Molecular Mechanism of Action of Microtubule-Stabilizing Anticancer Agents

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Science  01 Feb 2013:
Vol. 339, Issue 6119, pp. 587-590
DOI: 10.1126/science.1230582


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. We determined high-resolution crystal structures of αβ-tubulin in complex with two unrelated MSAs, zampanolide and epothilone A. Both compounds were bound to the taxane pocket of β-tubulin and used their respective side chains 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. Further, our results offer fundamental structural insights into the control mechanisms of microtubule dynamics.

The binding of microtubule-stabilizing agents (MSAs) such as paclitaxel (Taxol, Bristol-Myers Squibb) to microtubules is generally thought to shift the assembly equilibrium of tubulin toward the polymeric state and to block cell entry into mitosis by suppressing microtubule dynamics (1, 2). However, MSAs are also known to induce microtubule polymerization under conditions in which tubulin does not assemble spontaneously, suggesting a role in tubulin activation (3, 4). To provide insights into the interactions of MSAs with tubulin and microtubules at the molecular level, we crystallized the complex between αβ-tubulin (T), the stathmin-like protein RB3 (R), and tubulin tyrosine ligase (TTL) in the presence of either zampanolide (Zampa) or epothilone A (EpoA) (Fig. 1A) and determined the structures of the corresponding protein-ligand complexes (T2R-TTL-Zampa and T2R-TTL-EpoA) at 1.8 and 2.3 Å resolution, respectively, by x-ray crystallography (fig. S1A and table S1) (5). The two tubulin heterodimers in the T2R-TTL-MSA complexes were aligned in a head-to-tail fashion and assumed a curved conformation. Their overall structures superimposed well with the ones obtained in the absence of a MSA or of tubulin in complex with RB3 alone (6) [root mean square deviation (RMSD) ranging from 0.1 to 0.6 Å over more than 650 Cα atoms], which suggests that the binding of MSAs and TTL does not induce major structural changes in the T2R complex. Both Zampa and EpoA were deeply buried in a pocket formed by predominantly hydrophobic residues of helix H7; β strand S7; and the loops H6-H7, S7-H9 [designated the M-loop (7)], and S9-S10 of β-tubulin—this pocket is commonly known as the taxane pocket (Fig. 1, B to D) (8, 9).

Fig. 1

Tubulin-Zampa and tubulin-EpoA complex structures. (A) Chemical structure of Zampa and EpoA. (B) Overall view of the complex formed between tubulin (gray surface; M-loop in yellow) and Zampa (green spheres). The dashed box depicts the area shown in more detail in (C). (C and D) Close-up views of the interaction network observed between Zampa [green sticks, (C)] or EpoA [light green sticks, (D)] and β-tubulin (gray cartoon). Interacting residues of β-tubulin are shown in stick representation. Oxygen and nitrogen atoms are red and blue, respectively, and carbon atoms are green (Zampa and EpoA) or gray and yellow (β-tubulin). Hydrogen bonds are depicted as black dashed lines. The covalent bond between the C-9 atom of Zampa and the NE2 atom of His229 of β-tubulin is indicated by an orange stick. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

In the T2R-TTL-Zampa complex, the C-9 atom of Zampa was covalently bound to the NE2 atom of His229 of β-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 T2R-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 β-tubulin, respectively. The binding 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(9, 11). In contrast, the density of EpoA in our tubulin-EpoA x-ray crystal structure is very well defined and allowed the orientation of the ligand as well as its conformation to be defined unambiguously (fig. S1C).

A comparison of the tubulin-Zampa taxane pocket with that of tubulin-EpoA showed that its conformation is very similar in both complex structures (RMSD of 0.4 Å over 55 Cα atoms) and revealed that the side chains of Zampa and EpoA superimposed well (fig. S2B). In contrast, completely different sets of interactions were established by the two MSAs to anchor their macrolide core structures in the taxane pocket, with the planes of the macrocycles oriented at a ~90° angle.

