Crystal Structure Determination of FtsZ fromMethanococcus jannaschii

FtsZ is the polymer-forming protein of bacterial cell division. It is part of a ring in the middle of the dividing cell that is required for constriction of cell membrane and cell envelope to yield two daughter cells. FtsZ is a GTPase and is the only bacterial protein showing significant sequence homology to the eukaryotic tubulins. FtsZ can polymerize into tubes, sheets, and ringsin vitroand is ubiquitous in eubacteria and archaea. Full-length FtsZ1 fromMethanococcus jannaschiihas been over expressed inEscherichia coli,employing the hyperthermophilic properties of the protein. Crystals grown from PEG400 and ethanol belong to spacegroup I2₁3 witha=b=c= 159.1 Å. Isomorphous replacement using one Hg derivative yielded a interpretable electron density map at 4 Å resolution. The structure for residues 23–356 and one GDP has been refined to anR_freeof 0.28 (R_f = 0.20) at 2.8 Å resolution. FtsZ consists of two domains with a connecting core helix. The N-terminal domain and the core helix contain all residues involved in nucleotide binding and resemble the fold of dinucleotide-binding proteins. The structures of tubulin and FtsZ show striking similarity; together with the functional similarities, this provides a strong indication that FtsZ is a true homolog of tubulin.     

Conformational Flexibility of A Highly Conserved Helix Controls Cryptic Pocket Formation in FtsZ

Mycobacterium tuberculosis is responsible for more than 1.6 million deaths each year. One potential antibacterial target in M. tuberculosis is filamentous temperature sensitive protein Z (FtsZ), which is the bacterial homologue of mammalian tubulin, a validated cancer target. M. tuberculosis FtsZ function is essential, with its inhibition leading to arrest of cell division, elongation of the bacterial cell and eventual cell death. However, the development of potent inhibitors against FtsZ has been a challenge owing to the lack of structural information. Here we report multiple crystal structures of M. tuberculosis FtsZ in complex with a coumarin analogue. The 4-hydroxycoumarin binds exclusively to two novel cryptic pockets in nucleotide-free FtsZ, but not to the binary FtsZ-GTP or GDP complexes. Our findings provide a detailed understanding of the molecular basis for cryptic pocket formation, controlled by the conformational flexibility of the H7 helix, and thus reveal an important structural and mechanistic rationale for coumarin antibacterial activity.     

原核生物微管蛋白家族研究进展

自从1992年确定细菌分裂的关键蛋白Fts Z属于微管蛋白家族以来,越来越多的细菌细胞骨架蛋白被发现。原核生物中的微管同源蛋白主要有Fts Z、Cet Z、Tub Z和Btub A/B等。它们与微管蛋白具有相似的三级结构,可以结合鸟嘌呤-5′-三磷酸(Guanosine triphosphate,GTP)自聚合成不同的线状原丝纤维结构:单线状原丝纤维、双螺旋纤维结构或聚集成束状结构,在细菌细胞分裂、维持细胞形态、质粒分离等诸多重要生理功能中起着重要作用。     

Tethering the Z ring to the membrane through a conserved membrane targeting sequence in FtsA

The cytokinetic Z ring is required for bacterial cell division. It consists of polymers of FtsZ, the bacterial ancestor of eukaryotic tubulin, linked to the cytoplasmic membrane. Formation of a Z ring in Escherichia coli occurs as long as one of two proteins, ZipA or FtsA, is present. Both of these proteins bind FtsZ suggesting that they might function to tether FtsZ filaments to the membrane. Although ZipA has a transmembrane domain and therefore can function as a membrane anchor, interaction of FtsA with the membrane has not been explored. In this study we demonstrate that FtsA, which is structurally related to eukaryotic actin, has a conserved C-terminal amphipathic helix that is essential for FtsA function. It is required to target FtsA to the membrane and subsequently to the Z ring. As FtsA is much more widely conserved in bacteria than ZipA, it is likely that FtsA serves as the principal membrane anchor for the Z ring.     

