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2024

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NSMB | HKU research team reveals the enzymatic mechanism by which tubulin acetyltransferase modifies K40 in the lumen!

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Acetylation of α-tubulin K40 is the only post-translational modification that occurs more preferentially in the lumen of hollow cylindrical microtubules. However, how tubulin acetyltransferase enters luminal K40 in micron-long microtubules remains unknown.

On November 4, 2024, Shih-Chieh Ti, Zhai Yuanliang and Wai Hei Lam of the University of Hong Kong jointly communicatedNature Structural & Molecular Biology（IF=12.5）magazine published online entitled“Tubulin acetyltransferases access and modify the microtubule luminal K40 residue throughs anchors in taxane-binding pockets”The research paper,The study revealedThe enzymatic mechanism by which tubulin acetyltransferase modifies K40 in the lumen is shown.

A tubulin acetyltransferase spans the luminal lattice, catalyzing the docking of the core to two alpha-tubulin proteins, and the C- terminal domain of the enzyme occupies two taxane-binding pockets of beta-tubulin. The microtubule-stabilizing chemotherapeutic drug paclitaxel inhibits the luminal accessibility and enzymatic activity of tubulin acetyltransferase. Characterization with recombinant tubulin mimicking pre-and post-acetylated K40 revealed cross-talk between the microtubule acetylation state and acetyl-CoA, a cofactor in enzyme turnover.This study provides important insights into the conserved multivalent interactions of α-and β-tubulin, which are involved in limiting the acetylation of the microtubule lumen.

 

Microtubules are hollow cylindrical polymers of α/β-tubulin heterodimers containing post-translational modifications on the exposed outer and confined inner surfaces. As the only enzyme with a preference for intraluminal substrates, tubulin acetyltransferase targets luminal alpha-tubulin K40 to produce acetylated microtubules. Microtubule acetylation is an evolutionarily conserved post-translational modification that is enriched in cellular structures such as cilia and flagella, spindles, platelet border zones, and neuronal microtubule arrays. Dysregulation of microtubule acetylation is associated with mechanosecognition defects, brain malformations, cancer metastasis, taxane-induced cytotoxicity, and impaired platelet formation and activation. However, the molecular mechanism by which tubulin acetyltransferase enters and recognizes microtubule luminal residues remains a major unresolved issue.

Tubulin acetyltransferases consist of two evolutionarily conserved structures: an N-terminal structure catalytic core and a C- terminal flexible tail domain (CTD). Truncation of CTD was associated with decreased microtubule acetylation in cells, implying a role for CTD in the interaction of tubulin acetyltransferase with microtubules. However, the mechanism by which luminal enzymes specifically recognize residues on the inner surface of microtubules is unclear.

Tubulin acetyltransferase mediates the conversion of two substrates, acetyl-CoA and K40, to two products, CoA and acetylated K40. As a dual-substrate enzyme, tubulin acetyltransferase can catalyze the reaction either by forming a triplex with all substrates (I. e., a sequential mechanism) or by releasing the first product before binding to the second substrate (I. e., a ping-pong mechanism). Determining the catalytic mechanism of the dual-substrate enzyme is challenging because the reported acetyltransferase mechanism may depend on the assay design and protein domain . Current structural and kinetic analyses of the catalytic core of tubulin acetyltransferase suggest that microtubule acetylation employs a ternary complexation mechanism. However, the enzymatic mechanism of microtubule acetylation is unclear, and the interaction between microtubule acetyltransferase, microtubule and acetyl-CoA is unclear.

Here, the researchers investigated the mechanistic basis for full-length tubulin acetyltransferase entering and targeting the lumen of microtubules. The results show that tubulin acetyltransferase binds to microtubules in an evolutionarily conserved multivalent manner, with one enzyme molecule spanning four adjacent protofilaments (PFs), each consisting of head-to-tail aligned α/β-tubulin heterodimers.In particular, the tubulin acetyltransferase CTD is anchored in the taxane-binding pocket of both β-tubulin proteins and plays a key role in the processibility and luminal accessibility of this enzyme.By using recombinant tubulin to simulate pre-acetylated and post-acetylated α-tubulin, it further reveals how tubulin acetyltransferase senses the acetylation state of microtubules and promotes the cycle of α-tubulin acetylation in microtubules.Overall, this study has important implications for the relationship between the level of α-tubulin acetylation and paclitaxel-induced cancer cell cytotoxicity, as well as the development of tubulin acetyltransferase inhibitors to treat disease.

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