A hallmark of both the tubulin-Zampa and tubulin-EpoA complex structures was the presence of a short helix formed by residues Arg278-Tyr283 in the M-loop of β-tubulin (Fig. 2A). This segment was largely disordered in the absence of a MSA (Fig. 2B). In contrast, the other secondary structure elements of the taxane pocket superimposed well between the ligand-bound and -unbound states, suggesting that the binding of a MSA is not required to structure these parts of the pocket (RMSD of 0.2 Å over 77 Cα atoms). The helical conformation of the M-loop induced upon ligand binding can be explained by the various hydrophobic and polar contacts established between the side chains of Zampa and EpoA, respectively, and residues of the M-loop (Fig. 1, C and D). The helix was further stabilized by a characteristic intramolecular hydrogen bonding network formed by residues of the M-loop and helix H9 of β-tubulin (Fig. 2C).

Fig. 2

Conformation of the M-loop of β-tubulin. (A and B) 2mFo-DFc (gray mesh, contoured at 1.0σ) and mFo-DFc (green and red mesh, ±3.0σ) electron density maps of the region surrounding the M-loop of β-tubulin in the T2R-TTL-Zampa (A) and T2R-TTL (B) complexes. (C) Close-up view of the Zampa-induced intramolecular interaction network that contributes to the stabilization of the M-loop helix.

The "curved" structure of tubulin in the tubulin-RB3 complex corresponds to the conformation of unassembled, free tubulin (12, 13). In contrast, a "straight" conformation of tubulin is found in microtubules (8, 14). To assess possible structural differences between the taxane pocket in unassembled tubulin and microtubules, we compared models of β-tubulin in the curved (T2R-TTL-Zampa) and straight (14) conformational states. Superimposition of these structures showed that the overall architecture of the taxane pocket is only slightly affected by the curved-to-straight structural rearrangements (RMSD of 1.1 Å over 73 Cα atoms) (Fig. 3A). This observation is in agreement with biochemical studies suggesting that some MSAs can bind to unassembled and/or oligomeric forms of tubulin (10, 15). The stronger binding of MSAs to microtubules can be explained by the disordered nature of the M-loop in unassembled tubulin in comparison to its structured state in microtubules (7, 16).

Fig. 3

Lateral tubulin interactions in microtubules. (A) Superimposition of the taxane pocket (right) and M-loop–contacting secondary structure elements across protofilaments (left) in curved (T2R-TTL-Zampa; gray) and straight (PDP ID 1JFF; light blue) β-tubulin. (B) Cryo-EM map at 8.2 Å resolution of a microtubule viewed from its luminal side (gray surface; Electron Microscopy Data Bank map 1788). Two chimeric molecules composed of straight β-tubulin (cartoon representation) and elements shaping the taxane pocket in the curved tubulin-Zampa complex (A) are fitted in the map. (C) Close-up view of the lateral β-tubulin contact model shown in (B). (D) Proposed molecular mechanism of action of MSAs on tubulin and microtubules. (1) Binding of a MSA (rhomboid) to the taxane-site structures the disordered M-loop of β-tubulin (dashed line) into a helix (cylinder). (2) The MSA-stabilized M-loop promotes tubulin polymerization. (3) The M-loop helices of α- and β-tubulin are also formed in the context of the microtubule in the absence of a ligand. (4) All taxane-site MSAs bind to tubulin in the microtubule to stabilize lateral contacts. For more details, see text.

