Mechanism of phosphate release from actin filaments.

After ATP-actin monomers assemble filaments, the ATP's [Formula: see text]-phosphate is hydrolyzedwithin seconds and dissociates over minutes. We used all-atom molecular dynamics simulations to sample the release of phosphate from filaments and study residues that gate release. Dissociation of phosphate from Mg2+ is rate limiting and associated with an energy barrier of 20 kcal/mol, consistent with experimental rates of phosphate release. Phosphate then diffuses within an internal cavity toward a gate formed by R177, as suggested in prior computational studies and cryo-EM structures. The gate is closed when R177 hydrogen bonds with N111 and is open when R177 forms a salt bridge with D179. Most of the time, interactions of R177 with other residues occlude the phosphate release pathway. Machine learning analysis reveals that the occluding interactions fluctuate rapidly, underscoring the secondary role of backdoor gate opening in Pi release, in contrast with the previous hypothesis that gate opening is the primary event.

Cracked actin filaments as mechanosensitive receptors.

Actin filament networks are exposed to mechanical stimuli, but the effect of strain on actin filament structure has not been well established in molecular detail. This is a critical gap in understanding because the activity of a variety of actin-binding proteins has recently been determined to be altered by actin filament strain. We therefore used all-atom molecular dynamics simulations to apply tensile strains to actin filaments and find that changes in actin subunit organization are minimal in mechanically strained, but intact, actin filaments. However, a conformational change disrupts the critical D-loop to W-loop connection between longitudinal neighboring subunits, which leads to a metastable cracked conformation of the actin filament whereby one protofilament is broken prior to filament severing. We propose that the metastable crack presents a force-activated binding site for actin regulatory factors that specifically associate with strained actin filaments. Through protein-protein docking simulations, we find that 43 evolutionarily diverse members of the dual zinc-finger-containing LIM-domain family, which localize to mechanically strained actin filaments, recognize two binding sites exposed at the cracked interface. Furthermore, through its interactions with the crack, LIM domains increase the length of time damaged filaments remain stable. Our findings propose a new molecular model for mechanosensitive binding to actin filaments.

Mechanism of Phosphate Release from Actin Filaments.

After ATP-actin monomers assemble filaments, the γ-phosphate is hydrolyzed from ATP within seconds and dissociates from the filament over several minutes. We used all-atom well-tempered metadynamics molecular dynamics simulations to sample the release of phosphate from filaments along with unbiased molecular dynamics simulations to study residues that gate release. Dissociation of phosphate from Mg2+ is rate limiting and associated with an energy barrier of 20 kcal/mol, consistent with experimental rates of phosphate release. Phosphate then diffuses in an internal cavity toward a gate formed by R177 suggested in prior computational studies and cryo-EM structures. The gate is closed when R177 hydrogen bonds with N111 and is open when R177 forms a salt bridge with D179. Most of the time interactions of R177 with other residues occludes the phosphate release pathway. Machine learning analysis reveals that the occluding interactions fluctuate rapidly. These occluded states have not been documented in cryo-EM reconstructions.

Cracked actin filaments as mechanosensitive receptors.

Actin filament networks are exposed to mechanical stimuli, but the effect of strain on actin filament structure has not been well-established in molecular detail. This is a critical gap in understanding because the activity of a variety of actin-binding proteins have recently been determined to be altered by actin filament strain. We therefore used all-atom molecular dynamics simulations to apply tensile strains to actin filaments and find that changes in actin subunit organization are minimal in mechanically strained, but intact, actin filaments. However, a conformational change disrupts the critical D-loop to W-loop connection between longitudinal neighboring subunits, which leads to a metastable cracked conformation of the actin filament, whereby one protofilament is broken prior to filament severing. We propose that the metastable crack presents a force-activated binding site for actin regulatory factors that specifically associate with strained actin filaments. Through protein-protein docking simulations, we find that 43 evolutionarily-diverse members of the dual zinc finger containing LIM domain family, which localize to mechanically strained actin filaments, recognize two binding sites exposed at the cracked interface. Furthermore, through its interactions with the crack, LIM domains increase the length of time damaged filaments remain stable. Our findings propose a new molecular model for mechanosensitive binding to actin filaments.

