Acetylation at α-tubulin K40 is the sole post-translational modification preferred to occur inside the lumen of hollow cylindrical microtubules. However, how tubulin acetyltransferases access the luminal K40 in micrometer-long microtubules remains unknown. Here, we use cryo-electron microscopy and single-molecule reconstitution assays to reveal the enzymatic mechanism for tubulin acetyltransferases to modify K40 in the lumen. One tubulin acetyltransferase spans across the luminal lattice, with the catalytic core docking onto two α-tubulins and the enzyme’s C-terminal domain occupying the taxane-binding pockets of two β-tubulins. The luminal accessibility and enzyme processivity of tubulin acetyltransferases are inhibited by paclitaxel, a microtubule-stabilizing chemotherapeutic agent. Characterizations using recombinant tubulins mimicking preacetylated and postacetylated K40 show the crosstalk between microtubule acetylation states and the cofactor acetyl-CoA in enzyme turnover. Our findings provide crucial insights into the conserved multivalent interactions involving α- and β-tubulins to acetylate the confined microtubule lumen.

α-微管蛋白 K40 位点的乙酰化是唯一优选发生在空心圆柱形微管腔内的翻译后修饰。然而，微管蛋白乙酰转移酶如何进入微米长微管中的管腔 K40 仍然未知。在这里，我们使用冷冻电子显微镜和单分子重建测定来揭示微管蛋白乙酰转移酶修饰管腔中 K40 的酶促机制。一个微管蛋白乙酰转移酶跨越管腔晶格，催化核心对接在两个 α-微管蛋白上，酶的 C 端结构域占据两个 β-微管蛋白的紫杉烷结合口袋。微管蛋白乙酰转移酶的管腔可及性和酶持续合成能力被紫杉醇（一种微管稳定化疗剂）抑制。使用模拟前乙酰化和后乙酰化 K40 的重组微管蛋白进行表征，显示微管乙酰化状态与酶周转中辅因子乙酰辅酶 A 之间的串扰。我们的研究结果为涉及 α 和 β-微管蛋白以乙酰化局限微管腔的保守多价相互作用提供了重要见解。