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Structure of Bacterial Tubulin Btuba B: Evidence for Horizontal Gene Transfer

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Structure of Bacterial Tubulin Btuba B: Evidence for Horizontal Gene Transfer Structure of bacterial tubulin BtubA͞B: Evidence for horizontal gene transfer Daniel Schlieper*, Marı´a A. Oliva†, Jose´ M. Andreu†, and Jan Lo¨ we*‡ *Laboratory of Molecular Biology, Medical Research Council, Hills Road, Cambridge CB2 2QH, United Kingdom; and †Centro de Investigaciones Biolo´ gicas, Consejo Superior de Investigaciones Cientı´ficas, Ramiro de Maeztu 9, 28040 Madrid, Spain Edited by Robert Haselkorn, University of Chicago, Chicago, IL, and approved May 19, 2005 (received for review April 6, 2005) ␣␤-Tubulin heterodimers, from which the microtubules of the ble into protofilaments, like ␣␤-tubulin and FtsZ. Their crystal cytoskeleton are built, have a complex chaperone-dependent fold- structures show such far-reaching similarities to ␣␤-tubulin as to ing pathway. They are thought to be unique to eukaryotes, indicate that BtubA͞B were transferred from a eukaryotic cell whereas the homologue FtsZ can be found in bacteria. The excep- by horizontal gene transfer. tions are BtubA and BtubB from Prosthecobacter, which have higher sequence homology to eukaryotic tubulin than to FtsZ. Here Materials and Methods we show that some of their properties are different from tubulin, Protein Expression and Purification. BtubA from P. dejongeii DSM such as weak dimerization and chaperone-independent folding. 12251 was expressed with an N-terminal thioredoxin fusion protein However, their structure is strikingly similar to tubulin including partner and a C-terminal hexahistidine tag in E. coli C41(DE3) surface loops, and BtubA͞B form tubulin-like protofilaments. Pre- cells, induced with 1 mM isopropyl ␤-D-thiogalactoside for3hat sumably, BtubA͞B were transferred from a eukaryotic cell by 37°C. BtubA-trx (predicted molecular weight, 64,356) was purified horizontal gene transfer because their high degree of similarity to by using Ni-NTA agarose (Qiagen, Valencia, CA) resin (buffer A: eukaryotic genes is unique within the Prosthecobacter genome. 50 mM Tris⅐HCl͞300 mM NaCl, pH 7.0; buffer B: buffer A plus 1 M imidazole) and further purified using a S200 Sephacryl (Amer- cytoskeleton ͉ tubulin family ͉ Prosthecobacter dejongeii ͉ sham Pharmacia) size-exclusion column (20 mM Tris⅐HCl͞1mM Verrucomicrobia ͉ polymerization azide͞1 mM EDTA, pH 7.5). Typically, 34 mg was obtained from 12 liters of culture. enomic sequencing of the bacterium Prosthecobacter dejongeii For bicistronic coexpression, genes btubA and btubB were cloned Ghas revealed the genes btubA and btubB that share higher by leaving the intergenic region intact and not adding any extra sequence similarity with eukaryotic ␣␤-tubulin than with the bac- residues. Expression conditions were as above. The proteins were terial tubulin-homologue FtsZ, raising questions about the evolu- purified by using Q Sepharose (buffer A: 20 mM Tris⅐HCl͞1mM tionary origins of these genes (1). Historically, bacteria were azide͞5 mM magnesium acetate, pH 8.5; buffer B: buffer A plus 1 thought not to contain a cytoskeleton, a view that has only recently M NaCl) and size-exclusion chromatography (S300 Sephacryl, been overturned (2, 3). Tubulin and the bacterial tubulin- Amersham Pharmacia; 20 mM Tris⅐HCl͞1 mM azide͞1mM homologue FtsZ are thought to have evolved from a common EDTA, pH 7.5). The separated BtubA (predicted pI, 5.2; molecular ancestor as is indicated by a similar structure of the monomer and weight, 51,265) and BtubB (predicted pI, 6.1; molecular weight, protofilament. In addition, they share a polymerization-dependent 46,417) proteins were kept apart or pooled. Twelve liters of culture GTPase activation mechanism (4). It has been argued, however, typically yielded 100 mg of protein. that the evolutionary distance between these proteins (and also Selenomethionine-substituted proteins were produced by the actin and the bacterial actin homologue MreB) seems extraordi- feed-back inhibition method (13), and proteins were purified as narily large considering that tubulin is amongst the most conserved described above with 5 mM DTT or ␤-mercaptoethanol. proteins in eukaryotes (5, 6). ␣␤-Tubulin differ from the single subunit bacterial protein FtsZ in that they form stable heterodimers Nucleotide Analysis. The guanine nucleotide of Btub proteins was with nonexchangeable GTP being trapped in the interface. ␣␤- extracted with perchloric acid, measured spectrophotometrically, Tubulin contain a C-terminal domain, absent in FtsZ, that forms and analyzed by HPLC (14). The protein concentration was deter- the outside of microtubules (7), structures that have never been mined from absorption spectra in 6 M guanidinium hydrochloride, reported for FtsZ. after subtraction of the nucleotide contribution, employing the There have been several reports showing microtubule-like