Formin Cdc12's specific actin assembly properties are tailored for cytokinesis in fission yeast.

Formins generate unbranched actin filaments by a conserved, processive actin assembly mechanism. Most organisms express multiple formin isoforms that mediate distinct cellular processes and facilitate actin filament polymerization by significantly different rates, but how these actin assembly differences correlate to cellular activity is unclear. We used a computational model of fission yeast cytokinetic ring assembly to test the hypothesis that particular actin assembly properties help tailor formins for specific cellular roles. Simulations run in different actin filament nucleation and elongation conditions revealed that variations in formin's nucleation efficiency critically impact both the probability and timing of contractile ring formation. To probe the physiological importance of nucleation efficiency, we engineered fission yeast formin chimera strains in which the FH1-FH2 actin assembly domains of full-length cytokinesis formin Cdc12 were replaced with the FH1-FH2 domains from functionally and evolutionarily diverse formins with significantly different actin assembly properties. Although Cdc12 chimeras generally support life in fission yeast, quantitative live-cell imaging revealed a range of cytokinesis defects from mild to severe. In agreement with the computational model, chimeras whose nucleation efficiencies are least similar to Cdc12 exhibit more severe cytokinesis defects, specifically in the rate of contractile ring assembly. Together, our computational and experimental results suggest that fission yeast cytokinesis is ideally mediated by a formin with properly tailored actin assembly parameters.

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.

Sarcoplasmic Reticulum Ca2+ Release Uses a Cascading Network of Intra-SR and Channel Countercurrents.

In muscle, Ca2+ release from the sarcoplasmic reticulum (SR) into the cytosol is mediated through the ryanodine receptors (RyRs) and sustained by countercurrents that keep the SR membrane potential near 0 mV. Likewise, Ca2+ reuptake by the sarco/endoplasmic reticulum Ca2+ ATPase pump requires countercurrent. Although evidence has suggested that TRIC K+ channels and/or RyR K+ influx provide these countercurrents, the exact sources have not yet been determined. We used an equivalent circuit compartment model of a cardiac SR, the surrounding cytosol, and the dyadic cleft to probe the sources of countercurrent during a complete cardiac cycle. By removing and relocating TRIC K+ channels, as well as limiting when they are active, we explored the various possible sources of SR countercurrent under many conditions. Our simulations indicate that no single channel type is essential for countercurrent. Rather, a cascading network of countercurrents is present with anion fluxes within the SR redistributing charges throughout the full SR volume. This allows ion channels in the entire SR membrane, far from the Ca2+ fluxes through the RyRs in the junctional SR and sarco/endoplasmic reticulum Ca2+ ATPase pump in the nonjunctional SR, to mediate countercurrents that support Ca2+ release and reuptake. This multifactorial network of countercurrents allows Ca2+ release to be remarkably robust.

Sarcoplasmic reticulum Ca2+, Mg2+, K+, and Cl- concentrations adjust quickly as heart rate changes.

During systole, Ca2+ is released from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) while, simultaneously, other ions (specifically K+, Mg2+, and Cl-) provide counter-ion flux. These ions move back into the SR during diastole through the SERCA pump and SR K+ and Cl- channels. In homeostasis, all ion concentrations in different cellular regions (e.g., junctional and non-junctional SR, dyadic cleft, and cytosol) are the same at the beginning and end of the cardiac cycle. Here, we used an equivalent circuit compartment model of the SR and the surrounding cytoplasm to understand the heart rate dependence of SR ion homeostasis. We found that the Ca2+, Mg2+, K+, and Cl- concentrations in the SR and the cytoplasm self-adjust within just a few heartbeats with only very small changes in Mg2+, K+, and Cl- concentrations and membrane voltages (just a few percent). However, those small changes were enough to compensate for the large heart-rate-dependent changes in SR and cytoplasmic Ca2+ concentrations in the new steady state. The modeling suggests that ion adaptation to increases in heart rate is inherent to the system and that physiological changes that increase contractility and cardiac output are accommodated by the same self-adjusting mechanism of producing small changes in ion driving forces. Our findings also support the long-held hypothesis that SR membrane potentials are small (~1-2mV).