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.
Bibliographical noteFunding Information:
We greatly appreciate the financial support from the University of Minnesota through the Vince and McKnight Endowed Chairs (to G.I.G.). We thank Florenz Sasse for performing cell cytotoxicity and tubulin assembly assays. The authors recognize the Center for Mass Spectrometry and Proteomics at the University of Minnesota and various supporting agencies, including NSF Major Research Instrumentation Grant 9871237, NSF Grant CHE-0078192, NIH Multiuser Grant RR15808, 2001-2002, NSF-MRI Grant 0215759, State of Minnesota Infrastructure Grant, and University of Minnesota support from the College of Agriculture, Food and Environmental Sciences, the College of Biological Sciences, the Minnesota Graduate School, the Minnesota Medical Foundation, the Department of Biochemistry, Molecular Biology and Biophysics, the Office of the Vice President for Health Sciences, and the Vice President for Research. See http://www.cbs.umn.edu/research/resources/msp/about. We also thank the Minnesota Supercomputing Institute for their support for the computational study.