Linda A. Amos

Crystal structure of the bacterial cell-division protein FtsZ.

Bacterial cell division ends with septation, the constriction of the cell wall and cell membranes that leads to the formation of two daughter cells,. During septation, FtsZ, a protein of relative molecular mass 40,000 which is ubiquitous in eubacteria and is also found in archaea and chloroplasts, localizes early at the division site to form a ring-shaped septum. This septum is required for the mechanochemical process of membrane constriction. FtsZ is a GTPase, with weak sequence homology to tubulins. The nature of FtsZ polymers in vivo is unknown, but FtsZ can form tubules, sheets and minirings in vitro,. Here we report the crystal structure at 2.8 Å resolution of recombinant FtsZ from the hyperthermophilic methanogen Methanococcus jannaschii. FtsZ has two domains, one of which is a GTPase domain with a fold related to one found in the proteins p21ras and elongation factor EF-Tu. The carboxy-terminal domain, whose function is unknown, is a four-stranded β-sheet tilted by 90° against the β-sheet of the GTPase domain. The two domains are arranged around a central helix. GDP binding is different from that typically found in GTPases and involves four phosphate-binding loops and a sugar-binding loop in the first domain, with guanine being recognized by residues in the central connecting helix. The three-dimensional structure of FtsZ is similar to the structure of α- and β-tubulin.

Prokaryotic origin of the actin cytoskeleton.

It was thought until recently that bacteria lack the actin or tubulin filament networks that organize eukaryotic cytoplasm. However, we show here that the bacterial MreB protein assembles into filaments with a subunit repeat similar to that of F-actin—the physiological polymer of eukaryotic actin. By elucidating the MreB crystal structure we demonstrate that MreB and actin are very similar in three dimensions. Moreover, the crystals contain protofilaments, allowing visualization of actin-like strands at atomic resolution. The structure of the MreB protofilament is in remarkably good agreement with the model for F-actin, showing that the proteins assemble in identical orientations. The actin-like properties of MreB explain the finding that MreB forms large fibrous spirals under the cell membrane of rod-shaped cells, where they are involved in cell-shape determination. Thus, prokaryotes are now known to possess homologues both of tubulin, namely FtsZ, and of actin.

Tubulin and FtsZ form a distinct family of GTPases

Tubulin and FtsZ share a common fold of two domains connected by a central helix. Structure-based sequence alignment shows that common residues localize in the nucleotide-binding site and a region that interacts with the nucleotide of the next tubulin subunit in the protofilament, suggesting that tubulin and FtsZ use similar contacts to form filaments. Surfaces that would make lateral interactions between protofilaments or interact with motor proteins are, however, different. The highly conserved nucleotide-binding sites of tubulin and FtsZ clearly differ from those of EF-Tu and other GTPases, while resembling the nucleotide site of glyceraldehyde-3-phosphate dehydrogenase. Thus, tubulin and FtsZ form a distinct family of GTP-hydrolyzing proteins.

F-actin-like filaments formed by plasmid segregation protein ParM

It was the general belief that DNA partitioning in prokaryotes is independent of a cytoskeletal structure, which in eukaryotic cells is indispensable for DNA segregation. Recently, however, immunofluorescence microscopy revealed highly dynamic, filamentous structures along the longitudinal axis of Escherichia coli formed by ParM, a plasmid‐encoded protein required for accurate segregation of low‐copy‐number plasmid R1. We show here that ParM polymerizes into double helical protofilaments with a longitudinal repeat similar to filamentous actin (F‐actin) and MreB filaments that maintain the cell shape of non‐spherical bacteria. The crystal structure of ParM with and without ADP demonstrates that it is a member of the actin family of proteins and shows a domain movement of 25° upon nucleotide binding. Furthermore, the crystal structure of ParM reveals major differences in the protofilament interface compared with F‐actin, despite the similar arrangement of the subunits within the filaments. Thus, there is now evidence for cytoskeletal structures, formed by actin‐like filaments that are involved in plasmid partitioning in E.coli.

