The structure of bacterial ParM filaments

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The prokaryotic origins of the actin cytoskeleton have been firmly established, but it has become clear that the bacterial actins form a wide variety of different filaments, different both from each other and from eukaryotic F-actin. We have used electron cryomicroscopy (cryo-EM) to examine the filaments formed by the protein crenactin (a crenarchaeal actin) from Pyrobaculum calidifontis, an organism that grows optimally at 90°C. Although this protein only has ∼20% sequence identity with eukaryotic actin, phylogenetic analyses have placed it much closer to eukaryotic actin than any of the bacterial homologs. It has been assumed that the crenactin filament is double-stranded, like F-actin, in part because it would be hard to imagine how a single-stranded filament would be stable at such high temperatures. We show that not only is the crenactin filament single-stranded, but that it is remarkably similar to each of the two strands in F-actin. A large insertion in the crenactin sequence would prevent the formation of an F-actin-like double-stranded filament. Further, analysis of two existing crystal structures reveals six different subunit-subunit interfaces that are filament-like, but each is different from the others in terms of significant rotations. This variability in the subunit-subunit interface, seen at atomic resolution in crystals, can explain the large variability in the crenactin filaments observed by cryo-EM and helps to explain the variability in twist that has been observed for eukaryotic actin filaments.helical polymers | variable twist | cytoskeletal filaments | crenactin A ctin is one of the most highly conserved as well as abundant eukaryotic proteins. From chickens to humans, an evolutionary separation of ∼350 million years, there are no amino acid changes in the skeletal muscle isoform of actin (1). There are at least six different mammalian isoforms that are quite similar to each other, and all seem to have diverged from a common ancestral actin gene (2). In contrast, we now know that bacteria have actin-like proteins that share a common fold (3-5) but have vanishingly little sequence similarity both among themselves and to eukaryotic actin (6).Two recent crystal structures of a crenarchaeal actin, crenactin (7, 8), raise interesting questions about the structure of the crenactin filament and its evolutionary relationship to F-actin. In both crystals (with two different space groups) crenactin forms a single-stranded filament with eight subunits per ∼420-Å righthanded turn, with a rise and rotation per subunit, therefore, of ∼53 Å and 45°, respectively. In contrast, in F-actin there is a rise and rotation of ∼55 Å and ∼27°along each of the two long-pitch right-handed strands. It was stated (9) that outside of the crystal the crenactin filaments are double-stranded, based upon the suggestion (8) that a single-stranded filament would unlikely be stable and that power spectra from crenactin filaments showed a strong layer line at ∼1/(210 Å), half of the repeat in the crystals.We have been able to ...

supporting

confidence: 58%

Archaeal actin from a hyperthermophile forms a single-stranded filament

Braun

Orlova

Valegård

et al. 2015

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“…Atoms in P1 ParA residues 318 and 349 are represented as red spheres, and these correspond to residues 189 and 218 in Soj which have been shown to be crucial for sequence-independent DNA-binding (7). Atoms in P1 ParA residue 351 are shown as blue spheres, and this corresponds to residue 340 of SopA which has also been shown to be essential for sequence-independent DNA-binding (8). The HTH-domain is involved in both lateral (A, blue arrowhead) and longitudinal (B, red arrows) contacts within the ParA2-dsDNA-ATP filament.…”

Section: Discussionmentioning

confidence: 99%

“…Type I systems have ATPases that share a deviant Walker box ATP-binding motif and are further classified into two subgroups based on the presence (type Ia) or absence (type Ib) of an additional N-terminal domain (3,6). Type II systems have ATPases that are structurally homologous to eukaryotic actin (7,8). With some exceptions, chromosomal par systems encode ATPases similar to the plasmid type Ib ATPases but form their own subgroup based on amino acid sequence similarities (3,4).…”

mentioning

confidence: 99%

ParA2, aVibrio choleraechromosome partitioning protein, forms left-handed helical filaments on DNA

