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Title

Molecular Recognition of Epothilones by Microtubules and Tubulin Dimers Revealed by Biochemical and NMR Approaches

Other TitlesThe molecular recognition of epothilones by microtubules and tubulin dimers revealed by biochemical and NMR approaches
AuthorsCanales, Ángeles ; Nieto, Lidia ; Rodríguez-Salarichs, Javier; Andreu, José Manuel ; Jiménez-Barbero, Jesús ; Díaz, José Fernando
Issue Date13-Feb-2014
PublisherAmerican Chemical Society
CitationACS Chem. Biol. 9 (4)1033–1043 (2014)
AbstractThe 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 four 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 analogs has been quantitatively explained using the newly determined binding pose and the COMBINE methodology.
Description36 p.-6 fig.+ 9 p.-2 fig.-4 tab. (support. inf.)
Publisher version (URL)http://dx.doi.org/10.1021/cb400673h
URIhttp://hdl.handle.net/10261/153306
DOI10.1021/cb400673h
ISSN1554-8929
E-ISSN1554-8937
Appears in Collections:(CIB) Artículos
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