Tubulin-like protofilaments in Ca2+-induced FtsZ sheets

The 40 kDa protein FtsZ is a major septum-forming component of bacterial cell division. Early during cytokinesis at midcell, FtsZ forms a cytokinetic ring that constricts as septation progresses. FtsZ has a high propensity to polymerize in vitro into various structures, including sheets and filaments, in a GTP-dependent manner. Together with limited sequence homology, the occurrence of the tubulin signature motif in FtsZ and a similar three-dimensional structure, this leads to the conclusion that FtsZ is the bacterial tubulin homologue. We have polymerized FtsZ1 from Methanococcus jannaschii in the presence of millimolar concentrations of Ca2+ ions to produce two-dimensional crystals of plane group P2221. Most of the protein precipitates and forms filaments approximately 23.0 nm in diameter. A three-dimensional reconstruction of tilted micrographs of FtsZ sheets in negative stain between 0 and 60 degrees shows protofilaments of FtsZ running along the sheet axis. Pairs of parallel FtsZ protofilaments associate in an antiparallel fashion to form a two-dimensional sheet. The antiparallel arrangement is believed to generate flat sheets instead of the curved filaments seen in other FtsZ polymers. Together with the subunit spacing along the protofilament axis, a fitting of the FtsZ crystal structure into the reconstruction suggests a protofilamant structure very similar to that of tubulin protofilaments.     

Crystal structure of the cell division protein FtsA from Thermotoga maritima

Bacterial cell division requires formation of a septal ring. A key step in septum formation is polymerization of FtsZ. FtsA directly interacts with FtsZ and probably targets other proteins to the septum. We have solved the crystal structure of FtsA from Thermotoga maritima in the apo and ATP-bound form. FtsA consists of two domains with the nucleotide-binding site in the interdomain cleft. Both domains have a common core that is also found in the actin family of proteins. Structurally, FtsA is most homologous to actin and heat-shock cognate protein (Hsc70). An important difference between FtsA and the actin family of proteins is the insertion of a subdomain in FtsA. Movement of this subdomain partially encloses a groove, which could bind the C-terminus of FtsZ. FtsZ is the bacterial homologue of tubulin, and the FtsZ ring is functionally similar to the contractile ring in dividing eukaryotic cells. Elucidation of the crystal structure of FtsA shows that another bacterial protein involved in cytokinesis is structurally related to a eukaryotic cytoskeletal protein involved in cytokinesis.     

Bacterial division: the fellowship of the ring

The Z ring, composed of the tubulin homolog FtsZ, is essential for bacterial cell division. Recently a new protein, ZapA, has been discovered that localizes to the Z ring and stabilizes it, probably by promoting the bundling of FtsZ protofilaments.     

Activation of cell division protein FtsZ. Control of switch loop T3 conformation by the nucleotide gamma-phosphate

The effect of bound nucleotide on the conformation of cell division protein FtsZ from Methanococcus jannaschii has been investigated using molecular dynamics and site-directed mutagenesis. The molecular dynamics indicate that the gamma-phosphate of GTP induces a conformational perturbation in loop T3 (Gly88-Gly99 segment), in a position structurally equivalent to switch II of Ha-ras-p21. In the simulated GTP-bound state, loop T3 is pulled by the gamma-phosphate into a more compact conformation than with GDP, related to that observed in the homologous proteins alpha- and beta-tubulin. The existence of a nucleotide-induced structural change in loop T3 has been confirmed by mutating Thr92 into Trp (T92W-W319Y FtsZ). This tryptophan (12 A away from gamma-phosphate) shows large differences in fluorescence emission, depending on which nucleotide is bound to FtsZ monomers. Loop T3 is located at a side of the contact interface between two FtsZ monomers in the current model of FtsZ filament. Such a structural change may bend the GDP filament upon hydrolysis by pushing against helix H8 of next monomer, thus, generating force on the membrane during cell division. A related curvature mechanism may operate in tubulin activation.     

The prokaryotic cytoskeleton: a putative target for inhibitors and antibiotics?

