Tubulin tyrosine ligase structure reveals adaptation of an ancient fold to bind and modify tubulin.
Tubulin tyrosine ligase (TTL) catalyzes the post-translational C-terminal tyrosination of α-tubulin. Tyrosination regulates recruitment of microtubule-interacting proteins. TTL is essential. Its loss causes morphogenic abnormalities and is associated with cancers of poor prognosis. We present the first crystal structure of TTL (from Xenopus tropicalis), defining the structural scaffold upon which the diverse TTL-like family of tubulin-modifying enzymes is built. TTL recognizes tubulin using a bipartite strategy. It engages the tubulin tail through low-affinity, high-specificity interactions, and co-opts what is otherwise a homo-oligomerization interface in structurally related ATP grasp-fold enzymes to form a tight hetero-oligomeric complex with the tubulin body. Small-angle X-ray scattering and functional analyses reveal that TTL forms an elongated complex with the tubulin dimer and prevents its incorporation into microtubules by capping the tubulin longitudinal interface, possibly modulating the partition of tubulin between monomeric and polymeric forms.
PubMed ID: 22020298
PMC ID: PMC3342691
Article link: Nat Struct Mol Biol.
Grant support: ZIA NS003122-01 NINDS NIH HHS
Genes referenced: mapre3 ttl
Article Images: [+] show captions
|Figure 2. Active site architecture and α–tubulin C-terminal peptide recognition. (a) Conserved interactions in the TTL active site, colored as in Fig. 1a (b) Nucleotide (ventral) and (c) dorsal views of the TTL molecular surface color-coded for electrostatic potential55 (red, negative; blue, positive, ranging from −7 kBT to 7 kBT) (d) Tyrosination of a 14-residue α–tubulin C-terminal peptide by TTL and structure-guided TTL mutants (N≥2; Supplementary Fig. 3). The 14-residue peptide (VDSVEGEGEEEGEE) serves as an optimal TTL peptide substrate42. Error bars indicate s.e.m. and are frequently smaller than the symbols.|
|Figure 3. Molecular determinants for tubulin tyrosination. (a) TTL molecular surface with conserved residues important for tubulin tyrosination shown in stick atomic representation (b) Normalized relative tyrosination activity with α-tubulin peptide (blue) or tubulin (red) substrates of structure-guided TTL mutants (N≥3). Error bars indicate s.e.m.|
|Figure 4. Gel filtration studies and sedimentation velocity ultracentrifugation analysis of TTL binding to tubulin. (a–e) Complex formation between TTL or TTL mutants and tubulin in various nucleotide conditions monitored by gel filtration chromatography. Binding is monitored by the disappearance of the slower mobility peak (corresponding to uncomplexed TTL) when TTL or TTL mutants are incubated with excess tubulin (68 µM tubulin and 34 µM TTL or TTL mutant). The gels correspond to peak fractions indicated in green. (f) Sedimentation coefficient distributions (c(s)) obtained for 10 µM TTL and 4.8 µM tubulin (g) c(s) for TTL–tubulin complexes at varying concentrations. All complexes are at 1:1 stoichiometry, except that shown in the cyan trace, which denotes a 1:4 tubulin:TTL ratio. The sedimentation coefficient distributions obtained with mixtures of 1:1 tubulin:TTL ratio at increasing total concentrations show two peaks, a small s value peak corresponding to free TTL, with the area of the peak decreasing with increasing protein concentration, and the high s value peak representing the position of the TTL–tubulin complex reaction boundary. At lower concentrations, the position of the higher s value peak corresponds to that of the tubulin dimer alone and increases in s value with increasing concentration. By analyzing the shift in position of the complex peak with increasing protein concentration the affinity constant of TTL for tubulin was determined to be ~1 µM (method described in56; Methods). When TTL is in excess so that all tubulin is in complex with TTL, a narrow symmetrical boundary of TTL–tubulin is observed.|
|Figure 5. Model of the TTL–tubulin complex from small-angle X-ray scattering. (a) Pair-distance probability distributions, [P(r)], computed from the experimental SAXS data for the TTL–tubulin complex and the crystal structure of the tubulin dimer (1JFF.pdb44) (b) Agreement of the experimental scattering for the TTL–tubulin complex with the scattering profiles of the models obtained from 14 ab initio simulations using Gasbor57 (χavg= 1.49 ± 0.09). The average normalized spatial discrepancy (NSD) for 14 models is 1.314, indicating good agreement (c,d) Composite (cyan mesh) and filtered (solid grey surface) SAXS envelopes for the TTL–tubulin complex. The composite structure consists of the aligned, superimposed and summed models from 14 independent simulations, whereas the filtered model corresponds to the most probable density map. The docked αβ-tubulin dimer is shown in ribbon representation, α–tubulin, yellow, β-tubulin, dark red. The last 2 residues of the α-tubulin atomic model are colored red and mark the beginning of the α-tail. The resolution of the reconstruction does not allow the unambiguous determination of the relative orientation of the tubulin dimer along its long axis. (e,f) Ribbon structure of the αβ-tubulin dimer in two different possible orientations in the SAXS reconstruction. The unstructured region of the α–tubulin tail absent from the tubulin crystal structure was modeled as a fully extended strand to illustrate its maximal possible span, and is colored red. Panel f shows the α-tubulin tail is unlikely to reach the active site of TTL if the interface of TTL with the globular core of the tubulin heterodimer is through β-tubulin. For all panels mean ±sd|
|Figure 6. TTL inhibits spontaneous polymerization of purified tubulin in vitro and attenuates MT growth rates in vivo. (a) Tubulin polymerization determined via turbidity (at 350 nm) in the absence or presence of TTL at various concentrations. For each experiment tubulin concentration was 20 µM. (b) Tubulin polymerization determined via turbidity (at 350 nm) in the absence or presence of various TTL mutants. For each experiment tubulin concentration was 20 µM and the concentration of the indicated TTL mutants was 20 µM. (c) Spinning disc confocal image of a U2OS cell expressing GFP-TTL (top panel) and TTL E331Q mutant (bottom panel) and the plus-end tracker EB3 fused to the fluorescent protein mKusabira-Orange. Scale bar corresponds to 10 µm. (d) Histograms of growth velocities of all tracked MTs in a wild-type and a GFP-TTL expressing cell (left panel) and a wild-type and a GFP-TTL E331Q expressing cell (right panel). Distributions comprise 1427, 983 and 858 measurements for the wild-type, GFP-TTL and GFP-TTL E331Q cell, respectively. (e) MT growth rates in wild-type, GFP-TTL and TTL E331Q expressing cells: 25th percentile (bottom line), median (middle thick line), 75th percentile (top line). The average growth rate is 13.04 ± 0.07 µm min−1 (mean±s.d.; N= 4225 tracks from 4 cells) for wild-type, 10.86 ± 0.08 µm min−1 for GFP-TTL expressing cells (N=3787 from 4 cells) and 10.24 ± 0.12 µm min−1 for GFP-TTL E331Q expressing cells (N=5222 from 5 cells). Statistical significance of the difference was determined by a permutation t-test.|
|Figure 7. Model for TTL action. TTL binds to monomeric tubulin and prevents its incorporation into the MT lattice by occluding the longitudinal interface of the tubulin heterodimer (left panel). TTL acts preferentially on the soluble tubulin pool and detyrosination takes place on MTs. This results in an asymmetry in the distribution of Tyr-tubulin within the MT, with tyrosinated tubulin enriched at the growing plus end where it can aid in the recruitment of proteins sensitive to the tubulin tyrosination status such as plus-end tracking proteins (+TIPS) (right panel).|