Cryo-EM structures reveal how phosphate release from Arp3 weakens actin filament branches formed by Arp2/3 complex.

Arp2/3 complex nucleates branched actin filaments for cell and organelle movements. Here we report a 2.7 Å resolution cryo-EM structure of the mature branch junction formed by S. pombe Arp2/3 complex that provides details about interactions with both mother and daughter filaments. We determine a second structure at 3.2 Å resolution with the phosphate analog BeFx bound with ADP to Arp3 and ATP bound to Arp2. In this ADP-BeFx transition state the outer domain of Arp3 is rotated 2° toward the mother filament compared with the ADP state and makes slightly broader contacts with actin in both the mother and daughter filaments. Thus, dissociation of Pi from the ADP-Pi transition state reduces the interactions of Arp2/3 complex with the actin filaments and may contribute to the lower mechanical stability of mature branch junctions with ADP bound to the Arps. Our structures also reveal that the mother filament in contact with Arp2/3 complex is slightly bent and twisted, consistent with the preference of Arp2/3 complex binding curved actin filaments. The small degree of twisting constrains models of actin filament mechanics.

Cryo-EM structure of the bacterial effector protein SipA bound to F-actin reveals a unique mechanism for filament stabilization.

The bacterial pathogen Salmonella spp. modulates cellular processes by delivering effector proteins through its type III secretion systems. Among these effectors, SipA facilitates bacterial invasion and promotes intestinal inflammation. The mechanisms by which this effector carries out these functions are incompletely understood although SipA's ability to modulate actin dynamics is central to some of these activities. Here we report the cryo-EM structure of SipA bound to filamentous actin. We show that this effector stabilizes actin filaments through unique interactions of its carboxy terminal domain with four actin subunits. Furthermore, our structure-function studies revealed that SipA's actin-binding activity is independent from its ability to stimulate intestinal inflammation. Overall, these studies illuminate critical aspects of Salmonella pathogenesis, and provide unique insight into the mechanisms by which a bacterial effector modulates actin dynamics.

Cryo-EM structures of both ends of the actin filament explain why the barbed end elongates faster than the pointed end.

Actin filament ends are the sites of subunit addition during elongation and subunit loss during depolymerization. Prior work established the kinetics and thermodynamics of the assembly reactions at both ends but not the structural basis of their differences. Cryo-EM reconstructions of the barbed end at 3.1 Šresolution and the pointed end at 3.5 Šreveal distinct conformations at the two ends. These conformations explain why barbed ends elongate faster than pointed ends and why pointed ends rapidly dissociate the γ-phosphate released from ATP hydrolysis during assembly. The D-loop of the penultimate subunit at the pointed end is folded onto the terminal subunit, precluding its binding incoming actin monomers, and gates on the phosphate release channels of both subunits are wide open. The samples were prepared with FH2 dimers from fission yeast formin Cdc12. The barbed end reconstruction has extra density that may be partial occupancy by the FH2 domains. Significance Statement: Cells depend cytoplasmic filaments assembled from the protein actin for their physical integrity, as tracks for myosin motor proteins and movements of the whole cell and internal organelles. Actin filaments elongate and shrink at their ends by adding or dissociating single actin molecules. We used cryo-electron microscopy to determine the structures of the two ends of actin filaments at 3.5 Šresolution for the slowly growing pointed end and 3.1 Šfor the rapidly growing barbed end. These structures reveal why barbed ends grow faster than the pointed ends, why the rate at the pointed end is not diffusion-limited and why the pointed end has a low affinity for the γ-phosphate released from bound ATP inside the filament.

Mechanism of actin filament branch formation by Arp2/3 complex revealed by a high-resolution cryo-EM structureof the branch junction.

We reconstructed the structure of actin filament branch junctions formed by fission yeast Arp2/3 complex at 3.5 Å resolution from images collected by electron cryo-microscopy. During specimen preparation, all of the actin subunits and Arp3 hydrolyzed their bound adenosine triphosphate (ATP) and dissociated the γ-phosphate, but Arp2 retained the γ-phosphate. Binding tightly to the side of the mother filament and nucleating the daughter filament growing as a branch requires Arp2/3 complex to undergo a dramatic conformational change where two blocks of structure rotate relative to each other about 25° to align Arp2 and Arp3 as the first two subunits in the branch. During branch formation, Arp2/3 complex acquires more than 8,000 Å2 of new buried surface, accounting for the stability of the branch. Inactive Arp2/3 complex binds only transiently to the side of an actin filament, because its conformation allows only a subset of the interactions found in the branch junction.

Cryo-electron microscopy structures of pyrene-labeled ADP-Pi- and ADP-actin filaments.

