The structure of a 15-stranded actin-like filament from Clostridium botulinum

Abstract

Microfilaments (actin) and microtubules represent the extremes in eukaryotic cytoskeleton cross-sectional dimensions, raising the question of whether filament architectures are limited by protein fold. Here, we report the cryoelectron microscopy structure of a complex filament formed from 15 protofilaments of an actin-like protein. This actin-like ParM is encoded on the large pCBH Clostridium botulinum plasmid. In cross-section, the ~26 nm diameter filament comprises a central helical protofilament surrounded by intermediate and outer layers of six and eight twisted protofilaments, respectively. Alternating polarity of the layers allows for similar lateral contacts between each layer. This filament design is stiffer than the actin filament, and has likely been selected for during evolution to move large cargos. The comparable sizes of microtubule and pCBH ParM filaments indicate that larger filament architectures are not limited by the protomer fold. Instead, function appears to have been the evolutionary driving force to produce broad, complex filaments.

An actin-like filament fromClostridium botulinumexhibits a novel mechanism of filament dynamics

“…After phosphate release, CBg-ParM remained as a filament without undergoing bulk depolymerization judged by constant light scattering intensity (Fig. 1C), unlike previously studied ParMs (Garner et al ., 2004; Jiang et al ., 2016; Koh et al ., 2019). Supernatant CBg-ParM concentrations in a sedimentation assay indicated that the critical concentrations for polymerization were similar for the different nucleotides, determined to be 2.3 ± 0.1 μM, 2.8 ± 0.1 μM, 1.2 ± 0.1 μM, and 3.6 ± 0.4 μM with ATP, ADP, GTP, and GDP, respectively.…”

“…Unlike other ParMs, CBg-ParM did not depolymerize after phosphate release (Fig. 7) (Garner et al ., 2004; Jiang et al ., 2016; Koh et al ., 2019). Instead of depolymerization, the filament underwent a significant structural change.…”

“…However, many ParMRC systems show different features, indicating a large diversity in this family of DNA segregation systems. For instance, another ParM from C. botulinum forms a 15-stranded filament with completely different inter-strand interactions (Koh et al ., 2019). A ParM from Bacillus thuringiensis forms a double-stranded non-polar filament without ParR and a four-stranded filament with ParR, indicating that ParR acts as a template of the filament like gammatubulin for microtubules (Jiang et al ., 2016).…”

“…However, ParMRC systems are highly divergent (Bharat et al ., 2015; Jiang et al ., 2016; Koh et al ., 2019; Popp et al , 2012) and a wide range of biochemical and structural features of its constituent molecules, in different bacteria, remain to be elucidated. Here, we report a novel ParM, CBg-ParM, from Clostridium botulinum , which shows several unique features.…”

Catching a Walker in the Act—DNA Partitioning by ParA Family of Proteins

“…A search of the microbial genomes revealed as many as 40 different families of actin-like proteins, mostly found on plasmids (Derman et al, 2009 ). A few have been shown to assemble into polymeric structures and filaments with varying architectures (Becker et al, 2006 ; Derman et al, 2009 ; Popp et al, 2012 ; Jiang et al, 2016 ; Koh et al, 2019 ). However, the ParMRC system is the most extensively studied among the DNA partitioning systems.…”