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.2012 Jun 15;287(25):21121-9.
doi: 10.1074/jbc.M112.341016. Epub 2012 Apr 18.

Novel actin-like filament structure from Clostridium tetani

Affiliations

Novel actin-like filament structure from Clostridium tetani

David Popp et al. J Biol Chem..

Abstract

Eukaryotic F-actin is constructed from two protofilaments that gently wind around each other to form a helical polymer. Several bacterial actin-like proteins (Alps) are also known to form F-actin-like helical arrangements from two protofilaments, yet with varied helical geometries. Here, we report a unique filament architecture of Alp12 from Clostridium tetani that is constructed from four protofilaments. Through fitting of an Alp12 monomer homology model into the electron microscopy data, the filament was determined to be constructed from two antiparallel strands, each composed of two parallel protofilaments. These four protofilaments form an open helical cylinder separated by a wide cleft. The molecular interactions within single protofilaments are similar to F-actin, yet interactions between protofilaments differ from those in F-actin. The filament structure and assembly and disassembly kinetics suggest Alp12 to be a dynamically unstable force-generating motor involved in segregating the pE88 plasmid, which encodes the lethal tetanus toxin, and thus a potential target for drug design. Alp12 can be repeatedly cycled between states of polymerization and dissociation, making it a novel candidate for incorporation into fuel-propelled nanobiopolymer machines.

