Near-Atomic Resolution for One State of F-Actin

Self Cite

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 ...

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Archaeal actin from a hyperthermophile forms a single-stranded filament

Braun

Orlova

Valegård

et al. 2015

Self Cite

“…1), a polymerization defect was predictable. Recent advances in electron cryomicroscopy have led to the highest resolution structures (3.7-4.7 Å) of the actin filament to date (9,11). In these structures, R258 (R256 in protein) forms a salt bridge with E197 (E195 in protein) in the same protomer, which, in turn, forms a salt bridge with K115 (K113 in protein) on the cross-strand actin ( Fig.…”

Section: R258c Mutation Decreases Contractile Function By Perturbing mentioning

confidence: 99%

“…Upon polymerization, the actin protomer undergoes a conformational change: The two major domains, which are twisted in monomeric G-actin, become flatter with respect to one another in F-actin. Another feature of F-actin is that it can adopt multiple states in which the protomers adopt varied twists and tilts with respect to one another, as well as having different loop conformations (8)(9)(10)(11).…”

mentioning

confidence: 99%

Vascular disease-causing mutation R258C in ACTA2 disrupts actin dynamics and interaction with myosin

Fagnant

Bookwalter

et al. 2015

Point mutations in vascular smooth muscle α-actin (SM α-actin), encoded by the gene ACTA2, are the most prevalent cause of familial thoracic aortic aneurysms and dissections (TAAD). Here, we provide the first molecular characterization, to our knowledge, of the effect of the R258C mutation in SM α-actin, expressed with the baculovirus system. Smooth muscles are unique in that force generation requires both interaction of stable actin filaments with myosin and polymerization of actin in the subcortical region. Both aspects of R258C function therefore need investigation. Total internal reflection fluorescence (TIRF) microscopy was used to quantify the growth of single actin filaments as a function of time. R258C filaments are less stable than WT and more susceptible to severing by cofilin. Smooth muscle tropomyosin offers little protection from cofilin cleavage, unlike its effect on WT actin. Unexpectedly, profilin binds tighter to the R258C monomer, which will increase the pool of globular actin (G-actin). In an in vitro motility assay, smooth muscle myosin moves R258C filaments more slowly than WT, and the slowing is exacerbated by smooth muscle tropomyosin. Under loaded conditions, small ensembles of myosin are unable to produce force on R258C actin-tropomyosin filaments, suggesting that tropomyosin occupies an inhibitory position on actin. Many of the observed defects cannot be explained by a direct interaction with the mutated residue, and thus the mutation allosterically affects multiple regions of the monomer. Our results align with the hypothesis that defective contractile function contributes to the pathogenesis of TAAD.actin | myosin II | smooth muscle | thoracic aortic aneurysms | vascular disease T horacic aortic aneurysms and dissections (TAAD) are the 18th most common cause of death in individuals in the United States (1). The high degree of mortality is partly due to the fact that aneurysms tend to be asymptomatic until a lifethreatening acute aortic dissection occurs. Familial TAAD is an autosomal dominant disorder with variable penetrance, which is characterized by enlargement or dissection of the thoracic aorta (reviewed in 2). The most prevalent genetic cause of familial TAAD, responsible for ∼15% of all cases, are mutations in vascular smooth muscle α-actin (SM α-actin), encoded by the gene ACTA2. More than 40 mutations in ACTA2 have been identified to date (3-5). Intriguingly, ACTA2 mutations also differentially predispose individuals to occlusive vascular diseases, such as premature coronary artery disease and strokes (6). ACTA2 mutations thus can lead to either dilation of large elastic arteries like the aorta or occlusion of smaller muscular arteries.SM α-actin is the most abundant protein in vascular smooth muscle cells, constituting ∼40% of the total protein and ∼70% of the total actin, with the rest composed of β-and γ-cytoplasmic actin. Actin is critical for contraction and force production by smooth muscle cells, as well as for their proliferation and migration. Dissected aortas show s...

“…E167 forms a noncovalent ionic bridge with K61 of an adjacent subunit in high-resolution actin filament models derived from electron cryomicroscopy (34,35), raising the possibility that the observed effects of the A-to-E substitution are simply a consequence of introducing a stabilizing ionic bridge. Several observations argue against this interpretation.…”

Section: Significancementioning

confidence: 99%

Site-specific cation release drives actin filament severing by vertebrate cofilin

Kang

Bradley

Cao

et al. 2014

Actin polymerization powers the directed motility of eukaryotic cells. Sustained motility requires rapid filament turnover and subunit recycling. The essential regulatory protein cofilin accelerates network remodeling by severing actin filaments and increasing the concentration of ends available for elongation and subunit exchange. Although cofilin effects on actin filament assembly dynamics have been extensively studied, the molecular mechanism of cofilin-induced filament severing is not understood. Here we demonstrate that actin filament severing by vertebrate cofilin is driven by the linked dissociation of a single cation that controls filament structure and mechanical properties. Vertebrate cofilin only weakly severs Saccharomyces cerevisiae actin filaments lacking this "stiffness cation" unless a stiffness cation-binding site is engineered into the actin molecule. Moreover, vertebrate cofilin rescues the viability of a S. cerevisiae cofilin deletion mutant only when the stiffness cation site is simultaneously introduced into actin, demonstrating that filament severing is the essential function of cofilin in cells. This work reveals that site-specific interactions with cations serve a key regulatory function in actin filament fragmentation and dynamics.cytoskeleton | persistence length | mechanics | electron cryomicroscopy A ctin polymerization powers the directed motility of eukaryotic cells and some pathogenic bacteria (1-3). Actin assembly also plays critical roles in endocytosis, cytokinesis, and establishment of cell polarity. Sustained motility requires filament disassembly and subunit recycling. The essential regulatory protein cofilin severs actin filaments (4-6), which accelerates actin network reorganization by increasing the concentration of filament ends available for subunit exchange (7).Cofilin binding alters the structure and mechanical properties of filaments, which effectively introduces local "defects" that compromise filament integrity and promote severing (5). Filaments with bound cofilin have altered twist (8, 9) and are more compliant in both bending and twisting than bare filaments (10-13). It has been suggested that deformations in filament shape promote fragmentation at or near regions of topological and mechanical discontinuities, such as boundaries between bare and cofilin-decorated segments along partially decorated filaments (5,12,(14)(15)(16)(17)(18).Cations modulate actin filament structure and mechanical properties (19) and cofilin dissociates filament-associated cations (20), leading us to hypothesize that cation-binding interactions regulate filament severing by cofilin. Cations bind filaments at two discrete and specific sites positioned between adjacent subunits along the long-pitch helix of the filament (19, 21). These cation binding sites are referred to as "polymerization" and "stiffness" sites based on their roles in filament assembly and mechanics, respectively. These discrete sites bind both monovalent and divalent cations with a range of affinities (low millimolar f...