Structural transitions in the GTP cap visualized by cryo-EM of catalytically inactive microtubules
Microtubules (MTs) are polymers of /{beta}-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes, and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here we use cryo-EM and TIRF microscopy of GTP hydrolysis-deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in -tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice, but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wildtype GDP-MTs. End binding proteins of the EB family have the ability to compact both mutant GTP-lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wildtype MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these first high-resolution structures of truly GTP-bound MTs add mechanistic insight to our understanding of MT dynamic instability. Significance StatementMicrotubules (MTs) are non-equilibrium polymers that switch between states of growth and shrinkage. This property is critical for their function and is a consequence of GTP hydrolysis in the MT. The structure of the stable GTP part of the MT (the GTP cap) has previously been inferred from MTs polymerized with non-hydrolyzable GTP analogs. Here, we report the first high-resolution structures of MTs truly containing GTP, polymerized from mutated, hydrolysis-deficient tubulins. We find that GTP-MTs have an "expanded lattice" and a "closed seam", structural characteristics possibly responsible for stabilizing the GTP cap. These results provide new insight into the structural transitions at growing MT ends, furthering our understanding of the bistable nature of MTs.