In the recent decade, our view on the organization of the bacterial cell has been revolutionized by the identification of cytoskeletal elements. Most bacterial species have structural homologs of actin and tubulin that assemble into dynamic, filamentous structures at precisely defined sub-cellular locations. The essential cell division protein FtsZ forms a dynamic ring at mid-cell and is similar in its structure to tubulin. Proteins of the MreB family, which are structural homologs of actin, assemble into helical or straight filaments in the bacterial cytoplasm. As in eukaryotic cells, the bacterial cytoskeleton drives essential cellular processes such as cell division, cell wall growth, DNA movement, protein targeting, and alignment of organelles. Different high-throughput assays have been developed to search for inhibitors of components of the bacterial cytoskeleton. Cell-based assays for the detection of cell division inhibitors as well as FtsZ GTPase assays led to the identification of several compounds that inhibit the polymerization of FtsZ, by this blocking bacterial cell division. Such inhibitors might not only be valuable tools for basic research, but might also lead to novel therapeutic agents against pathogenic bacteria. For example, the polyphenol dichamanetin, the 2-alkoxycarbonylaminopyridine SRI-3072, and the benzophenanthridine alkaloid sanguinarine inhibit the GTPase activity of FtsZ and exhibit antimicrobial activity.     

Human S100B protein interacts with the Escherichia coli division protein FtsZ in a calcium-sensitive manner

S100B is a small, dimeric EF-hand calcium-binding protein abundant in vertebrates. Upon calcium binding, S100B undergoes a conformational change allowing it to interact with a variety of target proteins, including the cytoskeletal proteins tubulin and glial fibrillary acidic protein. In both cases, S100B promotes the in vitro disassembly of these proteins in a calcium-sensitive manner. Despite this, there is little in vivo evidence for the interaction of proteins such as tubulin with S100B. To probe these interactions, we studied the expression of human S100B in Escherichia coli and its interaction with the prokaryotic ancestor of tubulin, FtsZ, the major protein involved in bacterial division. Expression of S100B protein in E. coli results in little change in FtsZ protein levels, causes a filamenting bacterial phenotype characteristic of FtsZ inhibition, and leads to missed rounds of cell division. Further, S100B localizes to positions similar to those of FtsZ in bacterial filaments: the small foci at the poles, the mid-cell positions, and between the nucleoids at regular intervals. Calcium-dependent physical interaction between S100B and FtsZ was demonstrated in vitro by affinity chromatography, and this interaction was severely inhibited by the competitor peptide TRTK-12. Together these results indicate that S100B interacts with the tubulin homologue FtsZ in vivo, modulating its activity in bacterial cell division. This approach will present an important step for the study of S100 protein interactions in vivo.     

Folding, stability and polymerization properties of FtsZ chimeras with inserted tubulin loops involved in the interaction with the cytosolic chaperonin CCT and in microtubule formation

To attain its native conformation, the cytoskeletal protein tubulin needs the concourse of several molecular chaperones, among others the cytosolic chaperonin CCT. It has been previously described that denatured tubulin interacts with CCT in a quasi-folded conformation using several loops located throughout its sequence. These loops are also involved in microtubule formation and are absent in its prokaryote homologue FtsZ, which in vitro folds by itself and does not interact with CCT. Several FtsZ/tubulin chimeric proteins were generated by inserting consecutively one, two or three of the CCT-binding domains of tubulin into the corresponding sequence of FtsZ from Methanococccus jannaschii. The insertion of any of the CCT-binding loops generates in the FtsZ/tubulin chimeras the ability to interact with CCT. The accumulation of CCT-binding loops induces in the FtsZ/tubulin chimeras unfolding and refolding properties that are more similar to tubulin than to its prokaryote counterpart. Finally, the insertion of some of these loops generates in the FtsZ/tubulin chimeras more complex polymeric structures than those found for FtsZ. These results reinforce the notion that CCT has coevolved with tubulin to deal with the folding problems encountered by the eukaryotic protein with the appearance of the new sequences involved in microtubule formation.     

Structural insights into FtsZ protofilament formation

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.     

Immediate GTP hydrolysis upon FtsZ polymerization

To understand the polymerization dynamics of FtsZ, a bacterial cell division protein similar to tubulin, insight is required into the nature of the nucleotide bound to the polymerized protein. In a previous study, we showed that the FtsZ polymers contain mostly GDP. A recent study challenged this result, suggesting that the polymerized FtsZ is in a GTP-bound state. Here, we show that, when radiolabelled [gamma-32P]-GTP is used to polymerize FtsZ, GTP is hydrolysed instantaneously. The FtsZ polymer contains both GDP and the radiolabelled inorganic phosphate.     

