits. Because FtsZ molecules are smaller in size, they do not require lateral interactions in protofilament production. Instead, FtsZ contains longitudinal bonds as much as 100 times the strength of those found in tubulin polymers for stabilization. Concerning polymerization, GTP is hydrolyzed within seconds, but the same process may take as long as one minute in FtsZ. It is an evolutionary necessity to hydrolyze GTP at a faster rate to compensate for the larger cell size, increased complexities on the eukaryotic cell, and increased role of tubulin. For example, tubulin is involved in organelle movement, which is not necessary for FtsZ in prokaryotes due to the lack of organelles. In this way, tubulin can be seen as evolutionarily advanced compared to its ancestral homolog, though the structure and function remains generally conserved (6).
Another comparison molecule is the KIF1A head-microtubule complex structure (1IA0) of Sus scrofa and Mus musculus, which contains the α and β subunits of tubulin with an additional polymer, kinesin-like protein KIF1A. In these species, the sequence and secondary structure remain greatly conserved with that of Bos taurus (8). KIF1A head-microtubule complex has a Z-score of 55.0, an E-value of 0.0, and 98% similarity when compared to tubulin (7). KIF1A serves as a motor domain that creates movement across microtubules generated by hydrolysis of ATP. Similar to tubulin, this complex is capable of binding a member of the taxane family, taxotere, in addition to ATP and magnesium ions. The structure of KIF1A head-microtubule complex provides an example that tubulin can be incorporated into a number of different complexes that generate movement across the cell and the performance of cellular processes, due greatly in part to its structural role and the ability to perform hydrolysis (8).
In mitosis, tubulin plays a significant role in the division of genetic material. Chromosomes associate with microtubules located on the centrosomes during prometaphase. Tubulin has “dynamic instability” whereby polymers as found in microtubules frequently elongate and shorten driven by hydrolysis of bound GTP into GDP. The growth and shrinking of the tubulin polymers as a result of adding or losing dimers, respectively, occurs as the associated chromosome moves along the track formed by the microtubule. The chromosome acts as a “sleeve” in which its binding sites are located, organized in a manner in which the microtubule interacts with more binding sites as it draws further into the sleeve. As the microtubule interacts with more binding sites, the free energy is reduced, but the microtubule must lose tubulin dimers as it loses available space within the sleeve. Eventually, the chromosome reaches the centrosome from which the microtubule is attached and the chromosome is aligned in the cell for equal division of genetic material (9).
Tubulin has two associated metal ions, magnesium and zinc. When tubulin bonds molecules of GTP, there are two magnesium ions present, one each at the N- and E-sites. In contrast, GDP-tubulin only has one associated magnesium ion. This is due to a loss of magnesium during hydrolysis of GTP, leaving a single ion in the GDP state. Magnesium ions have a strong affinity for the E-site when GTP is bonded and at the N-site, ions form associations with Asp-69 and Glu-71 via salt bridges. Zinc binding has an interesting effect on tubulin dimers as it induces the formation of large sheets of polymers. The association of antiparallel protofilaments forms the zinc-induced sheets, but there is no direct structure-function relationship other than some slight level of stability for the dimer. Zinc does not affect the shape of a single tubulin dimer, only how dimers interact with one another in a polymer (1).
In addition to the associated metal ions, calcium ions can affect the polymerization of tubulin dimers. Calcium inhibits formation of microtubules and triggers depolymerization. The process, however, does not affect GTP hydrolysis in inhibiting growth. The disassembly occurs in an endwise fashion, gradually removing subunits from the ends of the polymer. This provides calcium with the role of regulating the rates of polymerization and depolymerization while affording the cell with the ability to maintain a certain equilibrium of polymers rather than reducing the structure completely to isolated dimers (10).
