Archaeal actin from a hyperthermophile forms a single-stranded filament

Abstract

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 ...

Crenactin forms actin-like double helical filaments regulated by arcadin-2

“…Previous studies on the architecture of crenactin filaments were performed under high salt concentrations (>0.5 M KCl) (Braun et al, 2015; Izoré et al, 2014) that might not be entirely justified given Pyrobaculum calidifontis' environmental and laboratory growth conditions (Amo et al, 2002), although the intracellular osmolarity is currently not known. To exclude the possibility that such high salt concentration might have altered filament architecture, we carried out experiments in low-salt buffer (50 mM ammonium carbonate, 20 mM KCl, see Materials and methods).…”

“…Given these striking similarities between the actin and crenactin monomers, it has been puzzling that crenactin filaments were reported by electron microscopy to form single, rather than double helical, F-actin-like filaments (Braun et al, 2015). …”

In search of the primordial actin filament

“…Low-resolution electron micrographs (EMs) of crenactin filaments also indicated a structure that is consistent with either single-or double-stranded filaments (6). Now, Braun et al (2) definitively show in an 18-Å cryo-EM reconstruction that crenactin can form a single-stranded filament in vitro.…”

“…Relatedness of actins. The structures of actin protofilaments (2,(9)(10)(11)(12)(13)(14)(15) are shown below a maximumlikelihood phylogenetic tree of the actin protein sequences. The structures are aligned via the central protomer, which is shown as a ribbon (slate blue) with the nucleotide in red.…”

“…Many more archaeal genomes will be needed to make that assessment. Nevertheless, the fascinating discovery that crenactin forms a single protofilament (2) demonstrates that the eukaryotic actin-like fold is capable of supporting stable single-stranded filaments.…”