αβ Tubulin (PDB ID: 1jff) from Bos taurus
Created by: Young Jun Lee
Alpha-beta tubulin (PDB ID: 1jff), a heterodimer protein from Bos taurus, forms microtubules in eukaryotic cells by making bonds from head to tail into protofilaments. Each tubulin subunit weighs 50,262.78 Da and has isoelectric point of 4.91 (1). The weight and isoelectric point were obtained from Expasy, which is a research portal that provides access to different databases and software tools to calculate such values. Microtubules are filamentous structures that are involved in many aspects such as cell structure, motility, and transport (2). About 13 protofilaments line up in parallel to form the microtubule wall and give rise to a polymer with well defined polarity, the plus end and minus end (3). The protofilaments also form lateral contacts to make left handed helical, hollow tube. In most microtubule types, including those with 13 protofilaments, helix gives three rise per turn around the axis, producing what is termed a three-start helix (4). The protofilaments in crystal structure run antiparallel from zinc induced sheets, while protofilaments run in parallel in microtubules. This results in different lateral interaction between the dimers for the two structures. The tubulin dimers are polymerized and added to the plus end, but removed from minus end in a process called treadmilling, which is a process dependent on the energy source of GTP. Microtubules are embedded within the microtubule organizing centre (MTOC) within the cell, and it remain attached while the plus end grows into the cytoplasm (5). GTP is always attached to the α subunit, while at the β subunit, GTP is exchangeable for the microfilaments to polymerize. GTP is hydrolyzed to GDP once the tubulin dimer is added to the plus end. Microfilaments are important for the cell structure, and together with microfilament and intermediate filament creates cytoskeleton. Both are important for movement of different organelles and various structures through the cell, as well as segregation of the chromosomes during the cell division. Microtubules can congregate and grow large enough to form cilia and flagella.
αβ-tubulin grows by attachment of GTP, specifically to the E site, or the exchangeable site. In αβ-tubulin, the N-site, or non-exchange site, is hidden within the binding region of the two monomers, and the E-site at β subunit is exposed on the surface. GTP and GDP are exchangeable at the B subunit, either within the dimer or the plus ends, but not within the microtubule body (6). A combination of light microscopy and electron cryomicroscoppy showed that microtubule plus ends lengthen without the interruption from the catastrophic event of depolymerization (5). However, upon polymerization, the GTP bound at E site becomes non-exchangeable resulting in unstable structure. The cap of the GTP tubulin subunits at the ends stabilizes the protein, loss of which leads to depolymerization (3).
The residues involved in binding of GTP are highly conserved due to their important function. The residues directly involved in nucleotide binding are Asn-206 and Asn-228, both of which are involved in specificity for GTP. The specificity of tubulin for GTP is obtained by hydrogen bonding of the 2-exocyclic amino group in GTP to the hydroxyl groups of Asn-206 and Asn-228, and by hydrogen bonding of the 6-oxo group to the amino group of Asn-206 (3). There are major differences in the residue for N-site and E-site for α tubulin and β tubulin. α tubulin contains Asp-254, which is an ideal residue for the hydrolysis of the nucleotide at the E-site, while β tubulin contains Lys-254, which strengthens the monomer-monomer interaction by interaction with the GTP phosphate group at N-site. The hydrolysis of nucleotide at E-site and strengthening of monomer-monomer interaction leads to depolymerization and polymerization of tubulin structure.
Alpha and beta tubulin subunits have 40% sequence homolgy, and structures are very similar (4). Alpha tubulin consist of 13% alpha helices, 39% beta sheets, and 48% random coils. Beta tubulin consists of 13% alpha helices, 42% beta sheets, and 45% random coils (3). Each monomer is formed by two interacting β sheets surrounded by α helices. The tubulin monomers are compact structures consisting of three functional domains. The N- terminal domain forms a Rossman fold (five alpha helices and six parallel beta strands), at the base of which sits the GTP nucleotide (7). The intermediate domain, containing taxol binding site, comprises of three alpha helices, beta sheets, and two more helices. Taxol, one of the ligands, was added to stabilize the sheets against cold temperature depolymerization and aging, similar to the effects of taxol on microtubules (3). Taxol, or Paclitaxel, is a anti-cancer drug that is used for chemotherapy, and it is a treatment that is used in breast, lung, and other kinds of cancer. Taxol binds to the beta subunit and stabilize tubulin in place, rendering its cytoskeleton function. The N' group on taxol is not mandatory for binding and function, but 2-phenyl side chain is critical (3). There are different key residues involved in direct binding of taxol in the second domain of beta tubulin. Val-23 makes hydrogen bonds with N' and 3' phenyl rings, and Asp-26 hydrogen bonds with the nitrogen in the side chain. The most important residues for binding of taxane ring are Pro-274, Leu-275, Thr-276, Ser-277, and Arg-278 located in the M-loop (3).
