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Structure of bacterial tubulin BtubA/B: evidence for horizontal gene transfer - PubMed

  • ️Sat Jan 01 2005

Structure of bacterial tubulin BtubA/B: evidence for horizontal gene transfer

Daniel Schlieper et al. Proc Natl Acad Sci U S A. 2005.

Abstract

alphabeta-Tubulin heterodimers, from which the microtubules of the cytoskeleton are built, have a complex chaperone-dependent folding pathway. They are thought to be unique to eukaryotes, whereas the homologue FtsZ can be found in bacteria. The exceptions are BtubA and BtubB from Prosthecobacter, which have higher sequence homology to eukaryotic tubulin than to FtsZ. Here we show that some of their properties are different from tubulin, such as weak dimerization and chaperone-independent folding. However, their structure is strikingly similar to tubulin including surface loops, and BtubA/B form tubulin-like protofilaments. Presumably, BtubA/B were transferred from a eukaryotic cell by horizontal gene transfer because their high degree of similarity to eukaryotic genes is unique within the Prosthecobacter genome.

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Figures

Fig. 1.
Fig. 1.

Heterodimerization and GTP-dependent dynamic polymerization of BtubA/B. (A) Bicistronic expression of P. dejongeii BtubA/B in E. coli. After 3 h of induction, soluble BtubA/B protein makes up the majority of proteins in the lysate (Coomassie-stained polyacrylamide SDS gel). (B) Nucleotide-dependent pelleting and polymerization of BtubA/B. BtubA/B was incubated at 10 μM in 100 mM Pipes·NaOH (pH 7.0) with and without nucleotides and magnesium and centrifuged at 80,000 rpm. Polymerization depends on nucleotide and magnesium binding. The electron micrographs are taken of samples from the pelleting reactions just before centrifugation to demonstrate fiber formation in these reactions. (Negative stain, scale bar: 200 nm.) (C) BtubA/B dimers form only at high concentrations as shown by analytical ultracentrifugation. Shown are sedimentation velocity profiles (Left) and the corresponding sedimentation coefficient distributions (Right). BtubA/B at low concentration (10 μM) gave one main peak with a sedimentation coefficient s20,w = 3.5S (92%) (Upper), whereas BtubA/B at higher concentration (50 μM) gave two main peaks of 3.5S (48%, monomers) and 4.4S (48%, dimers), respectively (Lower), plus a minor peak of 6.8S (4%; 2% in other experiments, which could correspond to incipient association to larger oligomers or to protein aggregation). Buffer contained 100 mM Pipes·NaOH and 200 mM KCl (pH 6.8). (D) BtubA/B polymerizes as a mixed polymer. Different ratios of purified BtubA and BtubB were pelleted under the same conditions as in B. Maximum pelleting occurs at a ratio of 1:1, indicating that polymer formation depends on the BtubA/B interaction. A/B concentrations were 11.2/0.01, 9.2/2, 7.4/3.6, 5.4/5.4, 3.6/7.4, 2/9.2, and 0.01/11.2 μM, respectively. (E) BtubA/B polymers are dynamic. A 90° light-scattering assay at 350 nm with 10 μM BtubA/B and 0.5 mM GTP showed rapid polymerization and slow depolymerization. More rounds of polymerization can be induced by adding fresh GTP.

Fig. 2.
Fig. 2.

BtubA/B polymers have a longitudinal repeat similar to α, β-tubulin indicating protofilament formation. (A) Low-magnification micrograph showing BtubA/B filaments after polymerization in the presence of GTP. Protein at 10 μM was incubated for 30 min at ambient temperature with 100 mM Pipes·NaOH (pH 6.8), 5 mM MgCl2, 200 mM KCl, and 1 mM GTP and was negatively stained with 2% uranyl acetate. (B-D) BtubA/B double filaments. These are the most commonly formed filaments, presumably consisting of two BtubA/B protofilaments. Most filaments twist (B and C), indicated by arrowheads at the crossover points. Filament C has an average width of ≈109 Å. (Scale bar: 100 nM.) (E) Computed diffraction pattern of filament B. Layer lines are clearly visible at ≈42 Å, representing the subunit repeat along the protofilament axis. This repeat matches the repeat seen in the BtubA/B crystal structure and is close to that of tubulin (40 Å).

