Microtubule-stabilizing agents (MSAs) are efficacious chemotherapeutic drugs widely used for the treatment of cancer. Despite the importance of MSAs for medical applications and basic research, their molecular mechanisms of action on tubulin and microtubules remain elusive. Here we determined high-resolution crystal structures of aß-tubulin in complex with two unrelated MSAs, zampanolide and epothilone A. Both compounds were bound to the taxane-pocket of ß-tubulin and used their respective side chain to induce structuring of the M-loop into a short helix. Because the M-loop establishes lateral tubulin contacts in microtubules, these findings explain how taxane-site MSAs promote microtubule assembly and stability. They further offer fundamental structural insights into the control mechanisms of microtubule dynamics. Here we determined high-resolution crystal structures of -tubulin in complex with two unrelated MSAs, zampanolide and epothilone A. Both compounds were bound to the taxanepocket of -tubulin and used their respective side chain to induce structuring of the M-loop into a short helix. Because the M-loop establishes lateral tubulin contacts in microtubules, these findings explain how taxane-site MSAs promote microtubule assembly and stability. They further offer fundamental structural insights into the control mechanisms of microtubule dynamics.One sentence summary:Microtubule-stabilizing agents use a common mechanism to stabilize a major loop in tubulin that controls microtubule assembly and stability. suggesting that binding of MSAs and TTL does not induce significant structural changes in the T 2 R complex. Both Zampa and EpoA were deeply buried in a pocket formed by predominantly hydrophobic residues of helix H7, -strand S7, and the loops H6-H7, S7-H9 (designated the Mloop (7)) and S9-S10 of -tubulin; this pocket is commonly known as the 'taxane-pocket' (8, 9)In the T 2 R-TTL-Zampa complex, the C9 atom of Zampa was covalently bound to the NE2 atom of His229 of -tubulin (Fig. S1B), which is consistent with mass spectrometry data (10). In addition, two hydrogen bonds were formed between the OH20 group and the O1' atom of Zampa, and the main chain carbonyl oxygen and the NH group of Thr276, respectively. In the T 2 R-TTL-EpoA complex, the O1, OH3, OH7 and N20 groups of EpoA were hydrogen bonded to atoms of residues Thr276 (main chain NH), Gln281 (side chain amide nitrogen), Asp226 (side chain oxygen) and Thr276 (side chain hydroxyl group) of -tubulin, respectively. The binding 4 mode of EpoA in the tubulin-EpoA structure is fundamentally different from the one proposed based on electron crystallography data of zinc-stabilized tubulin sheets (Fig. S2A); however, the orientation of the ligand in the taxane-pocket was ambiguous in the electron crystallography structure because the density of the ligand in experimental omit maps was discontinuous and limited in quality (9, 11). In contrast, the density of EpoA in our tubulin-EpoA X-ray crystal structure is very well defined and allowed the o...
The interactions of microtubules with most compounds described as stabilizing agents have been studied. Several of them (lonafarnib, dicumarol, lutein, and jatrophane polyesters) did not show any stabilizing effect on microtubules. Taccalonolides A and E show paclitaxel-like effects in cells, but they were not able to modulate in vitro tubulin assembly or to bind microtubules, which suggests that other factors are involved in their cellular effects. The binding constants of epothilones, eleutherobin, discodermolide, sarcodictyins, 3,17beta-diacetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10)-triene, and dictyostatin to the paclitaxel site; the critical concentrations of ligand-induced assembly; and their cytotoxicity in carcinoma cells have been measured, and correlations between these parameters have been determined. The inhibition of cell proliferation correlates better with the binding enthalpy change than with the binding constants, suggesting that large, favorable enthalpic contribution to the binding is desired to design paclitaxel site drugs with higher cytotoxicity.
Laulimalide is a cytotoxic natural product that stabilizes microtubules. The compound enhances tubulin assembly, and laulimalide is quantitatively comparable to paclitaxel in its effects on the reaction. Laulimalide is also active in P-glycoprotein overexpressing cells, while isolaulimalide, a congener without the drug's epoxide moiety, was reported to have negligible cytotoxic and biochemical activity [Mooberry et al. (1999) Cancer Res. 59, 653-660]. We report here that laulimalide binds at a site on tubulin polymer that is distinct from the taxoid site. We found that laulimalide, while as active as paclitaxel, epothilone A, and eleutherobin in promoting the assembly of cold-stable microtubules, was unable to inhibit the binding of radiolabeled paclitaxel or of 7-O-[N-(2,7-difluoro-4'-fluoresceincarbonyl)-L-alanyl]paclitaxel, a fluorescent paclitaxel derivative, to tubulin. Confirming this observation, we demonstrated that microtubules formed in the presence of both laulimalide and paclitaxel contained near-molar quantities, relative to tubulin, of both drugs. Laulimalide was active against cell lines resistant to paclitaxel or epothilones A and B on the basis of mutations in the M40 human beta-tubulin gene. We also report that a laulimalide analogue lacking the epoxide moiety, while less active than laulimalide in biochemical and cellular systems, is probably more active than isolaulimalide. Further exploration of the role of the epoxide in the interaction of laulimalide with tubulin is therefore justified.
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