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Macromolecular accessibility of fluorescent taxoids bound at a paclitaxel binding site in the microtubule surface
José Fernando Díaz1, Isabel Barasoain, André A Souto
1Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, Madrid 28040, Spain. fer@cib.csic.es
Abstract:
The macromolecular accessibility of the paclitaxel binding site in microtubules has been investigated using a fluorescent taxoid and antibodies against fluorescein, which cannot diffuse into the microtubule lumen. The formation of a specific ternary complex of microtubules, Hexaflutax (7-O-{N-[6-(fluorescein-4'-carboxamido)-n-hexanoyl]-l-alanyl}paclitaxel) and 4-4-20 IgG (a monoclonal antibody against fluorescein) has been observed by means of sedimentation and electron microscopy methods. The kinetics of binding of the antibody to microtubule-bound Hexaflutax has been measured. The quenching of the observed fluorescence is fast (k+ 2.26 +/- 0.25 x 10(6) m(-1) s(-1) at 37 degrees C), indicating that the fluorescein groups of Hexaflutax are exposed to the outer solvent. The velocity of the reaction is linearly dependent on the antibody concentration, indicating that a bimolecular reaction is being observed. Another fluorescent taxoid (Flutax-2) bound to microtubules has also been shown to be rapidly accessible to polyclonal antibodies directed against fluorescein. A reduced rate of Hexaflutax quenching by the antibody is observed in microtubule-associated proteins containing microtubules or in native cellular cytoskeletons. It can be concluded that the fluorescent taxoids bind to an outer site on the microtubules that is shared with paclitaxel. Paclitaxel would be internalized in a further step of binding to reach the known luminal site, this step being blocked in the case of the fluorescent taxoids. Because the fluorescent ligands are able to induce microtubule assembly, binding to the outer site should be enough to induce assembly by a preferential binding mechanism.
Insights
Fluorescent taxoids bind to an outer microtubule site, distinct from paclitaxel's inner binding site. This outer binding is sufficient for microtubule assembly, suggesting a preferential binding mechanism for paclitaxel analogs.
Area of Science:
- Biochemistry
- Cell Biology
- Structural Biology
Background:
- Microtubules are essential cytoskeletal components involved in cell division and intracellular transport.
- Paclitaxel is a potent anti-cancer drug that stabilizes microtubules by binding to a specific site.
- The precise location and accessibility of the paclitaxel binding site within microtubules remain areas of investigation.
Purpose of the Study:
- To investigate the macromolecular accessibility of the paclitaxel binding site in microtubules.
- To determine if fluorescent paclitaxel analogs bind to the same site as paclitaxel.
- To elucidate the mechanism by which paclitaxel and its analogs induce microtubule assembly.
Main Methods:
- Utilized fluorescent taxoids (Hexaflutax and Flutax-2) and antibodies against fluorescein.
- Employed sedimentation and electron microscopy to observe complex formation.
- Measured the kinetics of antibody binding to microtubule-bound fluorescent taxoids.
- Assessed antibody accessibility in the presence of microtubule-associated proteins and cellular cytoskeletons.
Main Results:
- Fluorescent taxoids bind to an outer site on microtubules, accessible to antibodies.
- The binding kinetics indicate rapid exposure of fluorescein groups to the solvent.
- A reduced antibody quenching rate was observed in microtubules with associated proteins or in cellular cytoskeletons.
- Fluorescent taxoids can induce microtubule assembly, suggesting outer site binding is sufficient.
Conclusions:
- Fluorescent taxoids bind to an outer microtubule site shared with paclitaxel.
- Paclitaxel likely internalizes to a luminal site after initial outer binding, a step blocked for fluorescent taxoids.
- Binding to the outer site alone is sufficient to induce microtubule assembly via preferential binding.
