Cyclostreptin and microtubules: is a low-affinity binding site required?

Cyclostreptin (CS) is a recently discovered natural product with cytotoxic activity caused by microtubule stabilization. It is the only known microtubule-stabilizing agent (MSA) that covalently binds to tubulin. It also exhibits the fast-binding kinetics seen for other MSAs. Through careful peptide digestion and mass spectrometry analysis, Buey et al. found that two amino acids are labeled by CS: Asn228, near the known taxane-binding site, and Thr220, in the type I microtubule pore. This led Buey et al. to propose Thr220 resides at the site previously predicted to be a way station or low-affinity site. By using molecular dynamics simulations and structural considerations of the microtubule pore and tubulin dimer, we conclude that postulation of a low-affinity site is unnecessary to explain the available experimental data. An alternative explanation views the microtubule pore as a structural entity that presents a substantial kinetic barrier to ligand passage to the known taxane-binding site-an entry point to the microtubule lumen that becomes completely blocked if cyclostreptin is bound at Thr220. Simulations of the free dimer also suggest a common mechanism of microtubule stabilization for taxane site MSAs through their conformational effect on the M-loop. Such an effect explains the low tubulin polymerization caused by cyclostreptin in vitro despite its covalent attachment.