The Solution Structure of the N-Terminal Domain of Human Tubulin Binding Cofactor C Reveals a Platform for Tubulin Interaction

Human Tubulin Binding Cofactor C (TBCC) is a post-chaperonin involved in the folding and assembly of α- and β-tubulin monomers leading to the release of productive tubulin heterodimers ready to polymerize into microtubules. In this process it collaborates with other cofactors (TBC's A, B, D, and E) and forms a supercomplex with TBCD, β-tubulin, TBCE and α-tubulin. Here, we demonstrate that TBCC depletion results in multipolar spindles and mitotic failure. Accordingly, TBCC is found at the centrosome and is implicated in bipolar spindle formation. We also determine by NMR the structure of the N-terminal domain of TBCC. The TBCC N-terminal domain adopts a spectrin-like fold topology composed of a left-handed 3-stranded α-helix bundle. Remarkably, the 30-residue N-terminal segment of the TBCC N-terminal domain is flexible and disordered in solution. This unstructured region is involved in the interaction with tubulin. Our data lead us to propose a testable model for TBCC N-terminal domain/tubulin recognition in which the highly charged N-terminus as well as residues from the three helices and the loops interact with the acidic hypervariable regions of tubulin monomers

Comparison of the interacting face of TBCA, BAG1 and TBCC N-terminal domain.

<p>Ribbon displays of the similarly oriented spectrin-like domains of TBCA (left), BAG1 (middle), and TBCC (right) with the residues involved in the interaction with β-tubulin, the ATPase domain of Hsc70, and the 16-residue C-terminal β-tubulin peptide EMYEDDEEESESQGPK (435–450), respectively, shown in strong colours.</p

Solution structure of the TBCC N-terminal domain.

<p>A) Superposition of the 20 lowest-energy conformers. B) Ribbon display of a representative conformer of the family showing the limits of the helical segments and one of the possible orientations of the N-terminal tail (in green) with respect to the protein core. Two regions with helical propensity (33–44) and (49–55) are labelled. C) Hydrophobic contacts in the interior of the bundle. Different colours are used along the helix axis for the upper N-terminal side (yellow), the lower C-terminal side (magenta), the bottom part including loop 2 (green), the top part comprising loop 3 next to the disordered N-terminal region (orange). The salt bridge between E67 and R90 is highlighted in red. D) Distribution of aliphatic/aromatic and charged/polar residues along the helices. The hydrophobic side-chains are concentrated at the helical interfaces favouring the molecular packing, and most polar and charged residues are in contact with the solvent. Colour code as in C).</p

Surface properties of the TBCC N-terminal domain.

<p>A) The electrostatic surface is represented for four views corresponding to 90° rotations. The distribution of the negatively charged (red), positively charged (blue), and nonpolar residues (white) defines a highly charged surface, with two 90°-rotated faces concentrating mainly negative and positive charges (left), and the other two with more random charge distribution (right). B) Two 180° rotated views of the mapped chemical shift perturbation data. Residues affected by the interaction with αβ-tubulin dimer are coloured in yellow in the helices and in green for the N-terminal disordered segment.</p

The TBCC N-terminal domain is embedded at the centrosome.

<p>A) Mitotic HeLa cell doubly-immunostained with the anti-TBCC antibody purified against the TBCC N-terminal domain, and tubulin. TBCC (arrows) is not detected at the centrosome by the antibody purified against the N-terminus of TBCC (immunoglobulins recognizing the C-terminus are removed). This result suggests that the TBCC N-terminal domain centrosomal epitopes are masked in the centrosome. B) (left) High resolution confocal images of HeLa cells transfected with the TBCC N-terminal domain. Overexpression of the TBCC N-terminal domain produces accumulates of this protein at the perinuclear-centrosomal region (inset, arrow). (right) Confocal microscopy projection image of a mitotic HeLa cell transfected with the TBCC N-terminal domain and doubly immunostained against tubulin and TBCC. TBCC N-terminal domain overexpression produces mitotic aberration defects such as multipolar spindles, similar to those observed for the full-length construct.</p

Specificity of polyclonal and monoclonal anti-TBCC antibodies.

