Structure of the α-tubulin acetyltransferase, αTAT1, and implications for tubulin-specific acetylation

Abstract

Protein acetylation is an important posttranslational modification with the recent identification of new substrates and enzymes, new links to disease, and modulators of protein acetylation for therapy. α-Tubulin acetyltransferase (αTAT1) is the major α-tubulin lysine-40 (K40) acetyltransferase in mammals, nematodes, and protozoa, and its activity plays a conserved role in several microtubule-based processes. Here, we present the X-ray crystal structure of the human αTAT1/acetyl-CoA complex. Together with structure-based mutagenesis, enzymatic analysis, and functional studies in cells, we elucidate the catalytic mechanism and mode of tubulin-specific acetylation. We find that αTAT1 has an overall fold similar to the Gcn5 histone acetyltransferase but contains a relatively wide substrate binding groove and unique structural elements that play important roles in α-tubulin-specific acetylation. Conserved aspartic acid and cysteine residues play important catalytic roles through a ternary complex mechanism. αTAT1 mutations have analogous effects on tubulin acetylation in vitro and in cells, demonstrating that it is the central determining factor of α-tubulin K40 acetylation levels in vivo. Together, these studies provide general insights into distinguishing features between histone and tubulin acetyltransferases, and they have specific implications for understanding the molecular basis of tubulin acetylation and for developing small molecule modulators of microtubule acetylation for therapy.

Fig. 1.

Structure of the αTAT1/AcCoA complex. (A) Cartoon representation of the complex. α-helices (orange) are numbered 1–4, β-strands (blue) are numbered 1–7, loops are colored green, and the N and C termini are indicated. AcCoA is represented as sticks and colored according to element: carbon, yellow; nitrogen, blue; and oxygen, red. (B) Alignment of αTAT1 (cyan) to Tetrahymena Gcn5 (PDB ID code 1QSN; magenta) in the same orientation and AcCoA rendering for αTAT1 as in A. The functionally important α1-α2-loop, β4-β5-hairpin, and C-terminal regions of αTAT1 are labeled. (C) Sequence alignment of αTAT1 from Homo sapiens αTAT1 (Hs), Mus musculus (Mm), Rattus norvegicus (Rn), Danio rerio (Dr), C. elegans (Ce), and Tetrahymena thermophilia Gcn5 (TtGcn5). Numbering and secondary structural elements above the sequence alignment is for HsαTAT1. Magenta squares highlight residues mutated in these studies, green hexagons highlight conserved aspartic acid and cysteine residues important for catalysis, and cyan circles highlight residues that make contacts with the AcCoA cofactor through either their side chain or backbone atoms.

Fig. 2.

Catalytic site of αTAT1 and bisubstrate kinetics. (A) Cartoon representation of the catalytic site of αTAT1. The coloring scheme and labeling is the same as in Fig. 1, with the addition of sulfur colored in magenta. Residues surrounding the catalytic site are labeled for frame of reference. (B) Microtubule acetylation progress curves for WT, C120, and D157 mutant αTAT1. Curves fit to D157 mutants are shown as dashed lines because of a poor fit. (C) Bisubstrate kinetics of αTAT1 acetylation. Experiments were performed at four different AcCoA concentrations, 25, 12, 10, and 5 μM, and a double reciprocal plot of 1/velocity versus 1/[MT] (microtubule) is shown. A best fit of the plot displays a plot with the lines intersecting at a common x-intercept indicative of a ternary complex mechanism.

Fig. 3.

The αTAT1 a-tubulin substrate binding site and mutational analysis. (A) Electrostatic surface potential mapping of αTAT1 as generated by Pymol. Red, blue, and white represent acidic, basic, and hydrophobic patches, respectively. AcCoA is colored as in Fig. 1. (B and C) The ability of the αTAT1 mutants to acetylate microtubules was assessed by using an in vitro acetyltransferase assay. Experiments were performed at microtubule concentrations ranging from 0.5 μM to 50 μM to obtain kinetic parameters (k_cat and K_m) necessary to calculate catalytic efficiency (Table S2). Experiments were performed at least three times. Shown in A and B are the amount of acetylated microtubules formed by using 20 μM microtubules. B contains only β-hairpin mutants, and C contains mutants from the α1-α2 loop, C120, and C-terminal loop domains. Statistical analysis was performed by using an unpaired two-tailed t test in GraphPad Prism. *P < 0.05 and **P < 0.01. (D) Surface representation of αTAT1 highlighting acetylation defective (orange) and enhancing (magenta) mutants.

Fig. 4.

Cell-based studies of selected αTAT1 mutants. (A) Representative micrographs of αTAT1^−/− MEFs expressing comparable levels of GFP-αTAT1 variants. Transfected cells are outlined, GFP-αTAT1 staining is green, acetylated tubulin staining is red, and DNA is blue. Note that the untransfected αTAT1^−/− MEFs display no acetylated tubulin staining. For scale, each panel is approximately 120 μm in width. (B) The ability of the αTAT1 mutants to acetylate microtubules in vivo was assessed by measuring the intensity of the acetylated tubulin immunostaining in 45 cells per mutant (for the WT, D157N, D109A, and E111A enzymes) or in 15 cells (C120A, F105A, R69A, and N182A), and normalizing the value to the intensity of the GFP-αTAT1 signal. Statistical analysis was performed by using a two-sided Wilcoxon test in R. *P < 0.05, **P < 0.01, ****P < 0.0001.

Fig. 5.

Protein substrate binding sites of selected protein acetyltransferases. (A) Surface representation of TtGcn5 bound to histone H3 peptide (yellow cpk stick) and CoA (white cpk stick). (B) Surface representation of the N-terminal acetyltransferase Naa50p bound to CoA (white cpk stick) and N-terminal substrate peptide (yellow cpk stick). (C) Surface representation of αTAT1 bound to AcCoA (white cpk stick).