Tubulin heterodimers

A microtubule is a hollow tube of 25 nm diameter formed by 13 protofilaments. Each protofilament consists of polymerized a- and (3-tubulin heterodimers. Microtubules are polarized and have a plus and a minus end. 

When tubulin heterodimers are assembled into microtubules, they form linear protofilaments with the P-tubulin subunit of one tubulin molecule linking covalently with the a-subunit of the next. Direct examination by electron microscopy of tannic acid-treated specimens has shown that micrombules in neurons and the A-microtubules of cilia and flagella have 13 protofilaments arranged side to side to form a cylinder around what appears to be an empty lumen. 

With regard to microtubular ultrastructure, micro filaments (5-7 run in diameter) are composed of filamentous actin. The tubule-like structures are formed by a, P-tubulin heterodimers. The wall is composed of 13 parallel protofilaments. Various microtubule-associated proteins and motor proteins (kinesin and dynein) are bound to the wall. The microtubule is a polar structure, i.e., plus and minus ends. 

These bind to the a- and p-tubulin heterodimers, which block the GTP binding site and prevent the addition of further heterodimers so the whole micro tubular spindle cannot form. 

Fig. 5. 3D EM shows how kinesin and tau bind to microtubules. (A) Reconstruction of a microtubule decorated with kinesin heads (ochre). One kinesin head binds per afi-tubulin heterodimer (grey) and, due to its asymmetric form, can be used to distinguish between the subunits. (B) Inside view of a microtubule that was coassembled with gold-labeled tau and decorated with kinesin heads. The kinesin heads can be seen on the outside through the holes between the protofilaments. The labeled repeat motif of tau binds to the inside face of microtubule. The averaged nanogold density (yellow), which is attached to a repeat motif of tau through a linker, can only be seen near the Taxol binding site of -tubulin, but not on the a subunit (Kar et al, 2003a). The ribbon diagram of the refined zinc-sheet structure is also shown for reference (see Figure 3).

Paclitaxel and docetaxel have been shown to act as spindle poisons, causing cell division cycle arrest, based on a unique mechanism of action.7-10 These drugs bind to the P-subunit of the tubulin heterodimer, the key constituent protein of cellular microtubules (spindles). The binding of these drugs accelerates the tubulin polymerization, but at the same time stabilizes the resultant microtubules, thereby inhibiting their depolymerization. The inhibition of microtubule depolymerization between the prophase and anaphase of mitosis results in the arrest of the cell division cycle, which eventually leads to the apoptosis of cancer cells. 

Tubulin is a 110-kDa heterodimeric protein composed of two subunits a- and P- (-450 amino acids each) and it is the basic subunit of MT. MT are hollow cylindrical protein fibers formed by the lateral association of protofilaments, where each protofilament is a linear polymer of tubulin heterodimers that are bound head to tail. The parallel arrangement of protofilament forms the cylindrical MT wall. Typically, MT contain 13 protofilaments in cross-section aligned with the same polarity. The two ends of a MT are not identical. The minus end is crowned by a-tubulin subunits and serves as the site of nucleation at the centrosome, while the plus end is crowned by P-tubulin subunits and faces outward from the nucleus to the plasma membrane. The repeating subunits are held together in the polymers by non-covalent interactions [4-6],... 

As we have seen, the flexible and dynamic behavior of tubulin has consequences at two levels. Tubulin a/p heterodimers are flexible molecules in nature and change conformation in response to a series of stimuli, such as the polymerization status or the binding of ligands. Besides, tubulin heterodimers can self-assemble into MT and a number of other polymer forms like protofilaments, rings or spirals [5], The complexity of this dynamic behavior makes its structural studies both experimentally... 

HTI-286 binds to the tubulin heterodimer and induces formation of 13-membered tubulin rings [138], Photoaffinity labeling with analogues of HTI-286 mapped to residues of a-tubulin close to the interdimer interface [139], Competitive binding assays with dolastatin-10, cryptophycin A, vinblastine, PTX and colchicines, indicate that the hemiasterlins bind in a subsite of the Vinca domain, which is located at the interdimer interface [28],... 

Interestingly, the initial tubulin coordinates used in the study of HTI-286 are those of straight tubulin in 2D sheets (PDB entry 1JFF) [83], The X-ray structure of the T2R complex with vinblastine showed that the unpolymerized tubulin heterodimer is bent and that there are local conformational changes at the interdimer interface in comparison with straight tubulin [13], The discrepancy between the experimental [12] and computationally predicted [31] binding modes of colchicine has been attributed to the fact that the computational model was derived from... 

The stathmin family of proteins destabilize MT [8]. The X-ray structures of complexes of tubulin with the 91-residue long stathmin-like domain of RB3 reveal that the RB3-SLD caps two tubulin heterodimers, thereby preventing the incorporation of tubulin into MT [12, 13, 21]. Apparently, the longitudinal interactions are impeded by the union of the SLD N-terminal cap domain to the a-subunit. 

The exact binding site of vinca alkaloids remained unknown until 2005, when the crystal structure of vinblastine bound to tubulin complexed with colchicine and with the stathmin-like domain of RB3 was determined (PDB entry 1Z2B) [3], The structure revealed that vinblastine binds to curved tubulin at the interface between two a/p-tubulin heterodimers (interdimer interface, Fig. 4), introducing a wedge that interferes with tubulin assembly. The vinblastine binding site is defined by loop T7, helix H10 and strand S9 in the a subunit of the first heterodimer, and by helix H6 and loops T5 and H6-H7 in the p subunit of the second heterodimer. In microtubules, this region is located toward the inner lumen and is... 

A great step forward was made in 1999 when Nogales and co-workers published the first structure of the complex between paclitaxel and tubulin heterodimer, obtained by EC [46], On this basis, the interactions between the ligand molecule... 

The polymerization of tubulin has many similarities to that of actin. The protomer is the tubulin heterodimer. Each subunit in the heterodimer can bind a nucleotide in this case it is GTP. There is a lag period associated with nucleation. Once sufficient nuclei are available, explosive polymerization can proceed from both ends of the nuclei. Calcium ions and low temperature (4°C) inhibit polymerization, while magnesium ions and high temperature (37°C) stimulate it. As with actin, the microtubule is polar, and polymerization proceeds about three times more quickly from the plus end than the minus end. Polymerization is accompanied by hydrolysis of GTP, on the /3-tubulin only, at or soon after the addition of the a//3 protomer to the filament. GTP caps can therefore exist at both ends of the filament with the cap likely to be larger, on average, at the plus end than at the minus end. 

Tubulin-binding agents prepared by Pinney (4) consisting of dihydronaphthalene derivatives, (IV), exhibited potent tumor cell cytotoxicity by inhibiting the polymerization of a,(3-tubulin heterodimers into the microtubule structures and were used in the treatment of proliferation diseases. 

Paclitaxel acts by enhancing microtubule assembly and stabilizing microtubules (1,2). Microtubules consist of polymers of tubulin in dynamic equilibrium with tubulin heterodimers. Their principal function is the formation of the mitotic spindle during cell division, but they are also active in many interphase functions, such as cellular motility, intracellular transport, and signal transmission. Paclitaxel inhibits the depolymerization of tubulin, and the microtubules formed in the presence of paclitaxel are extremely stable and dysfunctional. This stabilization impairs the essential assembly and disassembly required for dynamic cellular processes, and death of the cell results through disruption of the normal microtubular dynamics required for interphase processes and cell division. In tumor cells, cytotoxicity is represented by the appearance of abnormal microtubular bundles, which accumulate during G2 and mitosis, blocking the cell cycle (3). 

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