<p>A) Human TBCC protein family. Schematic representation of the functional domains ascribed to human TBCC, RP2 and TBCCD1. The human proteins also possess a CARP domain present in CAPs (cyclase-associated proteins) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025912#pone.0025912-Freeman1" target="_blank">[58]</a>. TBCCD1 is related to TBCC and RP2 which functionally overlaps with TBCC <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025912#pone.0025912-Bartolini1" target="_blank">[6]</a>. The C-terminus known as the TBCC domain is shown in light blue, the CARP domain in magenta and the N-terminus domain (alpha module) in green. B) Both, the purified rabbit polyclonal anti-TBCC produced in our laboratories (left) and the commercial mouse monoclonal antibody also used in this study (right, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025912#s4" target="_blank">Methods</a>) recognised a single band in whole cell extracts.</p

TBCC is located at the centrosome.

<p>A) Confocal microscopy image of TBCC localization on interphase (left) and mitotic (right) HeLa cells. TBCC is mostly a cytoplasmic protein but concentrates at the centrosomes of HeLa cells (arrows). B) TBCC overexpression produces an increase of TBCC immunostaining at the spindle poles (arrows) and a higher rate of mitotic aberration defects such as multipolar spindles. C) (left) HeLa cells in prophase exhibiting a clear TBCC colocalization with the protein Nedd1, a classical centrosomal marker. High resolution confocal microscopy images of both centrosomes (#1,#2) are shown. (right) Confocal-microscopic image of HeLa cells where the microtubule cytoskeleton has been destroyed by cold and nocodazole treatment. Double-immunostaining against acetylated tubulin and TBCC revealed how, under these conditions, TBCC colocalizes with the centrioles that typically exhibit acetylated tubulin. D) (left) Triple immunostained HeLa cell displaying a primary cilium (arrow). (right) High resolution confocal image of the primary cilium and daughter centriole (arrow) immunostained with anti-acetylated tubulin (blue channel) and TBCC (green channel). These images show that TBCC is mostly localised at the base of the primary cilium, around the basal body.</p

TBCC depletion leads to mitotic failure and apoptosis.

<p>A) (left) HeLa cell culture treated with control RNAi for 72 h. A cell confluence of almost 90% is achieved. (right) Identical culture treated with TBCC RNAi for 72 h. TBCC gene interference produces a rise in cell death leading to a conspicuous cell depletion in the culture. B) Confocal-microscopy projection image of 72 h RNAi treated HeLa cell where a multipolar mitotic spindle is shown (spindle poles indicated by arrows). C) Western blot confirmation of TBCC silencing on whole cell lysates (50 µg/lane total protein). TBCC expression was compared to total α- and β-tubulins. TBCC depletion did not noticeably affect tubulin levels at this post-transfection time point. D) Distribution of the different mitotic cell stages observed in TBCC RNAi treated cultures and controls at different time points after TBCC RNAi treatment. These data show that TBCC RNAi treatment blocks cells mostly at metaphase, leading to a high rate of apoptotic cells. Data represent mean values and bars standard errors.</p

TBCC N-terminal interaction assays with αβ-tubulin dimer and C-terminal β-tubulin peptides.

<p>Superposition of ¹⁵N-HSQC spectra of the TBCC N-terminal domain free (blue) and in the presence of αβ-tubulin heterodimer (yellow). Selected perturbed residues are labelled, with green labels corresponding to amino acids in the N-terminal disordered region. <b>B</b>) Superposition of ¹⁵N-HSQC spectra of the TBCC N-terminal domain free (blue) and in the presence of an excess of the 16-residue C-terminal β-tubulin peptide EMYEDDEEESESQGPK (magenta). Selected perturbed residues are labelled. <b>C</b>) Superposition of ¹⁵N-HSQC spectra of the TBCC N-terminal domain free (blue) and in the presence of excess of the 20-residue C-terminal β-tubulin peptide ESNMNDLVSEYQQYQDATAD (grey). No significant perturbations are observed.</p

TBCC N-terminal domain heteronuclear NOEs for local backbone flexibility.

<p>Residues in the 30-residue N-terminal segment have lower than average NOE values, indicative of high backbone mobility in the ns-ps time-scale. Some flexibility is also found at the C-terminus of the domain and residues at the interhelical connecting loops. The dynamics of the helices α2, α3, α4 is more restricted.</p