Repeat motifs of tau bind to the insides of microtubules in the absence of taxol

The tau family of microtubule‐associated proteins has a microtubule‐binding domain which includes three or four conserved sequence repeats. Pelleting assays show that when tubulin and tau are co‐ assembled into microtubules, the presence of taxol reduces the amount of tau incorporated. In the absence of taxol, strong binding sites for tau are filled by one repeat motif per tubulin dimer; additional tau molecules bind more weakly. We have labelled a repeat motif with nanogold and used three‐dimensional electron cryomicroscopy to compare images of microtubules assembled with labelled or unlabelled tau. With kinesin motor domains bound to the microtubule outer surface to distinguish between α‐ and β‐tubulin, we show that the gold label lies on the inner surface close to the taxol binding site on β‐tubulin. Loops within the repeat motifs of tau have sequence similarity to an extended loop which occupies a site in α‐tubulin equivalent to the taxol‐binding pocket in β‐tubulin. We propose that loops in bound tau stabilize microtubules in a similar way to taxol, although with lower affinity so that assembly is reversible.

Three-dimensional image reconstruction of actin-tropomyosin complex and actin-tropomyosin-troponin T-troponin I complex

Abstract The structure of the actin-tropomyosin complex, which represents on active form of the thin filaments of skeletal muscle and the actin-tropomyosin-troponin T-troponin I complex, which represents an inhibited form, have been studied by three-dimensional reconstruction from electron micrographs. A model of the three-dimensional structure of the actin-tropomyosin complex obtained by averaging the twelve “best” sets of data showed that the structure of the helix was polar and that the actin-tropomyosin contact was relatively loose. The detailed shape of the actin monomer and tropomyosin strands could be observed. A model of the three-dimensional structure of the actin-tropomyosin-troponin T-troponin I complex obtained by averaging the nine “best” sets of data showed that the contact between the actin and tropomyosin was very close in the inhibited filament, where the position of tropomyosin differed by approximately 10 A from that in the active filament. The biological significance of the change in the extent of the actin-tropomyosin contact and of the movement of tropomyosin is discussed with reference to the mechanism of the regulation of muscle contraction by the tropomyosin-troponin-calcium system.

Harmonic analysis of electron microscope images with rotational symmetry

Abstract This paper describes a method of analysing images from electron micrographs of biological specimens believed to possess rotational symmetry. An objective analysis of the symmetry is possible because the method, which is computational, produces a rotational power spectrum of the image. We can then combine just those components which are consistent with the previously determined symmetry to produce a filtered image. The method is applied to the base plate of bacteriophage T4 and to discs of tobacco mosaic virus protein. The advantages of this new approach over the well-known Markham rotation technique are discussed.

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 ∼23.0 nm in diameter. A three‐dimensional reconstruction of tilted micrographs of FtsZ sheets in negative stain between 0 and 60° 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.

Microtubules and Maps

Microtubules are very dynamic polymers whose assembly and disassembly is determined by whether their heterodimeric tubulin subunits are in a straight or curved conformation. Curvature is introduced by bending at the interfaces between monomers. Assembly and disassembly are primarily controlled by the hydrolysis of guanosine triphosphate (GTP) in a site that is completed by the association of two heterodimers. However, a multitude of associated proteins are able to fine-tune these dynamics so that microtubules are assembled and disassembled where and when they are required by the cell. We review the recent progress that has been made in obtaining a glimpse of the structural interactions involved.

Interaction of tau protein with the dynactin complex

Tau is an axonal microtubule‐associated protein involved in microtubule assembly and stabilization. Mutations in Tau cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP‐17), and tau aggregates are present in Alzheimers disease and other tauopathies. The mechanisms leading from tau dysfunction to neurodegeneration are still debated. The dynein–activator complex dynactin has an essential role in axonal transport and mutations in its gene are associated with lower motor neuron disease. We show here for the first time that the N‐terminal projection domain of tau binds to the C‐terminus of the p150 subunit of the dynactin complex. Tau and dynactin show extensive colocalization, and the attachment of the dynactin complex to microtubules is enhanced by tau. Mutations of a conserved arginine residue in the N‐terminus of tau, found in patients with FTDP‐17, affect its binding to dynactin, which is abnormally distributed in the retinal ganglion cell axons of transgenic mice expressing human tau with a mutation in the microtubule‐binding domain. These findings, which suggest a direct involvement of tau in axonal transport, have implications for understanding the pathogenesis of tauopathies.