Filament structure of bacterial tubulin homologue TubZ

Abstract

Low copy number plasmids often depend on accurate partitioning systems for their continued survival. Generally, such systems consist of a centromere-like region of DNA, a DNA-binding adaptor, and a polymerizing cytomotive filament. Together these components drive newly replicated plasmids to opposite ends of the dividing cell. The Bacillus thuringiensis plasmid pBToxis relies on a filament of the tubulin/FtsZ-like protein TubZ for its segregation. By combining crystallography and electron microscopy, we have determined the structure of this filament. We explain how GTP hydrolysis weakens the subunit-subunit contact and also shed light on the partitioning of the plasmid-adaptor complex. The double helical superstructure of TubZ filaments is unusual for tubulin-like proteins. Filaments of ParM, the actin-like partitioning protein, are also double helical. We suggest that convergent evolution shapes these different types of cytomotive filaments toward a general mechanism for plasmid separation.

Fig. 1.

TubZ is a member of the tubulin/FtsZ family. (A) SDS-PAGE of purified TubZ proteins. TubZ: full-length TubZ with a C-terminal hexahistidine tag; TubZ1-421: TubZ (L2V in this protein only) truncated C-terminally at residue 421 and untagged; Trx-TubZ: N-terminal thioredoxin fusion to full-length TubZ with a C-terminal hexahistidine tag; TubZ-GFP: hexahistidine tagged C-terminal fusion of TubZ to GFP molecular mass in kDa. (B) Cartoon representation of TubZ1-421•GTPγS; chain D. Secondary structural elements are labeled according to (21). The N-terminal extension is pink, the GTPase domain green, helix 7 yellow, the C-terminal domain purple, and the C-terminal tail blue. (C) Stereoscopic Cα traces of bovine tubulin (orange) [Protein Data Bank (PDB) ID code 1JFF), M. jannaschii FtsZ (blue) (PDB ID code 1W5A), and TubZ1-421 (pink) superimposed using only the GTPase domain. The β-sheets, H7, and nucleotide are highlighted in the same color. The lower pair have been rotated by 90°.

Fig. 2.

Structure of the TubZ protofilament. (A) Protofilaments from the TubZ1-421•GTPγS and GDP crystal forms. Both crystal forms contain continuous protofilaments with 14 and 12 subunits per complete turn, respectively. The lengths of the unit cell and asymmetric unit are indicated. The color scheme is identical to Fig. 1B, excluding two gray monomers corresponding to the first subunits of the next unit cell in the GDP form. (B) Cartoons of the protofilament dimers of bovine tubulin (PDB ID code 1JFF), M. jannaschii FtsZ (PDB ID code 1W5A), and TubZ (this study, TubZ1-421•GTPγS chains D and E). In each case, the lower monomer was superimposed by using the GTPase domain alone. All subunits are colored as in Fig. 1B. (C) Stereoscopic representations of the TubZ active site in Mg²⁺•GTPγS, GDP, and apostates. Interacting residues and regions of backbone are shown as skeleton and Cα traces, respectively. The most representative interfaces have been selected for Mg²⁺•GTPγS and GDP states for clarity—see Fig. S5 for a superposition of all interfaces. The GTPase domain is green, the C-terminal domain purple, and the nucleotide blue.

Fig. 3.

TubZ forms filaments both in vitro and when overexpressed in E. coli. (A–D) Negatively stained electron micrographs of TubZ filaments assembled in vitro, with magnified insets. Two double filaments join to form a quadruple in A, marked with *, whereas B represents a quadruple and C and D double filaments of TubZ. Gyre lines are marked in white. (E and F) Pairs of straightened and filtered TubZ filaments. The filament in E is double and in F quadruple. The crossovers are annotated alongside. (G) A rotary shadowed TubZ filament as viewed from above. Right-handed gyres are indicated. (H) Slice through a tomogram of plunge-frozen E. coli cells overexpressing full-length TubZ. Filaments are visible as a bundle, magnified in the inset. The filament lateral spacing at this angle is shown. (I) Fourier transform of the bundles of TubZ filaments in the tomograms. The lateral and significant longitudinal spacings, including the ∼35 nm cross-over repeat, are annotated. (J) A 50 nm cryosection through plunge-frozen E. coli cells expressing TubZ. A cross-section through a bundle of filaments is visible, looking along the filaments. Lateral spacings in two different directions are annotated and suggest double helices.

Fig. 4.

Structure of TubZ double helical filaments. (A) Averaged Fourier transform of straightened TubZ filaments showing the helical lattice and its latitudinal and longitudinal spacings (nanometers). The reciprocal unit cell is shown in red dotted lines. (B) Surface map (Upper) and fit (Lower) of the GTPγS asymmetric unit into each of the TubZ filament maps. The tilt of the subunits and the reconstructed density is indicated. The protrusion adjacent the C terminus is marked (*), as is the extra density adjacent the N terminus and the protofilament interface (†). Native TubZ is blue, Trx-TubZ purple, and TubZ-GFP green. Note that for this fit and figure the crystal structure of the protofilament has not been adjusted in any way. The writhe of the crystal structure protofilament indicates the correct orientation, as do the positions of the extra densities generated by thioredoxin and GFP. (C) Cartoon and surface representation of the final, adjusted, and fitted TubZ model (42 Å rise and 21° twist per subunit). A double helical filament restricts growth to one dimension, and it is presumed that TubZ represents a special adaptation of the tubulin/ftsZ family to yield filaments that superficially resemble F-actin and ParM through convergent evolution.