The prokaryotic tubulin homolog FtsZ polymerizes into a ring structure essential for bacterial cell division. We have used refolded FtsZ to crystallize a tubulin-like protofilament. The N- and C-terminal domains of two consecutive subunits in the filament assemble to form the GTPase site, with the C-terminal domain providing water-polarizing residues. A domain-swapped structure of FtsZ and biochemical data on purified N- and C-terminal domains show that they are independent. This leads to a model of how FtsZ and tubulin polymerization evolved by fusing two domains. In polymerized tubulin, the nucleotide-binding pocket is occluded, which leads to nucleotide exchange being the rate-limiting step and to dynamic instability. In our FtsZ filament structure the nucleotide is exchangeable, explaining why, in this filament, nucleotide hydrolysis is the rate-limiting step during FtsZ polymerization. Furthermore, crystal structures of FtsZ in different nucleotide states reveal notably few differences.

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DivIVA is a conserved protein in Gram-positive bacteria that localizes at the poles and division sites, presumably through direct sensing of membrane curvature. DivIVA functions as a scaffold and is vital for septum site selection during vegetative growth and chromosome anchoring during sporulation. DivIVA deletion causes filamentous growth in Bacillus subtilis, whereas overexpression causes hyphal branching in Streptomyces coelicolor. We have determined the crystal structure of the N-terminal (Nt) domain of DivIVA, and show that it forms a parallel coiledcoil. It is capped with two unique crossed and intertwined loops, exposing hydrophobic and positively charged residues that we show here are essential for membrane binding. An intragenic suppressor introducing a positive charge restores membrane binding after mutating the hydrophobic residues. We propose that the hydrophobic residues insert into the membrane and that the positively charged residues bind to the membrane surface. A lowresolution crystal structure of the C-terminal (Ct) domain displays a curved tetramer made from two parallel coiledcoils. The Nt and Ct parts were then merged into a model of the full length, 30 nm long DivIVA protein.

Bacterial cell division in many organisms involves a constricting cytokinetic ring that is orchestrated by the tubulin-like protein FtsZ. FtsZ forms dynamic filaments close to the membrane at the site of division that have recently been shown to treadmill around the division ring, guiding septal wall synthesis. Here, using X-ray crystallography of Staphylococcus aureus FtsZ (SaFtsZ), we reveal how an FtsZ can adopt two functionally distinct conformations, open and closed. The open form is found in SaFtsZ filaments formed in crystals and also in soluble filaments of Escherichia coli FtsZ as deduced by electron cryomicroscopy. The closed form is found within several crystal forms of two nonpolymerizing SaFtsZ mutants and corresponds to many previous FtsZ structures from other organisms. We argue that FtsZ’s conformational switch is polymerization-associated, driven by the formation of the longitudinal intersubunit interfaces along the filament. We show that such a switch provides explanations for both how treadmilling may occur within a single-stranded filament and why filament assembly is cooperative.

␣␤-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 BtubA͞B form tubulin-like protofilaments. Presumably, BtubA͞B 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.cytoskeleton ͉ tubulin family ͉ Prosthecobacter dejongeii ͉ Verrucomicrobia ͉ polymerization G enomic sequencing of the bacterium Prosthecobacter dejongeii has revealed the genes btubA and btubB that share higher sequence similarity with eukaryotic ␣␤-tubulin than with the bacterial tubulin-homologue FtsZ, raising questions about the evolutionary origins of these genes (1). Historically, bacteria were thought not to contain a cytoskeleton, a view that has only recently been overturned (2, 3). Tubulin and the bacterial tubulinhomologue FtsZ are thought to have evolved from a common ancestor as is indicated by a similar structure of the monomer and protofilament. In addition, they share a polymerization-dependent GTPase activation mechanism (4). It has been argued, however, that the evolutionary distance between these proteins (and also actin and the bacterial actin homologue MreB) seems extraordinarily large considering that tubulin is amongst the most conserved proteins in eukaryotes (5, 6). ␣␤-Tubulin differ from the single subunit bacterial protein FtsZ in that they form stable heterodimers with nonexchangeable GTP being trapped in the interface. ␣␤-Tubulin contain a C-terminal domain, absent in FtsZ, that forms the outside of microtubules (7), structures that have never been reported for FtsZ.There have been several reports showing microtubule-like structures in bacteria (8). The best examples are so-called ''epixenosomes,'' bacteria growing as ectosymbionts on Euplotidium ciliates (9). These organisms belong to the Verrucomicrobia, a phylum of bacteria of uncertain lineage (10). Recently, two genes, called btubA and btubB, sharing Ϸ35% sequence identity with ␣-and ␤-tubulin have been found in the free-living species Prosthecobacter dejongeii (1), which are also part of Verrucomicrobia (11). These genes are cotranscribed from one operon, but on the basis of theoretical modeling, Prothecobacter tubulin BtubA and BtubB were predicted not to form heterodimers (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 manner (12).Here we show that, contrary to eukaryotic tubulin, soluble and functiona...