The dynamic behavior of microtubules is essential to their functions, as underlined by the large number of compounds that bind tubulin, alter dynamics and result in mitotic arrest. A growing number of cellular factors regulate this dynamic behavior during the cell cycle. Interestingly, dynamic instability is an instrinsic property of tubulin that can be observed in purified solutions and is linked to the binding and hydrolysis of GPT. Our studies of tubulin bound to taxol in polymerized, straight protofilaments, obtained by electron crystallography (Nogales et al., Nature 1998) established the structural basis of nucleotide exchange and polymerization-coupled hydrolysis (Nogales et al., Nature Struct Biol. 1998; Löwe et al., JMB 2001). An essential question to understand microtubule dynamics is whether the structure of tubulin is dictated by nucleotide, polymerization state, or both. My lab has recently obtained two structures corresponding to the start and end points in the polymerization and nucleotide hydrolysis cycles that illustrate the conformational consequences of the nucleotide state and how they relate to longitudinal and lateral assembly.

Using cryo-electron microscopy and a new iterative Fourier Bessel method (Wang and Nogales, JSB 2005) we have obtained a structure of GDP-tubulin in the absence of depolymerizers (Wang and Nogales, Nature 2005). This structure shows distinctive intra and inter dimer interactions and thus distinguishes the GTP and GDP interfaces. The bending of these interfaces in GDP-tubulin is incompatible with the formation of the lateral contacts in microtubules. So, how can binding of GTP result in the "straightening" of protofilaments observed in microtubules? To answer this question we have studied the self-assembly of GMPCPP-bound tubulin into helical ribbons that correspond to the structural intermediate in microtubule assembly that preceed microtubule closure into a tube (Wang and Nogales, Nature 2005). Our study supports the separation of the straightening process into two stages: one, nucleotide-dependent, that allows for lateral association into a curved sheet, and a later one that occurs upon microtubule closure. Most importantly, it provides a pseudo-atomic model of this sheet that illustrates a bimodal mechanism of lateral protofilament interaction preceding microtubule closure. We are now pursuing the idea that this new protofilament interface may be the binding site for a new class of microtubule-binding proteins.