Since the fluorescent reagent N-(1-pyrene)iodoacetamide was first used to label skeletal muscle actin in 1981, the pyrene-labeled actin has become the most widely employed tool to measure the kinetics of actin polymerization and the interaction between actin and actin-binding proteins. Here we report high-resolution cryo-electron microscopy structures of actin filaments with N-1-pyrene conjugated to cysteine 374 and either ADP (3.2 Å) or ADP-phosphate (3.0 Å) in the active site. Polymerization buries pyrene in a hydrophobic cavity between subunits along the long-pitch helix with only minor differences in conformation compared with native actin filaments. These structures explain how polymerization increases the fluorescence 20-fold, how myosin and cofilin binding to filaments reduces the fluorescence, and how profilin binding to actin monomers increases the fluorescence.

Structural basis for polarized elongation of actin filaments.

Actin filaments elongate and shorten much faster at their barbed end than their pointed end, but the molecular basis of this difference has not been understood. We use all-atom molecular dynamics simulations to investigate the properties of subunits at both ends of the filament. The terminal subunits tend toward conformations that resemble actin monomers in solution, while contacts with neighboring subunits progressively flatten the conformation of internal subunits. At the barbed end the terminal subunit is loosely tethered by its DNase-1 loop to the third subunit, because its monomer-like conformation precludes stabilizing contacts with the penultimate subunit. The motions of the terminal subunit make the partially flattened penultimate subunit accessible for binding monomers. At the pointed end, unique contacts between the penultimate and terminal subunits are consistent with existing cryogenic electron microscopic (cryo-EM) maps, limit binding to incoming monomers, and flatten the terminal subunit, which likely promotes ATP hydrolysis and rapid phosphate release. These structures explain the distinct polymerization kinetics of the two ends.

Mechanism of actin polymerization revealed by cryo-EM structures of actin filaments with three different bound nucleotides.

We used cryo-electron microscopy (cryo-EM) to reconstruct actin filaments with bound AMPPNP (ß,γ-imidoadenosine 5'-triphosphate, an ATP analog, resolution 3.1 Å), ADP-Pi (ADP with inorganic phosphate, resolution 3.1 Å), or ADP (resolution 3.6 Å). Subunits in the three filaments have similar backbone conformations, so assembly rather than ATP hydrolysis or phosphate dissociation is responsible for their flattened conformation in filaments. Polymerization increases the rate of ATP hydrolysis by changing the positions of the side chains of Q137 and H161 in the active site. Flattening during assembly also promotes interactions along both the long-pitch and short-pitch helices. In particular, conformational changes in subdomain 3 open up multiple favorable interactions with the DNase-I binding loop in subdomain 2 of the adjacent subunit. Subunits at the barbed end of the filament are likely to be in this favorable conformation, while monomers are not. This difference explains why filaments grow faster at the barbed end than the pointed end. When phosphate dissociates from ADP-Pi-actin through a backdoor channel, the conformation of the C terminus changes so it distorts the DNase binding loop, which allows cofilin binding, and a network of interactions among S14, H73, G74, N111, R177, and G158 rearranges to open the phosphate release site.

Conformational changes in Arp2/3 complex induced by ATP, WASp-VCA, and actin filaments.

We used fluorescence spectroscopy and EM to determine how binding of ATP, nucleation-promoting factors, actin monomers, and actin filaments changes the conformation of Arp2/3 complex during the process that nucleates an actin filament branch. We mutated subunits of Schizosaccharomyces pombe Arp2/3 complex for labeling with fluorescent dyes at either the C termini of Arp2 and Arp3 or ArpC1 and ArpC3. We measured Förster resonance energy transfer (FRET) efficiency (ETeff) between the dyes in the presence of the various ligands. We also computed class averages from electron micrographs of negatively stained specimens. ATP binding made small conformational changes of the nucleotide-binding cleft of the Arp2 subunit. WASp-VCA, WASp-CA, and WASp-actin-VCA changed the ETeff between the dyes on the Arp2 and Arp3 subunits much more than between dyes on ArpC1 and ArpC3. Ensemble FRET detected an additional structural change that brought ArpC1 and ArpC3 closer together when Arp2/3 complex bound actin filaments. VCA binding to Arp2/3 complex causes a conformational change that favors binding to the side of an actin filament, which allows further changes required to nucleate a daughter filament.

Ligand-induced architecture of the leptin receptor signaling complex.

Despite the crucial impact of leptin signaling on metabolism and body weight, little is known about the structure of the liganded leptin receptor (LEP-R) complex. Here, we applied single-particle electron microscopy (EM) to characterize the architecture of the extracellular region of LEP-R alone and in complex with leptin. We show that unliganded LEP-R displays significant flexibility in a hinge region within the cytokine homology region 2 (CHR2) that is connected to rigid membrane-proximal FnIII domains. Leptin binds to CHR2 in order to restrict the flexible hinge and the disposition of the FnIII "legs." Through a separate interaction, leptin engages the Ig-like domain of a second liganded LEP-R, resulting in the formation of a quaternary signaling complex. We propose that the membrane proximal domain rigidification in the context of a liganded cytokine receptor dimer is a key mechanism for the transactivation of Janus kinases (Jaks) bound at the intracellular receptor region.