Structural insight into the stabilization of microtubules by taxanes
- Paul Scherrer Institute, Switzerland
- CSIC-Centro de Investigaciones Biologicas, Spain
- Chinese Academy of Medical Sciences & Peking Union Medical College, China
- ETH Zurich, Switzerland
- Chuo University, Japan
- Univeristy of Alcalá, Spain
Abstract
Paclitaxel (Taxol®) is a taxane and a first-line chemotherapeutic drug that stabilizes microtubules. While the interaction of paclitaxel with microtubules is well described, the current lack of high-resolution structural information on a tubulin-taxane complex precludes a comprehensive description of the binding determinants that affect the drug's mechanism of action. Here, we solved the crystal structure of the core baccatin III moiety of paclitaxel lacking the C13 side chain in complex with tubulin at 1.9 Å resolution. Based on this information, we engineered two tailor-made taxanes with modified C13 side chains, solved their crystal structures in complex with tubulin, and analyzed their effects along with those of paclitaxel, docetaxel, and baccatin III on the microtubule lattice by X-ray fiber diffraction. We then compared high-resolution structures of ligand-bound tubulin and microtubule complexes with apo forms and used molecular dynamics simulations to understand the consequences of taxane binding to tubulin as well as to simplified protofilament and microtubule-lattice models. Our combined approach sheds light on three mechanistic questions. Firstly, taxanes bind better to microtubules as compared to unassembled tubulin due to a dual structural mechanism: Tubulin assembly is linked to a conformational reorganization of the bM loop, which otherwise occludes ligand access to the taxane site, while the bulky C13 side chains preferentially recognize the microtubule-assembled over the unassembled conformational state of tubulin. Second, the occupancy of the taxane site by a ligand has no influence on the straightness of tubulin protofilaments. Finally, the longitudinal expansion of the microtubule lattices arises from the accommodation of the taxane core within the site, a process that is, however, not related to the microtubule stabilization mechanism of taxanes, as all analogs tested expand the microtubule lattice, despite the fact that one of them, Baccatin III, is biochemically inactive. In conclusion, our combined experimental and computational approach allowed us to describe the tubulin-taxane interaction in atomic detail and assess the structural determinants for binding.
Data availability
Diffraction data have been deposited in PDB under the accession codes 8BDE (T2R-TTL-BacIII), 8BDF (T2R-TTL-2a) and 8BDG ((T2R-TTL-2b).
Article and author information
Author details
Daniel Lucena-Agell
Fernando Josa-Prado
Funding
Ministerio de Ciencia e Innovación (PID2019-104545RB-I00)
- J Fernando Díaz
Consejo Superior de Investigaciones Científicas (PIE 201920E111)
- J Fernando Díaz
Fundación Tatiana Pérez de Guzmán el Bueno (Proyecto de Investigación en Neurociencia 2020)
- J Fernando Díaz
European Union NextGenerationEU (H2020-MSCA-ITN-2019 860070 TUBINTRAIN)
- Andrea E Prota
- J Fernando Díaz
Swiss National Science Foundation (310030_192566)
- Michel O Steinmetz
JSPS KAKENHI (16K07328/17H03668)
- Shinji Kamimura
National Natural Science Foundation of China (30930108)
- Wei-Shuo Fang
Chinese Academy of Medical Sciences (2016-I2M-1-010)
- Wei-Shuo Fang
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2023, Prota et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Structural insight into the stabilization of microtubules by taxanes
eLife 12:e84791.
https://doi.org/10.7554/eLife.84791
Further reading
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
African trypanosomes are the causative agents of neglected tropical diseases affecting both humans and livestock. Disease control is highly challenging due to an increasing number of drug treatment failures. African trypanosomes are extracellular, blood-borne parasites that mainly rely on glycolysis for their energy metabolism within the mammalian host. Trypanosomal glycolytic enzymes are therefore of interest for the development of trypanocidal drugs. Here, we report the serendipitous discovery of a camelid single-domain antibody (sdAb aka Nanobody) that selectively inhibits the enzymatic activity of trypanosomatid (but not host) pyruvate kinases through an allosteric mechanism. By combining enzyme kinetics, biophysics, structural biology, and transgenic parasite survival assays, we provide a proof-of-principle that the sdAb-mediated enzyme inhibition negatively impacts parasite fitness and growth.
-
- Structural Biology and Molecular Biophysics
The relationship between protein dynamics and function is essential for understanding biological processes and developing effective therapeutics. Functional sites within proteins are critical for activities such as substrate binding, catalysis, and structural changes. Existing computational methods for the predictions of functional residues are trained on sequence, structural, and experimental data, but they do not explicitly model the influence of evolution on protein dynamics. This overlooked contribution is essential as it is known that evolution can fine-tune protein dynamics through compensatory mutations either to improve the proteins’ performance or diversify its function while maintaining the same structural scaffold. To model this critical contribution, we introduce DyNoPy, a computational method that combines residue coevolution analysis with molecular dynamics simulations, revealing hidden correlations between functional sites. DyNoPy constructs a graph model of residue–residue interactions, identifies communities of key residue groups, and annotates critical sites based on their roles. By leveraging the concept of coevolved dynamical couplings—residue pairs with critical dynamical interactions that have been preserved during evolution—DyNoPy offers a powerful method for predicting and analysing protein evolution and dynamics. We demonstrate the effectiveness of DyNoPy on SHV-1 and PDC-3, chromosomally encoded β-lactamases linked to antibiotic resistance, highlighting its potential to inform drug design and address pressing healthcare challenges.