The C-terminal is comprised entirely of alpha helices and overalps the other domains to form protofilament 'crest' to allow binding of microtubule associated proteins and motor proteins, which allows the function of MTOC (7). Tubulin is rich in aromatic residues, which contributes to structural stability and strength of nucleotide binding region. An important interaction in the region is the formation of hydrogen bond from β: Tyr-202 residue to Asn-167 and Glu-200 in a very hydrophobic region, which stabilizes the monomer structure.
Microtubules are formed by the interaction of the protofilaments between homologous (α-α, β-β) subunits by lateral contact (8). Microtubules that contain 13 protofilaments, the most common type for most of the cells, have reverse lateral contacts (α-β, β-α) resulting in a loss of helical symmetry and creating a seam that could possibly be related to function of microtubule polymerization. The stability of the ends of microtubules are determined by the lateral interactions at the very last monomer. The minus end is laterally attached to the alpha subunit, which gives stability to the structure. The plus end usually contains a GTP cap, and hydrolysis or dissociation of the dimers in the plus end will result in loss of the cap and weak lateral contact with GDP (8). The lateral interaction involves polar, ionic interactions of residues of α: Arg-214, Arg-215, and Asn-216 with residues α: Arg-156, Val-159, Asp-160, and Lys-163 (3).
Both the alpha and beta subunits undergo several isotypic forms and undergo a variety of post translational modifications (7). The C-terminals of both alpha and beta tubulins could be determinant for discrimination of the different isotopes because C-terminal is highly different in each type of isotopes. The isotypical structures of alpha and beta tubulin subunits fit variety types of cells requiring different functions.
Proteins with similar structures compared to αβ tubulin were found using BLAST program and DALI server. The DALI server finds protein with similar tertiary structure by comparing intramolecular distances to measure Z score, which if greater than 2 means protein has similar tertiary structure (9). The DALI server finds Z score by using a sum-of-pairs method, which produces a measure of similarity by comparing intramolecular distances. BLAST program finds a protein of similar primary structure compared to the protein query and assigns E values, which is considered significant if less than .05 (10). The E-value decreases with total sequence homology and increases with the presence of gaps. A similar protein found from BLAST (E=0.0) was the alpha beta tubulin of the Ovis aries bound to designed ankyrin repeat protein (PDB ID: 4DRX). αβ tubulin attached to designed ankyrin repeat protein (DARPin) caps the plus end of microtubule and favors disassembly of microtubules, resulting in curved structure like those seen in tubulin attached to stathmin like domain (11). The two structures have similar overall tertiary structure, but the attachment of the ankyrin repeat protein results in hetero-3-mer for alpha beta tubulin of the Ovis aries compared to hetero-2-mer for alpha beta tubulin of Bos taurus. Another simililar protein found from DALI server (Z = 76.7) was giberellin receptor GID1 of the Oryza sativa (PDB ID: 3ed1).
Magnesium is found at both the N-site and E-site, where GTP binds to the tubulin. There are two Mg 2+ found in GTP-tubulin, while one, strongly attached Mg2+ is found in GDP- tubulin. At the N-site, the magnesium ion stabilizes the dimer structure, and at E-site, magnesium ion is involved in binding of GTP. The concentration of magnesium ion goes down once GTP is hydrolyzed to GDP, showing the ion's involvement in GTP binding. Zinc ion (Zn2+) is important in finding the crystal structure of αβ-tubulin by forming zinc-induced sheets (3). The ion stabilizes the M-loop for better analysis of the structure and the lateral contact.
Epithilone, an anti-cancer agent such as taxol, binds to αβ tubulin (PDB ID: 1TVK) in similar manner as taxol, but each ligand binds to the binding site in unique ways. The cyclic ring in Epithilone is thought to occupy the same space as baccatin core of taxol, whereas the thiazole side chain superposes one of taxol's three phenyl rings (12). The thiazole of the benzyl phenyl residue of epothilone binds to the space unoccupied by taxol. Vineblastin, another anti-cancer drug, binds to αβ tubulin (PDB ID: 1Z2B) at the tips and stops the polymerization of microtubules at the plus ends. Vineblastin modifies the longitudinal interface of the tubulin molecules leading to constraint into curved assembly from steric inhibition (13). The drug introduces a wedge between the interface of two subunits, causing problems in polymerization. Zampanolide (PDB ID: 4I4T), similar anti-cancer drug, binds to αβ-tubulin to stop the microtubule function. Zampanolides bind to the same binding sites as taxanes, leading to conformational change of the M-loop, a critical structure for lateral interaction, into a helical structure (14). The structural change in M-loop results in curved structure of the αβ-tubulin proteins, leading to inhibition of the function of microtubules.