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...

Structural analysis of a complex of tubulin and tubulin tyrosine ligase (TTL) reveals insights into TTL’s enzymatic mechanism, how it discriminates between α- and β-tubulin, and its possible evolutionary origin.

The recent success of antibody-drug conjugates (ADCs) in the treatment of cancer has led to a revived interest in microtubuledestabilizing agents. Here, we determined the high-resolution crystal structure of the complex between tubulin and maytansine, which is part of an ADC that is approved by the US Food and Drug Administration (FDA) for the treatment of advanced breast cancer. We found that the drug binds to a site on β-tubulin that is distinct from the vinca domain and that blocks the formation of longitudinal tubulin interactions in microtubules. We also solved crystal structures of tubulin in complex with both a variant of rhizoxin and the phase 1 drug PM060184. Consistent with biochemical and mutagenesis data, we found that the two compounds bound to the same site as maytansine and that the structures revealed a common pharmacophore for the three ligands. Our results delineate a distinct molecular mechanism of action for the inhibition of microtubule assembly by clinically relevant agents. They further provide a structural basis for the rational design of potent microtubuledestabilizing agents, thus opening opportunities for the development of next-generation ADCs for the treatment of cancer.drug mechanism | microtubule-targeting agents | X-ray crystallography

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Laulimalide and peloruside A are microtubule-stabilizing agents (MSAs), the mechanism of action on microtubules of which is poorly defined. Here, using X-ray crystallography it is shown that laulimalide and peloruside A bind to a unique non-taxane site on β-tubulin and use their respective macrolide core structures to interact with a second tubulin dimer across protofilaments. At the same time, they allosterically stabilize the taxane-site M-loop that establishes lateral tubulin contacts in microtubules. Structures of ternary complexes of tubulin with laulimalide/peloruside A and epothilone A are also solved, and a crosstalk between the laulimalide/peloruside and taxane sites via the M-loop of β-tubulin is found. Together, the data define the mechanism of action of laulimalide and peloruside A on tubulin and microtubules. The data further provide a structural framework for understanding the synergy observed between two classes of MSAs in tubulin assembly and the inhibition of cancer cell growth.