Molecular recognition of epothilones by microtubules and tubulin dimers revealed by biochemical and NMR approaches

Angeles Canales, Lidia Nieto, Javier Rodríguez-Salarichs, Pedro A Sánchez-Murcia, Claire Coderch, Alvaro Cortés-Cabrera, Ian Paterson, Teresa Carlomagno, Federico Gago, José M Andreu, Karl-Heinz Altmann, Jesús Jiménez-Barbero, J Fernando Díaz

Research output: Contribution to journalArticlepeer-review

Abstract

The binding of epothilones to dimeric tubulin and to microtubules has been studied by means of biochemical and NMR techniques. We have determined the binding constants of epothilone A (EpoA) and B (EpoB) to dimeric tubulin, which are 4 orders of magnitude lower than those for microtubules, and we have elucidated the conformation and binding epitopes of EpoA and EpoB when bound to tubulin dimers and microtubules in solution. The determined conformation of epothilones when bound to dimeric tubulin is similar to that found by X-ray crystallographic techniques for the binding of EpoA to the Tubulin/RB3/TTL complex; it is markedly different from that reported for EpoA bound to zinc-induced sheets obtained by electron crystallography. Likewise, only the X-ray structure of EpoA bound to the Tubulin/RB3/TTL complex at the luminal site, but not the electron crystallography structure, is compatible with the results obtained by STD on the binding epitope of EpoA bound to dimeric tubulin, thus confirming that the allosteric change (structuring of the M-loop) is the biochemical mechanism of induction of tubulin assembly by epothilones. TR-NOESY signals of EpoA bound to microtubules have been obtained, supporting the interaction with a transient binding site with a fast exchange rate (pore site), consistent with the notion that epothilones access the luminal site through the pore site, as has also been observed for taxanes. Finally, the differences in the tubulin binding affinities of a series of epothilone analogues has been quantitatively explained using the newly determined binding pose and the COMBINE methodology.

Original languageEnglish
Pages (from-to)1033-43
Number of pages11
JournalACS chemical biology
Volume9
Issue number4
DOIs
Publication statusPublished - 18 Apr 2014

Keywords

  • Dimerization
  • Drug Stability
  • Epothilones/chemistry
  • Ligands
  • Magnetic Resonance Imaging
  • Microtubules/chemistry
  • Models, Molecular
  • Protein Binding
  • Thermodynamics
  • Tubulin/chemistry

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