Abstract
MSAs promote structuring of the β-tubulin M-loop to enhance microtubule assembly and stability.
Major finding: MSAs promote structuring of the β-tubulin M-loop to enhance microtubule assembly and stability.
Approach: The high-resolution crystal structures of tubulin–MSA complexes were determined.
Impact: Structural analysis may facilitate the development of MSAs with improved anticancer activity.
Microtubule-stabilizing agents (MSA) such as paclitaxel are extensively used chemotherapeutic drugs that target β-tubulin to inhibit mitotic cell division. The anticancer activity of MSAs has been attributed to their ability to stabilize microtubules by impairing depolymerization, but they may also activate microtubule polymerization. To better understand the molecular mechanisms by which these agents modulate microtubule dynamics, Prota and colleagues determined the structure of αβ-tubulin complexed with 2 unrelated MSAs, zampanolide or epothilone A, by high-resolution x-ray crystallography. In both cases, MSAs bound to hydrophobic residues in the taxane-pocket of β-tubulin with a similar conformation of side-chain interactions. MSA binding did not significantly alter the overall structure of the tubulin complex but promoted the formation of a short helix in the M-loop of β-tubulin, which is located within the taxane-pocket and is disordered in unassembled tubulin in the absence of MSA. This helical structuring was mediated by the hydrophobic or polar side-chain interactions of zampanolide or epothilone A, respectively, with the arginine 278–tyrosine 283 residues of the M-loop and was stabilized by β-tubulin intramolecular hydrogen bonding. This M-loop is required for the establishment of lateral tubulin contacts within the microtubule lattice and also intrinsically forms in the absence of MSA binding; modeling of the helix in the context of a microtubule showed that this conformational change favorably positioned M-loop residues to stabilize interactions with neighboring β-tubulin molecules. In addition, the structures of the taxane-pocket and M-loop from the curved conformation of unassembled tubulin bound to MSA were largely superimposable with those of the straight tubulin conformation in microtubules. These results demonstrate that MSAs trigger structural changes in tubulin that enhance microtubule assembly and provide insights that may enable the development of MSA derivatives with more potent activity.
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