Luminal Localization of α-tubulin K40 Acetylation by Cryo-EM Analysis of Fab-Labeled Microtubules

The αβ-tubulin subunits of microtubules can undergo a variety of evolutionarily-conserved post-translational modifications (PTMs) that provide functional specialization to subsets of cellular microtubules. Acetylation of α-tubulin residue Lysine-40 (K40) has been correlated with increased microtubule stability, intracellular transport, and ciliary assembly, yet a mechanistic understanding of how acetylation influences these events is lacking. Using the anti-acetylated tubulin antibody 6-11B-1 and electron cryo-microscopy, we demonstrate that the K40 acetylation site is located inside the microtubule lumen and thus cannot directly influence events on the microtubule surface, including kinesin-1 binding. Surprisingly, the monoclonal 6-11B-1 antibody recognizes both acetylated and deacetylated microtubules. These results suggest that acetylation induces structural changes in the K40-containing loop that could have important functional consequences on microtubule stability, bending, and subunit interactions. This work has important implications for acetylation and deacetylation reaction mechanisms as well as for interpreting experiments based on 6-11B-1 labeling.微管的αβ-微管蛋白亚基可发生多种进化保守的翻译后修饰（post-translational modifications, PTMs），这些修饰能够赋予细胞内不同亚群微管功能特异性。α-微管蛋白赖氨酸40（Lysine-40, K40）位点的乙酰化已被证实与微管稳定性提升、细胞内运输增强及纤毛组装相关，但目前仍缺乏关于乙酰化如何调控这些过程的机制性认知。本研究借助抗乙酰化微管蛋白抗体6-11B-1与冷冻电子显微镜（electron cryo-microscopy），证实K40乙酰化位点位于微管管腔内，因此无法直接影响微管表面的相关过程，包括驱动蛋白1（kinesin-1）的结合。令人意外的是，单克隆抗体6-11B-1可同时识别乙酰化与去乙酰化的微管。上述结果表明，乙酰化可诱导含K40位点的环区发生结构变化，这可能对微管稳定性、弯曲性及亚基相互作用产生重要的功能性影响。本研究不仅对乙酰化与去乙酰化反应机制具有重要参考价值，也为基于6-11B-1标记的实验解读提供了新的思路。