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Figures

FIGURE 1.
FIGURE 1.
Typical electron micrographs of Alp12 filaments.A, individual filaments were observed to be relatively short.Scale bar = 200 nm.B, the calculated averaged Fourier transform (from nine Alp12 filaments) showed many layer lines typical for a helical filament. The first layer line at ∼141 nm classifies the helical repeat.C, closer view of an Alp12 filament.Scale bar = 50 nm.
FIGURE 2.
FIGURE 2.
Filtered images of Alp12.A, applied mask in Fourier space.B, examples of filtered projection structures using the back Fourier transformation inA. The trench and strands become clearer.
FIGURE 3.
FIGURE 3.
Alp12 assembly kinetics in near-physiological high salt buffer.A, Alp12 polymerization followed by light scattering using a stopped-flow machine.Orange, 7.5 μm Alp12 and 1 mm GTP;red, 7.5 μm Alp12 and 1 mm ATP;yellow, 15 μm Alp12 and 1 mm GTP;green, 15 μm Alp12 and 1 mm ATP.Blue curves are the best fits obtained by DYNAFIT.B, the kinetic scheme used for the fit: monomers associate into dimers and then tetramers and then elongate.C, steady-state light scattering intensities plotted as a function of Alp12 concentration. Data points are averaged over three individual experiments.Green, Alp12-ATP;black, Alp12-GTP;red, Alp12-AMP-PNP. The intersection of the linearly approximated curves with thex axis defines the critical concentration.D, long-term light scattering observations. Due to mixing by hand, the initial 10–15 s could not be resolved, and the Alp12-ATP and Alp12-GTP curves start almost at the plateau of polymerization. Protein concentration was 8 μm.Green, 1 mm ATP;black, 1 mm GTP;blue, 1 mm AMP-PNP;red, 1 mm GMP-PNP. Note the slower polymerization in the presence of non-hydrolyzable nucleotides.
FIGURE 4.
FIGURE 4.
Repeated polymerization-depolymerization cycles.A, Alp12 (8 μm) could be cycled repeatedly through polymerization and depolymerization. At eachgreen arrow, 100 μm NTP was added. At thepurple arrows, 1 mm NTP was added.Red, ATP;blue, GTP.B, ATP and GTP were not completely exchangeable. The amount of nucleotide added 100 μm ATP (green arrows), 100 μm GTP (purple arrow), 1 mm GTP (orange arrow), and 1 mm ATP (blue arrow). Note that 100 μm GTP was not sufficient to repolymerize Alp12. The Alp12 concentration was 20 μm.
FIGURE 5.
FIGURE 5.
Assembly, disassembly, and phosphate release.A, typical stopped-flow polymerization curves of Alp12 polymerized by 1 mm ATP.Blue, 7.5 μm Alp12;red, 15 μm Alp12.B, corresponding Pi release.Blue, 7.5 μm Alp12;red, 15 μm Alp12. The first ∼10 s could not be followed due to mixing by hand. Note that Pi release consisted of two phases (phases 1 and 2).C, disassembly monitored by light scattering. The Alp12 concentration was 15 μm. The ATP concentrations initially added were 100 μm (red), 500 μm (green), and 1000 μm (blue). Note the fast decreasing phase (yellow arrow), which was similar for all ATP concentrations. The second disassembly phase (purple arrow) was slower and depended on ATP concentration.D, corresponding Pi release. The Alp12 concentration was 15 μm. The ATP concentrations were 100 μm (red), 500 μm (green), and 1000 μm (blue). Pi release increased until it reached a plateau. The time to reach the plateau took substantially longer at 500 μm ATP than at 100 μm ATP, yet the difference between 500 and 1000 μm ATP was small. Similar behavior was observed for the total amount of Pi released (plateau height).
FIGURE 6.
FIGURE 6.
Electron density map.A, the two antiparallel strands are coloredcyan andpink. B–E, each strand consists of two parallel protofilaments individually presented in different colors. Thecyan strand inA is thered andpurple protofilaments. Thepink strand inA is thecyan andgreen protofilaments.B, strand 1 face;C, strand 2 face;D, trench face;E, back face. A comparison ofB withC highlights the antiparallel nature of the two strands in the Alp12 filament.F, fitting of the model into the electron density map. The three-dimensional reconstruction was obtained from 91 filaments and 5582 particles, and the resolution was 19.7 Å. Seesupplemental Fig. S12 for an enlarged view.
FIGURE 7.
FIGURE 7.
Model of Alp12 filament. The interactions between monomers in the Alp12 protofilament are grossly similar to those in the actin protofilament. Thered monomers have been aligned, and the subdomains labeled are the barbed (+) and pointed (−) ends of actin. The Alp12 strand is composed of two parallel protofilaments in an arrangement that is not similar to actin. The Alp12 filament is composed of two antiparallel strands connected through subdomain 3, possibly through a β-sheet interaction (inset).
FIGURE 8.
FIGURE 8.
Alp12 interactions.A andB, charged interaction surfaces within the Alp12 protofilament. The two views are tilted relative to each other to see the electrostatic interactions at the interface. In particular, subdomain 2 of the lower monomer offers an acidic surface to a basic region on subdomain 1 of the upper monomer.C, Alp12 arrangement of three monomers within a parallel strand.D, three monomers within F-actin whereby thedark green monomer is in the same orientation as theorange Alp12 monomer inC. Alp12 uses different surfaces to form a strand from two protofilaments.
FIGURE 9.
FIGURE 9.
Three views of Alp12 filament.A, focusing on a parallel strand (yellow andred) with the second parallel strand (blue andcyan) related by an antiparallel manner.B, focusing on the trench.C, filament viewed end-on.
FIGURE 10.
FIGURE 10.
Model for plasmid segregation of Alp12.A, initially, a short filament of antiparallel strands, each consisting of two parallel protofilaments, forms (light green andyellow), which can capture a plasmid complex at each end (red).B, Alp12 and other bacterial plasmid segregation proteins (ParM-R1, pSK41-ParM, and AlfA) are polymerizing motors that push plasmids to opposite ends of the bacteria via a polymerization mechanism. The bound plasmid (similar to the ParR-parC complex in ParM-R1) prevents immediate disassembly of the filament,C, filaments that fail to capture plasmids or, after successful plasmid segregation and plasmid dissociation, the unbound filament ends are vulnerable to dynamic instability and filament dissociation (arrows). This scheme allows the Alp12 filament system to repetitively probe the cytoplasm to capture and segregate the pE88 plasmid.
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References

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