Fig. 3.
Fig. 3.

Crystal structures of BtubA and BtubA/B. (A) Crystal structure of BtubA at 2.5-Å resolution. BtubA's structure is closely related to tubulin. The fold is divided into the N-terminal nucleotide-binding domain (blue), separated by helix H7 (yellow) from the intermediate domain (orange) and two large helices forming the C-terminal domain (red) (4). (B) BtubA contains the C-terminal tubulin domain. Shown is a view rotated 90° around the y axis from A. The two large helices (red) at the C terminus of tubulin form the outside of microtubules (7) and make the biggest difference between tubulin and FtsZ. (C) Crystal structure of BtubA/B heterodimer (asymmetric unit of the crystals) at 3.2-Å resolution. BtubA/B form the same heterodimer as tubulin (24, 25). The protofilament axis is vertical. BtubA is situated at the plus (+) end (red), and BtubB is at the minus (-) end (blue). In the crystals, BtubA contains GDP, whereas BtubB has a sulfate ion in the nucleotide-binding site. The heterodimer is not completely straight; the two subunits are rotated by ≈15° around the z axis [same direction as in the tubulin-stathmin complex (26)], tangential to the microtubule wall. (D) The BtubA/B crystals contain a continuous double filament. The 6522 space group symmetry produces an antiparallel double filament with repeating BtubA/B units in the crystal packing. The bend per heterodimer is 60°, divided into 15° between BtubA and -B (intradimer; see C) and 45° between B and A (interdimer). (E) BtubA/B are very closely related to tubulin. Shown is the superposition of BtubA (black) (rmsd to BtubB, 1.34 Å; 36% sequence identity; 82% aligned) with α-tubulin (25) (green; rmsd 1.5 Å; 37% sequence identity; 85% aligned; Protein Data Bank ID code 1JFF), β-tubulin (25) (red; rmsd 1.71 Å; 35% sequence identity; 85% aligned; Protein Data Bank ID code 1JFF), and subunit B from the tubulin-stathmin complex (26) (blue; rmsd 1.3 Å; 35% sequence identity; 85% aligned; Protein Data Bank ID code 1SA0). Differences are small and mainly located in the T7-loop, the M-loop, which is involved in microtubule formation for tubulin (7), helix H6, and loop H1-S2, which are part of the protofilament contact. BtubA/B have a short S9-S10 loop that in α-tubulin covers the Taxol-binding pocket completely. (Figure was generated with

pymol

.)

Fig. 4.
Fig. 4.

Chaperone independent folding of BtubA/B in vitro.(A) BtubA/B refold in vitro in the presence of GDP. CD spectra showing the folding state of BtubA/B, in 5.5 M guanidinium hydrochloride (GdmCl) and after refolding by dilution and subsequent size exclusion chromatography. The unfolded spectrum was cut at 213 nm because of light absorption by guanidinium hydrochloride. (B) Refolded BtubA/B are monomeric. Size exclusion chromatography of refolded BtubA/B on a Sephacryl S300 column (Amersham Pharmacia) in 20 mM Tris·HCl, 1 mM azide, and 1 mM EDTA (pH 7.5). Refolded protein shows no aggregation. Never-unfolded protein shows the same chromatogram with BtubA and BtubB running separately (data not shown). (C) Refolded BtubA/B polymerizes. An electron micrograph shows filaments made from refolded BtubA/B protein. The filaments are indistinguishable from wild-type filaments. Conditions are as for Figs. 1B and 2. (Scale bar: 200 nm.)

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