The M-loop of both α- and β-tubulin is a crucial element for lateral tubulin contacts between protofilaments in microtubules in the absence of ligands (7, 16). To provide structural insights into lateral tubulin contacts, we modeled the helical conformation of the M-loop in the context of the microtubule lattice. For this purpose, we used the straight tubulin structure (14) and cryogenic electron microscopy (cryo-EM) reconstructions of microtubules at ~8 Å resolution (7, 16). In contrast to the non-native M-loop conformation in zinc-stabilized tubulin sheets (14), the MSA-stabilized helical M-loop conformation of β-tubulin adequately explains the corresponding density of EM reconstructions of microtubules (Fig. 3B). In our model, Tyr283 of the M-loop is inserted across protofilaments into a pocket shaped by the S2′-S2′′ β hairpin and the H2-S3 loop (residues Ala56, Thr57, Val62, Gln85, Arg88, Pro89, and Asp90) of a neighboring β-tubulin subunit (Fig. 3C), secondary structure elements that were not considerably affected by the curved-to-straight tubulin conformational transition (RMSD of 0.7 Å over 91 Cα atoms) (Fig. 3A). In addition, the M-loop residues Ser280, Gln282, Arg284, and Ala285 were favorably positioned to form additional contacts to the neighboring β-tubulin. The M-loop of α-tubulin in our tubulin-MSA complexes was also stabilized in a similar helical conformation, in this case due to a crystal contact (fig. S3). In combination with molecular dynamics simulations (17), these data collectively suggest that the disordered M-loops of both α- and β-tubulin exhibit an intrinsic propensity to form a helix that establishes lateral tubulin contacts in microtubules.

Our study provides fundamental structural information on the molecular mechanism of action of MSAs (Fig. 3D). Apart from additional global effects (1719), a common feature of tubulin activation by MSAs is the formation of a short helix in the M-loop of β-tubulin upon MSA binding. As M-loop structuring is a crucial prerequisite for lateral tubulin interactions, this effect explains how MSAs promote microtubule assembly and stabilization. Our data further suggest that the intramolecular interaction network that stabilizes the M-loop helix of both α- and β-tubulin also forms in microtubules in the absence of a ligand. We propose that the helical structuring of the M-loop facilitates the curved-to-straight conformational change that occurs upon incorporation of tubulin into microtubules. In this context, the binding of a MSA leads to tubulin preorganization according to the gross structural requirements of the assembly process, thus reducing the entropy loss associated with microtubule formation. In turn, our model implies that dissolution of the helical structure of the M-loops is an early molecular event in the process of microtubule disassembly.

The high-resolution structural information obtained for the tubulin-MSA complexes reported here opens the possibility for structure-guided drug engineering. Whereas the structure-activity relationship of epothilones has been explored extensively (20) and one epothilone derivative, ixabepilone, has been approved by the U.S. Food and Drug Administration for breast cancer treatment (21), little structure-activity work has been reported on Zampa (22). Zampa exhibits favorable properties that could make it an attractive lead compound (10). It is a very potent MSA that exerts its action through covalent binding to tubulin, which might provide superior activity in the case of P-glycoprotein–mediated multidrug resistance.

Supplementary Materials

Materials and Methods

Figs. S1 to S3

Table S1

References (2330)

References and Notes

  1. Materials and methods are available as supplementary materials on Science Online.
  2. Acknowledgments: We thank R. Kammerer, F. Winkler, and Y. Barral for critical reading of the manuscript; V. de Lucas de Segovia for providing calf brains for tubulin purification; and V. Olieric and M. Wang for excellent technical assistance with the collection of x-ray data at beamline X06SA and X06DA of the Swiss Light Source (Paul Scherrer Institut, Villigen, Switzerland). J.J.F. received a short-term fellowship from the European Molecular Biology Organization and a Professional Development Grant from the Genesis Oncology Trust. This work was supported by grants from the Ministerio de Economía y Competitividad (BIO2010-16351) and the Comunidad Autónoma de Madrid (S2010/BMD-2457) (to J.F.D.), a Ph.D. fellowship from the Roche Research Foundation (to D.Z.), and grants from the Swiss National Science Foundation (310030B_138659) and the Swiss initiative (BIP-2011/122) (to M.O.S.). Coordinates have been deposited at the Protein Data Bank (PDB) under identification (ID) nos. 4I4T (T2R-TTL-Zampa), 4I50 (T2R-TTL-EpoA), and 4I55 (T2R-TTL).
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