Microtubules have spatiotemporally complex posttranslational modification patterns. Tubulin tyrosine ligase-like (TTLL) enzymes introduce the most prevalent modifications on α-tubulin and β-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 α-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 β-tubulin subunit, TTLL6 modifies the α-tail of the longitudinally adjacent tubulin dimer. Spanning two tubulin dimers along and across protofilaments (PFs) ensures fidelity of recognition of both the α-tail and the microtubule. Moreover, TTLL6 reads out and is stimulated by glutamylation of the β-tail of the laterally adjacent tubulin dimer, mediating crosstalk between α-tail and β-tail. This positive feedback loop can generate localized microtubule glutamylation patterns. Our work uncovers general principles that generate tubulin chemical and topographic complexity.

微管具有时空复杂的翻译后修饰模式。微管蛋白酪氨酸连接酶样 (TTLL) 酶对 α-微管蛋白和 β-微管蛋白引入最常见的修饰。 TTTL 如何专门用于特定底物识别并最终生成修饰图案尚不清楚。 TTLL6 是一种与纤毛病有关的谷氨酰胺酶，优先修饰微管中的微管蛋白 α 尾。冷冻电子显微镜、动力学分析和单分子生物化学揭示了前所未有的四价识别，确保同时读出微管几何形状和翻译后修饰状态。通过与 β-微管蛋白亚基结合，TTLL6 修饰纵向相邻的微管蛋白二聚体的 α-尾。沿着原丝 (PF) 跨越两个微管蛋白二聚体可确保 α 尾和微管识别的保真度。此外，TTLL6 读出并受到横向相邻微管蛋白二聚体 β 尾的谷氨酰化的刺激，介导 α 尾和 β 尾之间的串扰。这种正反馈回路可以产生局部微管谷氨酰化模式。我们的工作揭示了产生微管蛋白化学和拓扑复杂性的一般原理。