Biology subjects

Chou, S. Z.

Publications and source records attributed to Chou, S. Z..

6 recordsLinked to original sources

bioRxiv2023

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 SipAs 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. The structure shows 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 SipAs actin-binding activity is independent of 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.

microbiology↗

bioRxiv2023

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 [A] resolution and the pointed end at 3.5 [A] reveal distinct conformations at the two ends. These conformations explain why barbed ends elongate faster than pointed ends and why pointed ends rapidly dissociate the {gamma}-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 StatementCells 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 [A] resolution for the slowly growing pointed end and 3.1 [A] for 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 {gamma}-phosphate released from bound ATP inside the filament.

biochemistry↗

bioRxiv2020

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

We report high resolution cryo-electron microscopy structures of actin filaments with N-1-pyrene conjugated to cysteine 374 and either ADP (3.2 [A]) or ADP-phosphate (3.0 [A]) 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.

biochemistry↗

bioRxiv2020

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 towards 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 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. Significance StatementEukaryotic cells utilize actin filaments to move, change shape, divide, and transport cargo. Decades of experiments have established that actin filaments elongate and shorten significantly faster from one end than the other, but the underlying mechanism for this asymmetry has not been explained. We used molecular dynamics simulations to investigate the structures of the actin filament ends in the ATP, ADP plus{gamma} -phosphate, and ADP nucleotide states. We characterize the structures of actin subunits at both ends of the filament, explain the mechanisms leading to these differences, and connect the divergent structural properties of the two ends to their distinct polymerization rate constants.

biophysics↗

bioRxiv2018

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

We used electron cryo-micrographs to reconstruct actin filaments with bound AMPPNP ({beta},{gamma}-imidoadenosine 5-triphosphate, an ATP analog), ADP-Pi (ADP with inorganic phosphate) or ADP to resolutions of 3.4 [A], 3.4 [A] and 3.6 [A]. Subunits in the three filaments have nearly identical 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 conformations of the three ATP phosphates and the side chains of Gln137 and His161 in the active site. Flattening also promotes interactions along both the long-pitch and short-pitch helices. In particular, conformational changes in subdomain 3 open up 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. Loss of hydrogen bonds after phosphate dissociation may account for the greater flexibility of ADP-actin filaments.\n\nSignificance StatementActin filaments comprise a major part of the cytoskeleton of eukaryotic cells and serve as tracks for myosin motor proteins. The filaments assemble from actin monomers with a bound ATP. After polymerization, actin rapidly hydrolyzes the bound ATP and slowly dissociates the {gamma}-phosphate. ADP-actin filaments then disassemble to recycle the subunits. Understanding how actin filaments assemble, disassemble and interact with numerous regulatory proteins depends on knowing the structure of the filament. High quality structures of ADP-actin filaments were available, but not of filaments with bound ATP- or with ADP and phosphate. We determined structures of actin filaments with bound AMPPNP (a slowly hydrolyzed ATP analog), ADP and phosphate and ADP by cryo-electron microscopy. These structures show how conformational changes during actin assembly promote ATP hydrolysis and faster growth at one end of the filament than the other.

biochemistry↗

bioRxiv2017

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

We used fluorescence spectroscopy and electron microscopy to determine how binding of ATP, nucleation-promoting factors (NPF), actin monomers and actin filaments change 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 Forster 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 clefts of the Arp subunits. 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 a different structural change that involves bringing ArpC1 and ArpC3 closer together when Arp2/3 complex bound actin filaments. Each of the ligands that activates Arp2/3 complex changes the structure in different ways, each leading progressively to fully activated Arp2/3 complex on the side of a filament.

biophysics↗