Characterizing the Epothilone Binding Site on β‑Tubulin by Photoaffinity Labeling: Identification of β‑Tubulin Peptides TARGSQQY and TSRGSQQY as Targets of an Epothilone Photoprobe for Polymerized Tubulin

Photoaffinity
labeling with an epothilone A photoprobe led to the identification
of the β-tubulin peptides TARGSQQY and TSRGSQQY as targets of
the photoprobe for polymerized tubulin. These peptides represent residues
274–281 in different β-tubulin isotypes. Placing the
carbene producing 21-diazo/triazolo moiety of the photoprobe in the
vicinity of the TARGSQQY peptide in a homology model of TBB3 predicted
a binding pose and conformation of the photoprobe that are very similar
to the ones reported for 1) the high resolution cocrystal structure of
epothilone A with an α,β-tubulin complex and for 2) a saturation
transfer difference NMR and transferred NOESY NMR study of dimeric
and polymerized tubulin. Our findings thus provide additional support for these
models as physiologically the most relevant among several modes of binding
that have been proposed for epothilone A in the taxane pocket of β-tubulin.