Structural basis for -tubulin-specific and modification state-dependent glutamylation

Microtubules have spatiotemporally complex posttranslational modification patterns. Tubulin tyrosine ligase-like (TTLL) enzymes introduce the most prevalent modifications on alpha-tubulin and beta-tubulin. How TTLLs specialize for specific substrate recognition and ultimately modification-pattern generation is largely unknown. TTLL6, a glutamylase implicated in ciliopathies, preferentially modifies tubulin alpha-tails in microtubules. Cryo-electron microscopy, kinetic analysis and single-molecule biochemistry reveal an unprecedented quadrivalent recognition that ensures simultaneous readout of microtubule geometry and posttranslational modification status. By binding to a beta-tubulin subunit, TTLL6 modifies the alpha-tail of the longitudinally adjacent tubulin dimer. Spanning two tubulin dimers along and across protofilaments (PFs) ensures fidelity of recognition of both the alpha-tail and the microtubule. Moreover, TTLL6 reads out and is stimulated by glutamylation of the beta-tail of the laterally adjacent tubulin dimer, mediating crosstalk between alpha-tail and beta-tail. This positive feedback loop can generate localized microtubule glutamylation patterns. Our work uncovers general principles that generate tubulin chemical and topographic complexity. Cryo-electron microscopy (cryo-EM), kinetic analysis and single-molecule biochemistry reveal how the tubulin tyrosine ligase-like 6 (TTLL6) glutamylase binds reads microtubule geometry and modification state of neighboring tubulins, enabling a spatial positive feedback loop for microtubule modification.