Molecular Basis for Age-Dependent Microtubule Acetylation

Abstract

Microtubules are decorated with chemically diverse and evolutionarily conserved posttranslational modifications that mark them for specialized functions in cells. Other than viruses and clathrin cages, microtubules are the only known hollow polymers in eukaryotic cells. Acetylation on alpha-tubulin Lys40 stands out as the only known tubulin posttranslational modification located in the microtubule lumen. It marks stable, long-lived microtubules and is required for polarity establishment and directional migration. Tubulin acetyltransferase (TAT) presents a puzzle: its activity is stimulated by microtubules, yet the luminal location of Lys40 hinders its access. TAT was proposed to access its substrate by diffusing from microtubule ends, engaging the Lys40 loop from the exterior of the microtubule or gaining access through openings or defects in the microtubule. To elucidate substrate access by TAT and uncover the molecular basis for the in vivo correlation between acetylation and microtubule age, we combined X-ray crystallography, electron microscopy, biochemical and single-molecule fluorescence analyses, and modeling. We report the first crystal structure of TAT in complex with a bisubstrate analog that together with structure-based functional analyses constrains the active enzyme to the microtubule lumen and reveal the structural determinants for Lys40 loop recognition. Unexpectedly, acetylation by TAT proceeds stochastically along the microtubule without a preference for ends despite the luminal location of the acetylation site. Single-molecule fluorescence imaging reveals that TAT efficiently and stochastically scans the microtubule, while modeling and kinetic analyses demonstrate that TAT catalytic activity, rather than diffusion through the lumen, is rate-limiting. Consistent with this, our 1.35A X-ray complex crystal structure reveals that the TAT active site is suboptimal for supporting the proton transfer required for acetylation. Thus, by virtue of its preference for the microtubule and its low catalytic rate, TAT alone can function as an enzymatic timer for microtubule lifetimes.