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

Abstract

Microtubules (MTs) are polymers of αβ-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-electron microscopy and total internal reflection fluorescence 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 wild-type 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 wild-type 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 structures of catalytically inactive MTs add mechanistic insight into the GTP state of MTs, the stability of the GTP- and GDP-bound lattice, and our overall understanding of MT dynamic instability.

Asymmetric requirement for α-tubulin over β-tubulin

“…We speculate that the tubulin assemblies formed when β-tubulin is in excess could involve interactions between β-tubulin monomers and/or between β-tubulin monomers and αβ-heterodimers. In this scenario, any β-β contacts along the longitudinal interface would lack the catalytic residues that α-tubulin normally provides to the exchangeable GTP-binding site (Anders and Botstein 2001; LaFrance et al 2022), and therefore these assemblies would be locked in a high affinity GTP-bound state. These hyperstable assemblies could then sequester tubulin heterodimers and select MAPs such as XMAP215/Stu2, depleting components from the cell’s microtubule networks.…”

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).…”

Alternative Approaches to Understand Microtubule Cap Morphology and Function

“…Indeed, MTs show different protofilament twists depending on the nucleotide content, which could differentially affect homotypic and heterotypic lateral contacts. It still remains to be found whether the mini-MT lattice expands as the MT does in the presence of certain nucleotide analogues (GMPCPP and GMPCP, , drugs bound at the taxane site, , and amino acid replacements at the α-tubulin catalytic loop) or as do others found in non-mammalian sources (where the protofilament number is also somewhat smaller , ).…”