A pathway to the design of even more effective versions of the powerful anti-cancer drug Taxol has been opened with the most detailed look ever at the assembly and disassembly of microtubules, tiny fibers of tubulin protein that form the cytoskeletons of living cells and play a crucial role in mitosis. Through a combination of high-resolution cryo-electron microscopy (cryo-EM) and new methodology for image analysis and structure interpretation, researchers with the Lawrence Berkeley National Laboratory (Berkeley Lab) and the University of California (UC) Berkeley have produced images of microtubule assembly and disassembly at the unprecedented resolution of 5 angstroms (Å). Among other insights, these observations provide the first explanation of Taxol’s success as a cancer chemotherapy agent.
“This is the first experimental demonstration of the link between nucleotide state and tubulin conformation within the microtubules and, by extension, the relationship between tubulin conformation and the transition from assembled to disassembled microtubule structure,” says Eva Nogales, a biophysicist with Berkeley Lab’s Life Sciences Division who led this research. “We now have a clear understanding of how hydrolysis of guanosine triphosphate (GTP) leads to microtubule destabilization and how Taxol works to inhibit this activity.”
During mitosis, the process by which a dividing cell duplicates its chromosomes and distributes them between two daughter cells, microtubules disassemble and reform into spindles across which the duplicate sets of chromosomes migrate. For chromosome migration to occur, the microtubules attached to them must disassemble, carrying the chromosomes in the process. The crucial ability of microtubules to transition from a rigid polymerized or “assembled” state to a flexible depolymerized or “disassembled” state – called “dynamic instability” – is driven by GTP hydrolysis in the microtubule lattice. Taxol prevents or dramatically slows down the unchecked cell division that is cancer by binding to a microtubule in such a manner as to block the effects of hydrolysis. However, until now the atomic details as to how microtubules transition from polymerized to depolymerized structures and the role that Taxol can play have been sketchy.
The tubulin protein is a heterodimer consisting of alpha (α) and beta (β) monomer subunits. It features two guanine nucleotide binding sites, an “N-site” on the α-tubulin that is buried, and an “E-site” on the β-tubulin that is exposed when the tubulin is depolymerized. Previous microtubule reconstruction studies were unable to distinguish the highly similar α-tubulin and β-tubulin from each other.
Nogales and her colleagues found that GTP hydrolysis and the release of the phosphate (GTP becomes GDP) leads to a compaction of the E-site and a rearrangement of the α-tubulin monomer that generates a strain on the microtubule that destabilizes its structure. Taxol binding leads to a reversal of this E-site compaction and α-tubulin rearrangement that restores structural stabilization.