extinction coefficients BtubA, 47,770 MϪ1⅐cmϪ1; BtubB, 38,780 structures in bacteria (8). The best examples are so-called MϪ1⅐cmϪ1; and BtubA-trx, 61,360 MϪ1⅐cmϪ1 at 280 nm. ‘‘epixenosomes,’’ bacteria growing as ectosymbionts on Euplo- tidium ciliates (9). These organisms belong to the Verrucomi- Pelleting Assays and Light Scattering. Each reaction contained 10 crobia, a phylum of bacteria of uncertain lineage (10). Recently, ␮M BtubA͞B (i.e., 10 ␮M BtubA and 10 ␮M BtubB protein in a two genes, called btubA and btubB, sharing Ϸ35% sequence state of association that depends on the solution conditions), 100 ␣ ␤ identity with - and -tubulin have been found in the free-living mM Pipes⅐NaOH (pH 7.0), 0 or 5 mM MgCl2,and0or1mM species Prosthecobacter dejongeii (1), which are also part of nucleotide (50 ␮l). After 30 min at room temperature, samples were Verrucomicrobia (11). These genes are cotranscribed from one centrifuged at 80,000 rpm for 20 min at 20°C in a TLA100 rotor operon, but on the basis of theoretical modeling, Prothecobacter (Beckman). The supernatants were removed and mixed with an tubulin BtubA and BtubB were predicted not to form het- equal volume of gel-loading buffer. The pellets were washed with erodimers (1). However, a recent study pelleting N-terminally His-tagged BtubA and -B showed that equimolar amounts of the two proteins assembled into protofilaments in a cooperative This paper was submitted directly (Track II) to the PNAS office. manner (12). Abbreviation: rmsd, rms deviation. Here we show that, contrary to eukaryotic tubulin, soluble and Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, functional BtubA and BtubB proteins can be expressed in www.pdb.org [PDB ID codes 2BTO (BtubA-trx) and 2BTQ (BtubAB)]. Escherichia coli and can be unfolded reversibly. No tag is ‡To whom correspondence should be addressed. E-mail: [email protected]. necessary. The purified proteins form heterodimers and assem- © 2005 by The National Academy of Sciences of the USA 9170–9175 ͉ PNAS ͉ June 28, 2005 ͉ vol. 102 ͉ no. 26 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0502859102 Table 1. Crystallographic data † ‡ § Crystal ␭, Å res., Å I͞␴I* Rm, % Multipl. Compl., % P. dejongeii BtubA-trx space group P321, a ϭ b ϭ 180.5 Å, c ϭ 84.2 Å PEAK (Se) 0.9793 3.1 19.7 (5.1) 0.076 (0.332) 8.3 99.9 (100.0) INFL (Se) 0.9796 3.1 20.3 (5.0) 0.072 (0.347) 8.4 99.9 (100.0) HREM (Se) 0.9184 3.1 19.0 (4.4) 0.078 (0.392) 8.2 99.9 (100.0) NATI 0.9340 2.5 12.5 (3.0) 0.083 (0.452) 4.9 99.9 (100.0) P. dejongeii BtubA͞B heterodimer space group P6522, a ϭ b ϭ 154.4 Å, c ϭ 256.3 Å PEAK (Se) 0.9790 4.0 11.4 (4.0) 0.157 (0.429) 8.1 99.7 (100.0) NATI 0.9340 3.2 12.8 (2.6) 0.090 (0.379) 3.6 99.0 (99.5) *Signal-to-noise ratio of merged intensities; values in parentheses are for the highest resolution shell. † Rm: ͚h͚i͉I(h, i) Ϫ I(h)͉͚͞h͚iI(h, i) were I(h, i) are symmetry-related in tensities and I(h) is the mean intensity of the reflection with unique index h. The highest resolution bin is in parentheses. ‡Multiplicity for unique (anomalously unique where appropriate) reflections. §Completeness for unique reflections; anomalous completeness is identical because inverse beam geometry was used. Values in parentheses are for the highest resolution bin. Correlation coefficients of anomalous differences at different wavelengths for the MAD experiment (cut at 4 Å): PEAK vs. INFL, 0.51; PEAK vs. HREM, 0.64; and INFL vs. HREM, 0.40. 50 ␮l of identical solution without protein and then solubilized in fuge with interference optics, using an An50Ti rotor at 50,000 rpm 100 ␮l of loading buffer. For electrophoresis, pellets and superna- at 20°C (buffer: 100 mM Pipes⅐NaOH͞200 mM KCl, pH 6.8). tants were loaded with a delay on the same SDS͞polyacrylamide Differential sedimentation coefficient distributions, c(s), were cal- gel. The presence or absence of filaments was verified by using culated with SEDFIT (15). Sedimentation equilibrium measurements electron microscopy as described below. were made as described in ref. 16. For static 90° light scattering, 400 ␮l of solution containing 10 ␮M BtubA͞B, 100 mM Pipes⅐NaOH (pH 6.8), 5 mM MgCl , 200 mM 2 Electron Microscopy. Fifty-microliter reactions were performed at KCl, and 0.5 mM GTP was measured in a PerkinElmer LS 50B room temperature for 30 min, containing 5 or 10 ␮M BtubA͞B, 100 luminescence spectrometer at 350 nm and 37°C (10-mm path ⅐ length). Measurements were taken every 2 s, and fresh GTP was mM Pipes NaOH (pH 6.8), 5 mM MgCl2, 0 or 200 mM KCl, and added after the signal approached baseline. 0.5–1 mM GTP. Ten microliters was then transferred to glow- discharged carbon-coated copper electron microscopic grids and Analytical Ultracentrifugation. Sedimentation velocity experiments negatively stained with 2% (wt͞vol) uranyl acetate. Images were were conducted in a Beckman Optima XL-I analytical ultracentri- taken in an 80 keV Phillips 208 electron microscope on film and Table 2.
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