New data on structures formed by the FtsZ protein during the cell division process in <Emphasis Type="Italic">Escherichia coli</Emphasis> obtained using the localization microscopy method

The FtsZ protein, a bacterial tubulin homolog, is one of the key proteins in bacterial cell division, forming a contractile Z-ring in the middle of the dividing cell. In the present study, immunofluorescence staining, in combination with the localization microscopy method, was used for visualization of the structures formed by unlabelled FtsZ in Escherrichia coli cells. The techniques employed allowed reconstruction of the multistep mechanism of formation of FtsZ structures during the cytokinesis process. New data were obtained confirming the hypothesis that FtsZ is a helixlike structure that constricts during the division, producing constriction between the daughter cells.     

Structural Change in FtsZ Induced by Intermolecular Interactions between Bound GTP and the T7 Loop

FtsZ is a prokaryotic homolog of tubulin and is a key molecule in bacterial cell division. FtsZ with bound GTP polymerizes into tubulin-like protofilaments. Upon polymerization, the T7 loop of one subunit is inserted into the nucleotide-binding pocket of the second subunit, which results in GTP hydrolysis. Thus, the T7 loop is important for both polymerization and hydrolysis in the tubulin/FtsZ family. Although x-ray crystallography revealed both straight and curved conformations of tubulin, only a curved structure was known for FtsZ. Recently, however, FtsZ from Staphylococcus aureus has been shown to have a very different conformation from the canonical FtsZ structure. The present study was performed to investigate the structure of FtsZ from Staphylococcus aureus by mutagenesis experiments; the effects of amino acid changes in the T7 loop on the structure as well as on GTPase activity were studied. These analyses indicated that FtsZ changes its conformation suitable for polymerization and GTP hydrolysis by movement between N- and C-subdomains via intermolecular interactions between bound nucleotide and residues in the T7 loop.     

Recent advances in the discovery and development of antibacterial agents targeting the cell-division protein FtsZ

With the emergence of multidrug-resistant bacterial strains, there is a dire need for new drug targets for antibacterial drug discovery and development. Filamentous temperature sensitive protein Z (FtsZ), is a GTP-dependent prokaryotic cell division protein, sharing less than 10% sequence identity with the eukaryotic cell division protein, tubulin. FtsZ forms a dynamic Z-ring in the middle of the cell, leading to septation and subsequent cell division. Inhibition of the Z-ring blocks cell division, thus making FtsZ a highly attractive target. Various groups have been working on natural products and synthetic small molecules as inhibitors of FtsZ. This review summarizes the recent advances in the development of FtsZ inhibitors, focusing on those in the last 5 years, but also includes significant findings in previous years.     

FtsZ and the division of bacterial cell

In this review we have tried to describe proteins and supermolecular structures which take part in the division of bacterial cell. The principal cell division protein of the most of prokaryotes is FtsZ, a homologue of eukaryotic tubulin. FtsZ just as tubulin is capable to bind and hydrolyze GTP. The division of bacterial cell begins with forming of so called divisome. The basis of such divisome is a contractile ring (Z ring); the ring encircles the cell about midcell. Z ring consists of a bundle of laterally bound protofilaments, which have been formed as a result of FtsZ polymerization. Z ring is rigidly bounded to cytozolic side of inner membrane with participation of FtsA, ZipA, FtsW and many other cell division proteins of divisome. The ring directs the process of cytokinesis transmitting power of constriction to membrane. Primary structures of members of the family of prokaryotic FtsZs differ from eukaryotic tubulines significantly except the region, where the site of GTP binding is placed. There is high degree of homology between structures of these proteins in the region. FtsZ is one of the most conservative proteins in prokaryotes, but ftsZ genes have not been found in completely sequenced genomes of several species of microorganisms. There are 2 species of mycoplasmas (Ureaplasma parvum and Mycoplasma mobile), Prostecobacter dejongeii, 10 species of chlamydia and 5 species of archaea among them. So these organisms divide without FtsZ. There are many open reading frames which encode proteins with unknown functions in genomes of U. parvum and M. mobile. The comparison of primary structures of these hypothetical proteins with structures of cell division proteins did not allow researchers to find similar proteins among them. We suppose that the process of cell division of these organisms should recruit proteins with function similar to FtsZ and having homologous with FtsZ or other cell division proteins spatial structures.     

