Molecular insight into c–c tubulin lateral interactions within the c-tubulin ring complex (c-TuRC)

Abstract

c-tubulin is essential for the nucleation and organization of mitotic microtubules in dividing cells. It is localized at the microtubule organizing centers and mitotic spindle fibres. The most well accepted hypothesis for the initiation of microtubule polymerization is that a/b-tubulin dimers add onto a c-tubulin ring complex (cTuRC), in which adjacent c-tubulin subunits bind to the underlying non-tubulin components of the cTuRC. This template thus determines the resulting microtubule lattice. In this study we use molecular modelling and molecular dynamics simulations, combined with computational MM-PBSA/ MM-GBSA methods, to determine the extent of the lateral atomic interaction between two adjacent c-tubulins within the cTuRC. To do this we simulated a c–c homodimer for 10 ns and calculated the ensemble average of binding free energies of -107.76 kcal/mol by the MM-PBSA method and of -87.12 kcal/mol by the MM-GBSA method. These highly favourable binding free energy values imply robust lateral interactions between adjacent c-tubulin subunits in addition to their end-interactions longitudinally with other proteins of cTuRC. Although the functional reconstitution of c-TuRC subunits and their stepwise in vitro assembly from purified components is not yet feasible, we nevertheless wanted to recognize hotspot amino acids responsible for key c–c interactions. Our free energy decomposition data from converting a compendium of amino acid residues identified an array of hotspot amino acids. A subset of such mutants can be expressed in vivo in living yeast. Because cTuRC is important for the growth of yeast, we could test whether this subset of the hotspot mutations support growth of yeast. Consistent with our model, c-tubulin mutants that fall into our identified hotspot do not support yeast growth.