Conformation of Microtubule-Bound Paclitaxel Determined by Fluorescence Spectroscopy and REDOR NMR

Abstract

The conformation of microtubule-bound paclitaxel has been examined by fluorescence and solid-state NMR spectroscopy. A fluorescent derivative of paclitaxel, 3'-N-debenzoyl-3'-N-(m-aminobenzoyl)paclitaxel (N-AB-PT), was prepared by semisynthesis. No differences in the microtubule-promoting activity between N-AB-PT and paclitaxel were observed, demonstrating that addition of the amino group did not adversely affect the ligand-receptor association. The distance between the fluorophore N-AB-PT and the colchicine binding site on tubulin polymers was determined through time-resolved measurements of fluorescence resonance energy transfer to be 29 +/- 2 A. The absorption and emission spectra of N-AB-PT bound to microtubules and in various solvents were measured. A plot of the Stokes shift as a function of solvent polarity was highly unusual. The Stokes shift increased linearly with solvent polarity in protic solvents, which is expected due to the nature of the fluorophore. In aprotic solvents, however, the Stokes shift was invariant with solvent polarity, indicating that the fluorophore was somehow shielded from the effects of the solvent. These data are best explained by considering the solution-state conformational properties of paclitaxel. It is known that paclitaxel adopts different conformations depending on the nature of the solvent, and these fluorescence data are consistent with the molecule adopting a "hydrophobic collapsed" conformation in protic solvents and an "extended" conformation in aprotic solvents. The Stokes shift of microtubule-bound N-AB-PT was within the protic solvent region, demonstrating that microtubule-bound paclitaxel is in a hydrophobic collapsed conformation. Microtubule-bound paclitaxel was also investigated by solid-state NMR. Paclitaxel was labeled with (19)F at the para position of the C-2 benzoyl substituent and with (13)C and (15)N in the side chain. Distances between the fluorine and carbon nuclei were determined by REDOR. The distance between the fluorine and the 3'-amide carbonyl carbon was 9.8 +/- 0.5 A, and the distance between the fluorine atom and the 3'-methine carbon was 10. 3 +/- 0.5 A. These spectroscopic data were used in conjunction with molecular modeling to refine the microtubule-bound conformation of paclitaxel and to suggest an alternative orientation of the ligand within the paclitaxel binding site.

The binding conformation of Taxol in β-tubulin: A model based on electron crystallographic density

“…PTX docked into EC ␤-TB appeared, which also favor the polar structure (29,30). The binding conformation derived from the present analysis belongs to neither extreme.…”

“…Three recent reports have used the 3.7-Å resolution EC coordinates of ␣␤-TB to derive a proteinbound conformation for either PTX or epothilone (29,30,48).…”

“…4a was derived by fitting numerous conformations followed by a treatment that remains fully faithful to the EC density of both the protein and the ligand. Of particular note is the Bane, Kingston, and Schaefer model (30) derived from a combination of solid-state rotational echo double resonance (REDOR) NMR and fluorescence spectroscopy [fluorescence resonance energy transfer (FRET)]. REDOR, performed on labeled and MT-bound PTX, is a conformational determinant permitting measurement of the distance between 19 F at the para-position of the C-2 benzoyl substituent and 13 C at the 3Ј-amide carbonyl and the C-3Ј methine carbons.…”

The bioactive Taxol conformation on β-tubulin: Experimental evidence from highly active constrained analogs

“…On the other hand, it can provide a conceptual model for the synthesis of simplified analogs that may well retain the full activity of the parent compound. Several experimental attempts to identify this conformation have been made by NMR measurement of internuclear distances within Taxol bound to microtubules (23,24), and by the synthesis of various conformationally restricted Taxol analogs (25)(26)(27)(28)(29)(30). In addition, studies have compared the conformations of Taxol and epothilone B by molecular modeling and other methods (29,(31)(32)(33).…”

Understanding tubulin–Taxol interactions: Mutations that impart Taxol binding to yeast tubulin

“…8), whereas studies with Taxol-resistant mammalian cell lines containing mutations in ␤-tubulin have identified specific amino acids that may be involved in Taxol binding (9)(10)(11)(12). These results, together with the 3.7-Å electron crystal structure of a tubulin polymer showing the electron density of bound Taxol (13), have been used to propose sites of interaction and conformations for bound Taxol (9,(14)(15)(16)(17). However, there has been no direct experimental evidence supporting any of the putative binding interactions.…”