Structural transitions in the GTP cap visualized by cryo-EM of catalytically inactive microtubules

Abstract

Microtubules (MTs) are polymers of alphabeta-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 α-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.

Preference of CAMSAP3 for expanded microtubule lattice contributes to stabilization of the minus end

“…4 µM D2 induced 3.1 ± 0.8% (mean ± S.D.) lattice expansion, which is comparable to TPX2/GMPCPP-induced double expansion (Zhang et al, 2018), the most expanded state to our knowledge (LaFrance et al, 2022).…”

“…In terms of microtubule stability, the lattice expansion/compaction of the tubulin periodicity along the longitudinal axis shows a clear correlation. The inhibition of microtubule depolymerization by the very slowly hydrolysable nucleotide guanosine-5'-[(α, β)methyleno]triphosphate (GMPCPP), treatment with the anti-cancer drug taxol (Alushin et al, 2014;Kamimura et al, 2016) or mutations in the GTPase site (LaFrance et al, 2022) forces microtubules to take conformations with the expanded lattice compared to the unstable GDP-bound state. Therefore, lattice expansion is thought to be a common conformational change needed for microtubule stabilization.…”

Increased microtubule lattice spacing correlates with selective binding of kinesin-1 in cells

“…These distinct lattice spacings could even be detected within the same tomogram (Supplemental Figure 2). Our results indicate that microtubule lattice spacings are diverse even far away from microtubule tips, where an altered lattice spacing is expected (Hyman et al, 1995; LaFrance et al, 2021). Within the field of view of our data, which is approximately 0.8 μm, we did not observe any microtubule ends.…”

“…In vitro GMPCPP-MT (Estevez-Gallego et al, 2020;Hyman et al, 1995;Kellogg et al, 2017;LaFrance et al, 2021;Vale et al, 1994;Zhang et al, 2018) IV.…”

“…Upon polymerization of free αβ-tubulin dimers into microtubules, GTP bound to β-tubulin is hydrolysed to GDP and the structural conformation of the αβ- tubulin dimer changes (Alushin et al, 2014). This GDP-bound state, which makes up the bulk of the microtubule core, generally has a compacted average monomer spacing of ∼41 Å that is distinct from that of the GTP-bound state at the tip (GTP-cap), which has an expanded lattice spacing of ∼42 Å as determined for hydrolysis deficient mutants and the slowly-hydrolysable GTP analogue guanylyl-(α,β)- methylene-diphosphonate (GMPCPP) (Hyman et al, 1995; LaFrance et al, 2021). Besides the nucleotide state, drugs, like the anti-cancer drug Taxol, can alter the lattice spacing of the microtubule (Kellogg et al, 2017).…”