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.
Highlights
Alp12 is a novel plasmid-encoded actin-like protein from Clostridium tetani
Filament Assembly and Dynamics—Initially, GFP-labeled Alp12 was expressed in yeast and was observed to form bundles of filaments by light microscopy
All force-generating motors known to be involved in segregating plasmids (ParMR1, pSK41-ParM, and AlfA) were found to be helical polymers composed of two protofilaments gently winding around each other [2, 7,8,9, 36]
Summary
Alp is a novel plasmid-encoded actin-like protein from Clostridium tetani. Results: Alp forms dynamically unstable filaments with an open helical cylinder structure composed of four protofilaments. Through fitting of an Alp 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 par locus of the R1 drug resistance plasmid encodes three components: a centromere-like site in the DNA (parC), a DNA-binding protein (ParR), and ParM (an Alp bearing NTPase activity) [5] In vivo, these components can form a linear assembly of multiple filaments [6], which positions pairs of plasmids at opposite ends of the rod-shaped bacteria by a polymerization mechanism, ensuring equal distribution of the plasmids between daughter cells [5]. We show by three-dimensional electron microscopy reconstructions that C. tetani Alp filaments have a unique polymer structure that is entirely different from F-actin and that Alp filaments display dynamic behavior similar to microtubules
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