Op18/stathmin caps a kinked protofilament-like tubulin tetramer

Abstract

Oncoprotein 18/stathmin (Op18), a regulator of microtubule dynamics, was recombinantly expressed and its structure and function analysed. We report that Op18 by itself can fold into a flexible and extended alpha-helix, which is in equilibrium with a less ordered structure. In complex with tubulin, however, all except the last seven C-terminal residues of Op18 are tightly bound to tubulin. Digital image analysis of Op18:tubulin electron micrographs revealed that the complex consists of two longitudinally aligned alpha/beta-tubulin heterodimers. The appearance of the complex was that of a kinked protofilament-like structure with a flat and a ribbed side. Deletion mapping of Op18 further demonstrated that (i) the function of the N-terminal part of the molecule is to 'cap' tubulin subunits to ensure the specificity of the complex and (ii) the complete C-terminal alpha-helical domain of Op18 is necessary and sufficient for stable Op18:tubulin complex formation. Together, our results suggest that besides sequestering tubulin, the structural features of Op18 enable the protein specifically to recognize microtubule ends to trigger catastrophes.

Fig. 1. CD spectroscopy and EM of recombinant human Op18. (A) CD spectra recorded at 5°C (•) and 25°C (○). (B) Thermal unfolding (•) and refolding (__) profiles recorded at 222 nm. Op18 was adjusted to a concentration of 25 μM in 5 mM sodium phosphate pH 7.4 containing 150 mM NaCl. (C) Electron micrographs of glycerol-sprayed/rotary metal-shadowed recombinant Op18 molecules at 20°C. Scale bars, 20 nm for the low magnification overview and 10 nm for the high magnification gallery.

Fig. 2. Tubulin sequestering activity of recombinant Op18. (A) Tubulin (15 μM) was incubated with various amounts of Op18 and polymerized to MTs as described in Materials and methods. After high-speed centrifugation, the supernatants (sup) and the pellets (pel) were separated and analysed on SDS–PAGE by Coomassie Blue staining. Only the tubulin protein bands (migrating at ∼50 kDa) are displayed. Bottom: tubulin control preparation without Op18. (B) TEM electron micrograph of glycerol-sprayed/rotary metal-shadowed tubulin subunits. (C) TEM electron micrograph of glycerol-sprayed/rotary metal-shadowed Op18:tubulin complexes. Scale bar for (B) and (C), 50 nm.

Fig. 3. (A) Contrast-reversed STEM ADF electron micrographs of negatively stained Op18:tubulin complexes. Bottom right image: representative average comprising 23 particles of a subclass obtained by multivariate statistical analysis of a set of 270 particles. Arrows point to the slightly thicker appearing tips of the complexes. Scale bars, 20 nm for the low magnification overview and 5 nm for the high magnification images. (B) Same average as the bottom right image in (A) displayed enlarged and with isodensity contours superimposed. Crosses mark the centres of mass. The flat (at lower particle radius) and ribbed side (at higher particle radius) suggest that they correspond to the outside and inside, respectively, of the MT wall.

Fig. 4. ¹⁵N,¹H HSQC spectra of unbound ¹⁵N-labelled Op18 (A) and tubulin-bound ¹⁵N-labelled Op18 (B). Assignments are indicated for the seven Op18 residues that are flexible in the Op18:tubulin complex. Lines in (B) indicate resonance shifts for Ala143, Asp144 and Glu145.

Fig. 5. Functional analysis of the STHM41–140 deletion mutant. (A) TEM image of negatively stained Op18:tubulin complexes obtained under double ring-forming conditions (i.e. 16 mM Mg²⁺, 1 mM GDP, 1 h at 4°C). (B) TEM image of glycerol-sprayed/rotary metal-shadowed STHM41–140:tubulin complexes obtained under MT polymerization conditions (i.e. 6 mM Mg²⁺, 0.5 mM GTP, 3.4 M glycerol, 1 h at 37°C). (C) TEM image of negatively stained STHM41–140:tubulin double ring oligomers obtained under double ring-forming conditions as described in (A). For each experiment, 15 μM of tubulin was incubated with equimolar amounts of Op18 or STHM41–140. Scale bars, 50 nm.

Fig. 6. Proposed mechanism of action of Op18 on tubulin and MTs. On the basis of the results presented here and the biochemical data available from the literature (for details, see Discussion), we derived a possible mechanism of how Op18 acts on tubulin and MTs. Typically, MTs are assembled from tubulin-GTP dimers (1). In the presence of Op18 (which can adopt an extended α-helical structure), assembly of MTs is inhibited. As proposed in (2), a straight intermediate containing four longitudinally aligned tubulin monomers is stabilized by interacting with the entire length of the C-terminal α-helical domain of Op18. Subsequent GTP hydrolysis on β-tubulin, which is stimulated by Op18, induces the preferred tubulin-GDP-driven relaxed conformational change at the inter-dimer interface (3). The resulting kinked Op18:tubulin structure is incompatible with the straight protofilament conformation found within the lattice of the MT wall, making the sequestered tubulins assembly incompetent. The ‘cap’ formed by the N-terminus of Op18 ensures the specificity and integrity of the complex by preventing further longitudinal Op18:tubulin complex aggregation. Alternatively, Op18 specifically recognizes the plus end of an MT (4). As for tubulin dimers (2), binding of Op18 to a straight tubulin-GTP-capped protofilament promotes GTP hydrolysis on β-tubulin. As a result, the curved and relaxed GDP state protofilament is formed (5). The conformational transition from straight to curved destabilizes the MT, thereby triggering a catastrophe (6). The GDP-containing ring-like MT disassembly products can either be recycled to tubulin-GTP subunits (7) or sequestered by free Op18 molecules. It should be noted that phosphorylation of Op18 on four distinct Ser residues, which regulates the activity of the protein both in vitro and in vivo, is not considered in the model. White spheres represent α-tubulin and grey spheres β-tubulin. The state of hydrolysis of the nucleotide bound to the exchangeable site of the β-tubulin subunit is indicated by small black (for GTP) or white (for GDP) circles. For simplicity, the non-exchangeable and never hydrolysed nucleotide bound to the α-tubulin subunit is not marked. The minus end of the MT is indicated by (–) and the plus end by (+). All drawings are to scale.