It is yet to be investigated whether both ligands target structurally related assembly switches.

Essential cell division protein FtsZ is an assembling GTPase which directs the cytokinetic ring formation in dividing bacterial cells. FtsZ shares the structural fold of eukaryotic tubulin and assembles forming tubulin-like protofilaments, but does not form microtubules. Two puzzling problems in FtsZ assembly are the nature of protofilament association and a possible mechanism for nucleated self-assembly of single-stranded protofilaments above a critical FtsZ concentration. We assembled two-dimensional arrays of FtsZ on carbon supports, studied linear polymers of FtsZ with cryo-electron microscopy of vitrified unsupported solutions, and formulated possible polymerization models. Nucleated self-assembly of FtsZ from Escherichia coli with GTP and magnesium produces flexible filaments 4-6 nm-wide, only compatible with a single protofilament. This agrees with previous scanning transmission electron microscopy results and is supported by recent cryo-electron tomography studies of two bacterial cells. Observations of double-stranded FtsZ filaments in negative stain may come from protofilament accretion on the carbon support. Preferential protofilament cyclization does not apply to FtsZ assembly. The apparently cooperative polymerization of a single protofilament with identical intermonomer contacts is explained by the switching of one inactive monomer into the active structure preceding association of the next, creating a dimer nucleus. FtsZ behaves as a cooperative linear assembly machine.

We have studied the assembly and GTPase of purified FtsZ from the hyperthermophilic archaeon Methanococcus jannaschii, a structural homolog of eukaryotic tubulin, employing wild-type FtsZ, FtsZ-His 6 (histidinetagged FtsZ), and the new mutants FtsZ-W319Y and FtsZ-W319Y-His 6 , with light scattering, nucleotide analyses, electron microscopy, and image processing methods. This has revealed novel properties of FtsZ. The GTPase of archaeal FtsZ polymers is suppressed in Na ؉ -containing buffer, generating stabilized structures that require GDP addition for disassembly. FtsZ assembly is polymorphic. Archaeal FtsZ(wt) assembles into associated and isolated filaments made of two parallel protofilaments with a 43 Å longitudinal spacing between monomers, and this structure is also observed in bacterial FtsZ from Escherichia coli. The His 6 extension facilitates the artificial formation of helical tubes and sheets. FtsZ-W319Y-His 6 is an inactivated GTPase whose assembly remains regulated by GTP and Mg 2؉. It forms twodimensional crystals made of symmetrical pairs of tubulin-like protofilaments, which associate in an antiparallel array (similarly to the known Ca 2؉ -induced sheets of FtsZ-His 6 ). In contrast to the lateral interactions of microtubule protofilaments, we propose that the primary assembly product of FtsZ is the double-stranded filament, one or several of which might form the dynamic Z ring during prokaryotic cell division.Bacterial cells place at their division site a Z-ring that assembles from FtsZ at the cytosolic side of the plasma membrane and recruits the other protein components of the septation machinery. The positioning of the Z-ring is in turn determined by the self-organizing Min system (1-3). Archaeal FtsZ from Methanococcus jannaschii and eukaryotic tubulin share the same structural framework and constitute a distinct family of protofilament-forming GTPases (4, 5). The structures of bacterial FtsZs are probably similar to the archaeal protein, based on FtsZ sequence conservation. The Escherichia coli Z-ring is very dynamic, with turnover in seconds, similar to or faster than a typical mitotic spindle (6). Although the position of the Z ring was visualized by immunoelectron microscopy (7), its structure is unknown. A number of studies have shown that purified bacterial FtsZ can assemble with GTP and divalent cations forming polymers that hydrolyze the nucleotide ␤-␥ phosphate bond and disassemble upon GTP consumption (8 -13), although the mechanism linking GTP hydrolysis and exchange to the polymer dynamics (14) remains controversial (15-17). FtsZ polymers have the characteristic tubulin ϳ40 Å axial spacing between consecutive monomers (18). At pH 6 and in the presence of high Ca 2ϩ and GTP concentrations, a small proportion of archaeal FtsZ from M. jannaschii formed crystalline sheets made of symmetric protofilament pairs associated in an antiparallel arrangement, whereas most of the protein precipitated into aggregates of helical cable-like tubes. Manual fitting of the structure of GDP-bound Ft...