Hui

Galkin²,

Xiong³

et al. 2010

Most bacterial chromosomes contain homologs of plasmid partitioning (par) loci. These loci encode ATPases called ParA that are thought to contribute to the mechanical force required for chromosome and plasmid segregation. In Vibrio cholerae, the chromosome II (chrII) par locus is essential for chrII segregation. Here, we found that purified ParA2 had ATPase activities comparable to other ParA homologs, but, unlike many other ParA homologs, did not form high molecular weight complexes in the presence of ATP alone. Instead, formation of high molecular weight ParA2 polymers required DNA. Electron microscopy and three-dimensional reconstruction revealed that ParA2 formed bipolar helical filaments on double-stranded DNA in a sequence-independent manner. These filaments had a distinct change in pitch when ParA2 was polymerized in the presence of ATP versus in the absence of a nucleotide cofactor. Fitting a crystal structure of a ParA protein into our filament reconstruction showed how a dimer of ParA2 binds the DNA. The filaments formed with ATP are left-handed, but surprisingly these filaments exert no topological changes on the right-handed B-DNA to which they are bound. The stoichiometry of binding is one dimer for every eight base pairs, and this determines the geometry of the ParA2 filaments with 4.4 dimers per 120 Å pitch left-handed turn. Our findings will be critical for understanding how ParA proteins function in plasmid and chromosome segregation.nucleoprotein filament | DNA segregation

“…Type I plasmid partitioning systems (4,5) are based on deviant Walker A ATPases (6,7), type II (8) on actin-like proteins (9), and type III (10)(11)(12) on tubulin/FtsZ-like proteins (13,14). Although the structure of the filament implicated in segregation has been determined for all three systems (9,(14)(15)(16), only examples of type I and II centromeric complexes have so far been resolved (17)(18)(19).…”

mentioning

confidence: 99%

Superstructure of the centromeric complex of TubZR C plasmid partitioning systems

Aylett

Löwe

2012

Bacterial plasmid partitioning systems segregate plasmids into each daughter cell. In the well-understood ParMRC plasmid partitioning system, adapter protein ParR binds to centromere parC, forming a helix around which the DNA is externally wrapped. This complex stabilizes the growth of a filament of actin-like ParM protein, which pushes the plasmids to the poles. The TubZRC plasmid partitioning system consists of two proteins, tubulin-like TubZ and TubR, and a DNA centromere, tubC, which perform analogous roles to those in ParMRC, despite being unrelated in sequence and structure. We have dissected in detail the binding sites that comprise Bacillus thuringiensis tubC, visualized the TubRC complex by electron microscopy, and determined a crystal structure of TubR bound to the tubC repeat. We show that the TubRC complex takes the form of a flexible DNA-protein filament, formed by lateral coating along the plasmid from tubC, the full length of which is required for the successful in vitro stabilization of TubZ filaments. We also show that TubR from Bacillus megaterium forms a helical superstructure resembling that of ParR. We suggest that the TubRC DNA-protein filament may bind to, and stabilize, the TubZ filament by forming such a ring-like structure around it. The helical superstructure of this TubRC may indicate convergent evolution between the actincontaining ParMRC and tubulin-containing TubZRC systems.FtsZ | X-ray crystallography | DNA segregation L ow copy number plasmids often encode their own segregation machinery, ensuring that copies are partitioned into each daughter cell. These plasmid-partitioning systems organize replicated plasmids and actively separate them. The known plasmidpartitioning systems are minimalist and elegant. They consist of just three components: a DNA centromere, an adapter protein that binds the centromere, forming the centromeric complex, and a nucleotide triphosphate-dependent filament-forming protein, which produces the force to move the plasmids (1, 2).Plasmid partitioning systems have been divided into types I-III, based upon the homology of their filament-forming proteins to known protein families (2, 3). Type I plasmid partitioning systems (4, 5) are based on deviant Walker A ATPases (6, 7), type II (8) on actin-like proteins (9), and type III (10-12) on tubulin/FtsZ-like proteins (13,14). Although the structure of the filament implicated in segregation has been determined for all three systems (9, 14-16), only examples of type I and II centromeric complexes have so far been resolved (17)(18)(19).The centromeric complex of the type II (actin-like) ParMRC plasmid partitioning system is formed by the binding of adapter protein ParR to parC, a series of parallel (direct), 11-base pair (bp) repeats (20). The superstructure this complex forms is helical, right-handed, and places the parC DNA on the exterior of the helix and ParR on the interior (18,19). This arrangement clusters the interaction surfaces between ParM and ParR, which are located at the tip of the ParM filament and ...