Studying the Structural Origins of Microtubule Dynamic Instability through Combining Computational Modeling and cryoEM

Abstract

Despite the importance of microtubule dynamic instability for their function in cell division and for the effect of anticancer drugs like taxol, understanding this macroscopic microtubule behavior at the structural level has been so far hindered by the limitations in cryoEM resolution available for this system. To gain mechanistic molecular understanding of the dynamic nature of microtubules, we have used real space refinement helical reconstruction to obtain cryoEM maps of microtubules in various ligand-bound states: GMPCPP, (a non-hydrolyzable GTP analog), GDP (dynamic microtubules), and GDP+taxol (drug-stabilized). These maps, ranging from 4.5 to 5 Angstrom resolution, represent the most detailed description of alpha and beta tubulin in the microtubule lattice to date. In order analyze the differences between the maps, we performed molecular refinement using the software Rosetta guided by the EM density, as well as information from available crystal structures. With this new approach, we were successful in obtaining well-converged ensembles of models that fit their respective maps significantly better than they fit the maps of other ligand-bound states. We find that the major differences between the GMPCPP and GDP bound microtubules is a compression along the longitudinal axis, accompanied by subtle structural rearrangements within the asymmetric dimer. This compression is not observed when comparing the GMPCPP and GDP+taxol bound structures, which are highly similar. The M-loop involved in lateral contacts between tubulins is modeled well in all of the maps by a short helical structure that is similar to that described recently in the zampanolide bound crystal structure of Steinmetz and colleagues. More detailed structural analysis is underway and will be presented at the conference.