Tubulin activity encourages drug R&D
structures of tubulin during the construction of microtubules. The
discovery is of use to drug researchers and developers as tubulin
is a target for anticancer drugs, which can prevent the transition
from growing to shrinking states.
The new models of tubulin transitional states will be used to understand how microtubules explore their cellular environment - a process crucial to the deployment of spindles during cell division, - and how drugs can be designed and targeted to put a stop to the growth of cancer cells.
Microtubules are active protein polymers critical to the structure and function of cells and the process of cell division. In a living cell their growing ends elongate during polymerisation and depolymerisation. Known as "dynamic instability," this ongoing rapid growth and shrinkage is key to the diverse workings of microtubules in the cell.
Lead researchers Eva Nogales and Hong-Wei Wang of the Department of Energy's Lawrence Berkeley National Laboratory, first worked on the depolymerisation process, watching what happens to microtubules when guanosine diphosphate (GDP) is bound to tubulin.
During this process tubulin protofilaments curve back sharply from the end of the microtubule, forming "peels." Wang found conditions for which the curls of GDP-bound tubulin close into tubes, with the protofilaments tightly wrapped around the tube axis. These tubes are double-layered, a property that contributes to their stability.
Next the researchers tackled the polymerisation process, observing when guanosine triphosphate, (GTP) is bound to tubulin molecules. If GDP somehow causes protofilaments to curl up tightly GTP does the opposite, causing protofilaments to straighten and assemble themselves into microtubules. But because GTP can rapidly hydrolyse into GDP, capturing transitional states of GTP-bound tubulin directly was impractical.
Instead the researchers worked with a similar molecule, GMPCPP, an analogue of GTP that binds to the same site but is not vulnerable to hydrolysis. Using Cryo-electron microscopy (cryo-EM) they were able to capture GMPCPP-bound tubulin during the initial steps of microtubule growth.
Cryo-EM is capable of capturing such polymer structures without having to break them down and crystallise single subunits for x-ray diffraction analysis. X-ray diffraction might allow higher resolution, but it would lose all information concerning contacts between the subunits during polymerisation. "With cryo-EM, by changing the parameters we could see how different polymer forms convert," says Nogales.
Raised to body temperature the protofilamentary ribbons immediately straighten and convert to normal microtubules, suggesting that the ribbons and gently curved sheets correspond to the polymerising protofilaments at the end of a growing microtubule.
"The key was to get the tubulin assemblies locked at the correct states suitable for structural analysis, but even more challenging was finding an algorithm to reconstruct the double-layered GDP-tubulin tubes," said Wang.
Due to the double-layered property, it was impossible for the team to use classical helical reconstruction methods.
"Unfortunately, there were no other methods available when we started the project. We had to spend over two years developing and implementing our own algorithm. Once it was ready, we were able to get the correct reconstruction in less than three months," he added.
Tubulin has long been a viable target in the fight against cancer and current drugs such as Taxol and the epothilones act as microtubule stabilisers. Tubulin loses its flexibility and the microtubules can no longer disassemble. That means cell division stops.
Since cancer is the result of uncontrolled cell division, it has long been recognised that microtubule stabilisers could prove valuable as cancer chemotherapy agents. Taxanes are also microtubule stabilisers that bind with tubulin and has been shown in clinical to be an effective treatment for a number of cancers including ovarian, breast, and lung.
However, Taxol has a number of unpleasant side effects on patients, and treatment for recurrent cancers is often unsuccessful. It is believed a tubulin-binding site called a pharmacophore, common to both families and with a specific set of chemical characteristics, could be found.
This would be the route through which safer and more effective artificial forms of the taxanes, epothilones, and other natural anticancer compounds could be synthesised.
Each year 10.9 million people worldwide are diagnosed with cancer and there are 6.7 million deaths from the disease. It is estimated that there are 24.6 million people alive who have received a diagnosis of cancer in the last five years.
"Nucleotide-dependent bending flexibility of tubulin regulates microtubule assembly," by Hong-Wei Wang and Eva Nogales, appears in the 16 June 2005 issue of Nature.
