Nucleotide-dependent bending flexibility of tubulin regulates microtubule assembly

Abstract

The atomic structure of tubulin in a polymerized, straight protofilament is clearly distinct from that in a curved conformation bound to a cellular depolymerizer. The nucleotide contents are identical, and in both cases the conformation of the GTP-containing, intra-dimer interface is indistinguishable from the GDP-containing, inter-dimer contact. Here we present two structures corresponding to the start and end points in the microtubule polymerization and hydrolysis cycles that illustrate the consequences of nucleotide state on longitudinal and lateral assembly. In the absence of depolymerizers, GDP-bound tubulin shows distinctive intra-dimer and inter-dimer interactions and thus distinguishes the GTP and GDP interfaces. A cold-stable tubulin polymer with the non-hydrolysable GTP analogue GMPCPP, containing semi-conserved lateral interactions, supports a model in which the straightening of longitudinal interfaces happens sequentially, starting with a conformational change after GTP binding that straightens the dimer enough for the formation of lateral contacts into a non-tubular intermediate. Closure into a microtubule does not require GTP hydrolysis.

Fig. 1. Cryo-EM reconstruction of double-layered tubes of GDP-bound tubulin and docking of crystallographic models

(a) 3-D densities for the inner (top) and outer (bottom) layers of the GDP-tubulin tubes. Inside view of the tubes on the left, outside view on the right. Dimer boundaries are indicated by yellow boxes. (b) The crystallographic structures of tubulin were manually docked into the cryo-EM densities of the outer and inner layers of the tubes. β-tubulins (1SA0) are shown in yellow, α-tubulins (1JFF) are shown in green for the lower dimer and in magenta for the top dimer. Major regions of discrepancy with the crystal structures are indicated in the last panel.

Fig. 1. Cryo-EM reconstruction of double-layered tubes of GDP-bound tubulin and docking of crystallographic models

(a) 3-D densities for the inner (top) and outer (bottom) layers of the GDP-tubulin tubes. Inside view of the tubes on the left, outside view on the right. Dimer boundaries are indicated by yellow boxes. (b) The crystallographic structures of tubulin were manually docked into the cryo-EM densities of the outer and inner layers of the tubes. β-tubulins (1SA0) are shown in yellow, α-tubulins (1JFF) are shown in green for the lower dimer and in magenta for the top dimer. Major regions of discrepancy with the crystal structures are indicated in the last panel.

Fig. 2. Intra- and inter-dimer bends in different tubulin polymers

(a) Two dimers in a microtubule (plus-end at the top). The red box marks the lower dimer. The dashed yellow boxes indicate the monomers shown in the end-on views in (b). (b) The microtubule (green), RB3-bound structure (violet), outer (orange-red) and inner layer of the GDP tubes (blue) were aligned on the first β subunit. The bottom superposition shows the displacements due to the intra-dimer bending, and the top shows the displacements due to the inter-dimer bending. (c) Relative magnitude (arrow length) and the radial and tangential components of each bend.

FIG. 3. Cryo-EM reconstruction of GMPCPP-tubulin tubes and docking of the crystallographic model

(a) 3-D densities of the GMPCPP tubulin tubes. Notice the association of protofilaments in pairs. (b) β-tubulin (1JFF) was manually docked into the density of the GMPCPP tube. The small outward curvature of the protofilaments is clearly seen in the Side View on the left (right surface corresponds to the outside of the microtubule). The Front View shows the lateral stagger between protofilaments, identical to that in microtubules. The End-on View shows more clearly the pairing of protofilaments. Within a pair the lateral contacts are indistinguishable from those in microtubules, but the lateral contact between pairs has been displaced towards the inside surface of the tube.

FIG. 3. Cryo-EM reconstruction of GMPCPP-tubulin tubes and docking of the crystallographic model

(a) 3-D densities of the GMPCPP tubulin tubes. Notice the association of protofilaments in pairs. (b) β-tubulin (1JFF) was manually docked into the density of the GMPCPP tube. The small outward curvature of the protofilaments is clearly seen in the Side View on the left (right surface corresponds to the outside of the microtubule). The Front View shows the lateral stagger between protofilaments, identical to that in microtubules. The End-on View shows more clearly the pairing of protofilaments. Within a pair the lateral contacts are indistinguishable from those in microtubules, but the lateral contact between pairs has been displaced towards the inside surface of the tube.

Fig. 4. GMPCPP tubes: comparison with and conversion into microtubules

(a) GMPCPP tube (yellow) and microtubule (green): Side View illustrating radial bending (blue arrow); End-on View showing how the lateral contact that results in the closure of microtubules is maintained within a protofilament pair, but is displaced between pairs (red arrow on inset). (b) Direct conversion of GMPCPP tubes into microtubules at 37 °C visualized by negative-stain electron microscopy (scale bar 100 nm). (c) Fluorescence microscopy of microtubules formed by mixing two populations of differentially labelled GMPCPP tubes before warming the solution, proving that conversion does not involve a depolymerization step (scale bar 10 μm).

Fig. 4. GMPCPP tubes: comparison with and conversion into microtubules

(a) GMPCPP tube (yellow) and microtubule (green): Side View illustrating radial bending (blue arrow); End-on View showing how the lateral contact that results in the closure of microtubules is maintained within a protofilament pair, but is displaced between pairs (red arrow on inset). (b) Direct conversion of GMPCPP tubes into microtubules at 37 °C visualized by negative-stain electron microscopy (scale bar 100 nm). (c) Fluorescence microscopy of microtubules formed by mixing two populations of differentially labelled GMPCPP tubes before warming the solution, proving that conversion does not involve a depolymerization step (scale bar 10 μm).

Fig. 4. GMPCPP tubes: comparison with and conversion into microtubules

(a) GMPCPP tube (yellow) and microtubule (green): Side View illustrating radial bending (blue arrow); End-on View showing how the lateral contact that results in the closure of microtubules is maintained within a protofilament pair, but is displaced between pairs (red arrow on inset). (b) Direct conversion of GMPCPP tubes into microtubules at 37 °C visualized by negative-stain electron microscopy (scale bar 100 nm). (c) Fluorescence microscopy of microtubules formed by mixing two populations of differentially labelled GMPCPP tubes before warming the solution, proving that conversion does not involve a depolymerization step (scale bar 10 μm).