Magnesium-induced linear self-association of the FtsZ bacterial cell division protein monomer. The primary steps for FtsZ assembly

The bacterial cell division protein FtsZ from Escherichia coli has been purified with a new calcium precipitation method. The protein contains one GDP and one Mg(2+) bound, it shows GTPase activity, and requires GTP and Mg(2+) to polymerize into long thin filaments at pH 6.5. FtsZ, with moderate ionic strength and low Mg(2+) concentrations, at pH 7.5, is a compact and globular monomer. Mg(2+) induces FtsZ self-association into oligomers, which has been studied by sedimentation equilibrium over a wide range of Mg(2+) and FtsZ concentrations. The oligomer formation mechanism is best described as an indefinite self-association, with binding of an additional Mg(2+) for each FtsZ monomer added to the growing oligomer, and a slight gradual decrease of the affinity of addition of a protomer with increasing oligomer size. The sedimentation velocity of FtsZ oligomer populations is compatible with a linear single-stranded arrangement of FtsZ monomers and a spacing of 4 nm. It is proposed that these FtsZ oligomers and the polymers formed under assembly conditions share a similar axial interaction between monomers (like in the case of tubulin, the eukaryotic homolog of FtsZ). Similar mechanisms may apply to FtsZ assembly in vivo, but additional factors, such as macromolecular crowding, nucleoid occlusion, or specific interactions with other cellular components active in septation have to be invoked to explain FtsZ assembly into a division ring.     

FtsZ and bacterial cell division

In this review we describe proteins and supermolecular structures which take part in the division of bacterial cells. FtsZ, a eukaryotic tubulin homolog is a key cell division protein in most prokaryotes. FtsZ, as well as tubulin, is capable of binding and hydrolyzing GTP. The division of a bacterial cell begins with the forming of a so-called divisome. The basis of such a divisome is a contractile ring (Z ring) which encircles the cell about midcell. The Z-ring consists of a bundle of laterally bound protofilaments formed in result of FtsZ polymerization. Z-ring is rigidly bounded to the cytosolic side of the inner membrane with the participation of FtsA, ZipA, FtsW and many other divisome cell division proteins. The ring directs the process of cytokinesis transmitting constriction power to the membrane. The primary structures of the prokaryotic FtsZ family members significantly differ from eukaryotic tubulins except for the sites of GTP binding. There is a high degree of structural homology between these proteins in the region. FtsZ is one of the most conserved proteins in prokaryotes. However, ftsZ genes have not been found in several species of microorganisms with completely sequenced genomes. They include two species of mycoplasmas (Ureaplasma parvum and Mycoplasma mobile), Prostecobacter dejongeii, 10 species of chlamydia and 5 species of archaea. Consequently, these organisms divide without FtsZ participation. The genomes of U. parvum and M. mobile have many open reading frames which encode proteins with unknown functions. A comparison of the primary structures of these hypothetical proteins did not identify any known cell division proteins. We hypothesize that the process of cell division in these organisms should involve proteins similar to FtsZ in function and homologous to FtsZ or other cell division proteins in structure.     

Structure of Mycobacterium tuberculosis FtsZ reveals unexpected, G protein-like conformational switches

We report three crystal structures of the Mycobacterium tuberculosis cell division protein FtsZ, as the citrate, GDP, and GTPgammaS complexes, determined at 1.89, 2.60, and 2.08A resolution. MtbFtsZ crystallized as a tight, laterally oriented dimer distinct from the longitudinal polymer observed for alphabeta-tubulin. Mutational data on Escherichia coli FtsZ suggest that this dimer interface is important for proper protofilament and "Z-ring" assembly and function. An alpha-to-beta secondary structure conformational switch at the dimer interface is spatially analogous to, and has many of the hallmarks of, the Switch I conformational changes exhibited by G-proteins upon activation. The presence of a gamma-phosphate in the FtsZ active site modulates the conformation of the "tubulin" loop T3 (spatially analogous to the G-protein Switch II); T3 switching upon gamma-phosphate ligation is directly coupled to the alpha-to-beta switch by steric overlap. The dual conformational switches observed here for the first time in an FtsZ link GTP binding and hydrolysis to FtsZ (and tubulin) lateral assembly and Z-ring contraction, and they are suggestive of an underappreciated functional analogy between FtsZ, tubulin and G-proteins.