The crystal structure of a cross-linked actin dimer suggests a detailed molecular interface in F-actin

The initiation of actin polymerization in cells requires actin filament nucleators. With the exception of formins, known filament nucleators use the Wiskott-Aldrich syndrome protein (WASP) homology 2 (WH2 or W) domain for interaction with actin. A common architecture, found in Spire, Cobl, VopL, and VopF, consists of tandem W domains that tie together three to four actin monomers to form a polymerization nucleus. Uncontrollable polymerization has prevented the structural investigation of such nuclei. We have engineered stable nuclei consisting of an actin dimer and a trimer stabilized by tandem W domain hybrid constructs and studied their structures in solution by x-ray scattering. We show that Spire-like tandem W domains stabilize a polymerization nucleus by lining up actin subunits along the long-pitch helix of the actin filament. Intersubunit contacts in the polymerization nucleus, thought to involve the DNase I-binding loop of actin, coexist with the binding of the W domain in the cleft between actin subdomains 1 and 3. The successful stabilization of filament-like multiactin assemblies opens the way to the crystallographic investigation of intersubunit contacts in the actin filament.actin nucleation ͉ cytoskeleton dynamics ͉ WH2 domain T he spontaneous polymerization of actin filaments is inhibited in cells by actin monomer-binding proteins such as profilin and thymosin-␤4 (T␤4). Cells use filament nucleators to stabilize actin polymerization nuclei, whose formation is rate-limiting during actin assembly (1). In addition to Arp2/3 complex and formins (2), a number of filament nucleators have been recently discovered: Spire (3), Cobl (4), VopL (5), VopF (6), and Lmod (7). With the exception of formins, all filament nucleators use the W domain for interaction with actin.The W domain is a short actin-binding motif of 17-27 aa (8). The N-terminal portion of the W domain forms a helix that binds in the cleft between actin subdomains 1 and 3 at the barbed end of the actin monomer (9). After this helix, the W domain presents an extended region that climbs toward the pointed end of the actin monomer. This portion of the W domain has variable length and sequence but comprises the conserved four-aa motif LKKT(V). T␤4, a member of the W domain family, contains an additional helix at the C terminus, which binds atop actin subdomains 2 and 4 and caps the pointed end (10).The W domain frequently occurs in tandem repeats. Tandem W domains constitute a common architecture among actin filament nucleators, such as Spire, Cobl, VopL, and VopF (Fig. 1A). The actin monomers bound to the individual W domains of these proteins are thought to come together to form a filamentlike nucleus for polymer assembly. This proposal, however, has not been directly demonstrated. An apparent contradiction is that the cleft between actin subdomains 1 and 3, where the N-terminal helix of the W domain binds, is also thought to participate in intersubunit contacts in the actin filament (11-13). Therefore, it remains to be demonstrated whether and...

Section: Discussionmentioning

confidence: 99%

X-ray scattering study of actin polymerization nuclei assembled by tandem W domains

Rębowski

Boczkowska

Hayes

et al. 2008

“…Thus far, none of the more than 80 actin crystal structures has captured the subunit-subunit interface present in F-actin. Although it was believed that one actin crystal structure did contain the interface between SD4 of one subunit and SD3 of a subunit above it (36), comparison with new atomic models (16,17) shows that the two interfaces are actually quite different, and one could not be simply converted to the other. The fact that the actual crenactin filament can be approximated by an 8 1 screw may be the most important factor allowing this filament to be crystalized, whereas F-actin cannot be approximated by a 2 1 screw despite early attempts to argue that a 2 1 screw axis in an actin crystal was the filament axis (37)(38)(39)(40).…”

Section: Discussionmentioning

confidence: 99%

Archaeal actin from a hyperthermophile forms a single-stranded filament

Braun

Orlova

Valegård

et al. 2015

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 ...

“…Such comparable behavior is expected given the strict conservation of polymerization site residues between both actin isoforms. High-resolution structures of actin dimers reveal that T203 and D288 can be within hydrogen-bonding distance (approximately 2.9 Å) (52,53). Therefore, we mutated the highly conserved T203 at the polymerization site, which could potentially participate in direct cation binding and/or orienting D288 through H-bonding for proper coordination geometry of the bound cation.…”

Section: Substitutions At Predicted Sites Modulate Cation-dependent Rmentioning

confidence: 99%

Identification of cation-binding sites on actin that drive polymerization and modulate bending stiffness

Kang

Bradley

McCullough

et al. 2012

Self Cite

148

The assembly of actin monomers into filaments and networks plays vital roles throughout eukaryotic biology, including intracellular transport, cell motility, cell division, determining cellular shape, and providing cells with mechanical strength. The regulation of actin assembly and modulation of filament mechanical properties are critical for proper actin function. It is well established that physiological salt concentrations promote actin assembly and alter the overall bending mechanics of assembled filaments and networks. However, the molecular origins of these salt-dependent effects, particularly if they involve nonspecific ionic strength effects or specific ion-binding interactions, are unknown. Here, we demonstrate that specific cation binding at two discrete sites situated between adjacent subunits along the long-pitch helix drive actin polymerization and determine the filament bending rigidity. We classify the two sites as "polymerization" and "stiffness" sites based on the effects that mutations at the sites have on salt-dependent filament assembly and bending mechanics, respectively. These results establish the existence and location of the cation-binding sites that confer salt dependence to the assembly and mechanics of actin filaments.ion-linkage | structural bioinformatics | persistence length | polyelectrolyte T he polymerization of the protein actin into double-stranded helical filaments powers many eukaryotic cell movements and provides cells with mechanical strength and integrity (1-4). Filament formation is favored when the total actin concentration exceeds the critical concentration (C c ) for assembly-defined as the monomer concentration at steady state for ATP-actin, or the dissociation constant for the reversible-equilibrium binding reaction of monomer binding to ADP-actin filament ends. Accordingly, the C c of ADP-actin is linked to the filament subunit interaction free energy such that lower C c values reflect greater thermodynamic stability (5).The effects of solution ionic conditions on the assembly and stability of actin filaments have been investigated for several decades (6-12). The actin C c and (monomer and filament) conformation depend on the nucleotide-associated divalent cation (Ca 2þ or Mg 2þ ) as well as the type and concentration of ions in solution (6, 7, 13-15), a behavior shared among characterized actins and their bacterial homologs (16). However, it is not firmly established if these salt effects on actin filament assembly and mechanics originate from nonspecific ion effects (e.g., electrostatic screening, counterion condensation, etc.) and/or specific ion binding interactions, potentially at discrete sites. Identification of saturable cation binding sites with different affinities favors specific and discrete binding sites on monomers (8-10, 17), but the location of these sites and their contributions to filament assembly and stiffness are unknown.Here we identify distinct cation-binding sites at subunit interfaces that regulate actin filament assembly and rigidity. Sit...