Two critical ligands of tubulin are the anticancer drugs taxol and epothilone A. Both drugs bind to the same binding pocket in the β-tubulin subunit, as shown by the ability of epothilone A to displace taxol upon binding to tubulin (11). However, taxol (molecular weight of 853.91 g/mol) interacts with additional residues because it is larger than epothilone A (molecular weight of 493.66 g/mol) and thus, each ligand manipulates the shared binding pocket differently. Both taxol and epothilone A share hydrogen bonds with Thr-274 and Arg-276 (in specific binding modes), but taxol also creates van der Waals interactions with Leu-215, Leu-217, Leu-228, Ala-231, Ser-234, Phe-270, and Pro-358. Epothilone A does not have any van der Waals interactions with tubulin and instead relies on hydrogen bonding, which determines the binding energy and likewise, the molecular stability. The key difference between epothilones and taxol is that epothilones are more beneficial in stabilizing microtubules and are more effective in treating cancer than taxanes, though there has not been sufficient research to substitute the use of taxol with epothilone in cancer treatment (3). Further, because epothilones are smaller molecules, they require less energy to bind. The interactions with chromosomes in which tubulin is involved are the processes through which the anticancer drugs function. Taxol or epothilone A binds to tubulin and inhibits its ability to alternate between growth and shrinking phases, rendering it unable to drive chromosome movement and thereby stopping cell proliferation in mitosis. For the purpose of cancer treatment, anticancer drugs can be bound to tubulin in tumor cells to prevent their rapid proliferation, which slows the overall spread of cancer by cell inhibition and programmed cell death (9).
The drug taxol binds to β-tubulin through an extensive series of hydrophobic interactions primarily with its phenyl rings. In helix H1, Val-23 contributes to the hydrophobic interaction with the N’ and 3’-phenyl rings, and Asp-26 is within hydrogen bonding distance. There are hydrophobic interactions with the 2-phenyl ring at Leu-217 and Leu-219 of helices H6 and H7 and at His-229 and Leu-230 of the core helix locus, where the nucleotide binding domain and C-terminal domain adjoin. The core helix contacts the 3’-phenyl group at Ala-233 and Ser-236, which is completed by a hydrophobic interaction from residue 272. The M-loop, the loop between the β-strand S7 and helix H9 that is important for lateral contacts, contributes interactions from residues 274-278 to the taxol binding pocket. Finally, interactions from residues 360, 369, 370, and 371 in the B9-B10 loop complete the pocket (1).
Epothilones are 16-membered macrocyclic lactones known to be more soluble in water and more successful in anticancer treatments than taxanes. By being more soluble in water, epothilones allow for in vivo treatment without significant hypersensitivity reactions (11). Both epothilone A and epothilone B are successful anticancer drugs and epothilone B binds tighter to tubulin due to the difference in a single substituent of a methyl group in place of a hydrogen atom, but epothilone A has been a larger focus of research due to its smaller size. Epothilones bind to β-tubulin (1TVK) through four different modes of interaction, but there is a proposed model that appears to be favored.
In the proposed model, epothilone interacts at many of the same residue positions as taxol, but the residues are utilized differently. The C-3 to C-7 backbone fragment of epothilone consists of a large concentration of hydrogen bonds due to the backbone fragment containing many binding opportunities with hydroxyl groups and double-bonded oxygen atoms. The thiazole group of epothilone A forms a hydrogen bond with the amide of Gly-360 that positions the thiazole close to the protein surface deep in the binding pocket, providing epothilone A with protection against solvation based on the presence of a hydrophobic sulfur atom and C-4, C-6, and C-8 methyl groups. There are also hydrogen bonds at the amide group of Thr-274 and carbonyl group of Pro-272 at the O-7 hydroxyl of epothilone, the amine of Arg-282 with the double-bonded O-5, and both amine groups of Arg-282 as well as the carbonyl of Gly-360 with the O-3 group. The hydrogen bonds with residues 272, 274, and 282 provide stability to the backbone region between C-3 and C-7 in epothilone A. Pro-272, Thr-274, and Arg-282 interactions with epothilone are significant as a locus conducive to bonding with electron donors and this region is also critical in altering the M-loop to create a stronger interaction among tubulin dimers, increasing the rate of polymerization (3).