ATP competes with PIP₂ for binding to gelsolin

<div><p>Gelsolin is a severing and capping protein that targets filamentous actin and regulates filament lengths near plasma membranes, contributing to cell movement and plasma membrane morphology. Gelsolin binds to the plasma membrane via phosphatidylinositol 4,5-bisphosphate (PIP₂) in a state that cannot cap F-actin, and gelsolin-capped actin filaments are uncapped by PIP₂ leading to filament elongation. The process by which gelsolin is removed from PIP₂ at the plasma membrane is currently unknown. Gelsolin also binds ATP with unknown function. Here we characterize the role of ATP on PIP₂-gelsolin complex dynamics. Fluorophore-labeled PIP₂ and ATP were used to study their interactions with gelsolin using steady-state fluorescence anisotropy, and Alexa488-labeled gelsolin was utilized to reconstitute the regulation of gelsolin binding to PIP₂-containing phospholipid vesicles by ATP. Under physiological salt conditions ATP competes with PIP₂ for binding to gelsolin, while calcium causes the release of ATP from gelsolin. These data suggest a cycle for gelsolin activity. Firstly, calcium activates ATP-bound gelsolin allowing it to sever and cap F-actin. Secondly, PIP₂-binding removes the gelsolin cap from F-actin at low calcium levels, leading to filament elongation. Finally, ATP competes with PIP₂ to release the calcium-free ATP-bound gelsolin, allowing it to undergo a further round of severing.</p></div

Model of the severing, capping, uncapping and inactivation/release cycle of gelsolin.

<p>The cartoon presents a model for the cycle of activation and function of gelsolin. Severing and capping: Elevated free calcium levels activate gelsolin, releasing ATP, leading to severing and capping of actin filaments. Depolymerization: Gelsolin-capped filaments will depolymerize from their pointed ends. Uncapping and polymerization: Gelsolin-capped actin filaments will be uncapped on encountering PIP₂ in the membrane, resulting in force being exerted on the membrane from the polymerization of the uncapped filaments. Inactivation and membrane release: Gelsolin will be released from PIP₂ and the membrane by competition with ATP. Following its release gelsolin is able to undergo subsequent cycles of severing, capping, uncapping and inactivation/release.</p

Synthetic Polyketide Enzymology: Platform for Biosynthesis of Antimicrobial Polyketides

Synthetic biology often employs enzymes in the biosynthesis of compounds for purposeful function. Here, we define synthetic enzymology as the application of enzymological principles in synthetic biology and describe its use as an enabling platform in synthetic biology for the purposeful production of compounds of biomedical and commercial importance. In particular, we demonstrated the use of synthetic polyketide enzymology as a means to develop lead polyketide based compounds for antimicrobial therapeutics, as exemplified by the modular coupling of acid:CoA ligases to type III polyketide synthases in the biosynthesis and development of polyketide-based biochemicals. Using wild-type and rationally designed mutants of a type III polyketide synthase isolated from Oryza sativa (OsPKS), we produced a chemically diverse library of novel polyketides and identified two bioactive antimicrobials, 4-hydroxy-6-[(1<i>E</i>)-2-(4-hydroxyphenyl)­ethenyl]-2<i>H</i>-pyran-2-one (bisnoryangonin) and 3,6,7-trihydroxy-2-(4-methoxybenzyl)-4<i>H</i>-1-benzopyran-4,5,8-trione (26OH), respectively, from a screen against a collection of Gram-positive and Gram-negative bacteria. The purification, crystallization, and structural resolution of recombinant OsPKS at 1.93 Å resolution are also reported. Using the described route of synthetic polyketide enzymology, a library of OsPKS mutants was generated as an additional means to increase the diversity of the polyketide product library. We expect the utility of synthetic enzymology to be extended to other classes of biomolecules and translated to various purposeful functions as the field of synthetic biology progresses

The interplay between calcium, magnesium, ATP and PIP₂ for binding to gelsolin.

<p><b>(a)</b> In the absence of cations, the anisotropy of ATP-N (0.5 μM) increased and saturated by ~ 5 μM gelsolin. Dashed line shows the fit to the data (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201826#pone.0201826.e002" target="_blank">Eq 2</a>). <b>(b)</b> Micromolar calcium levels (C, open triangles, inflection point at pCa 5.2) or millimolar magnesium levels (M, orange circles, inflection point at pMg 2.3) were able to reduce the anisotropy of ATP-N (0.5 μM) in the presence of gelsolin (5 μM), indicating the dissociation of ATP-N from gelsolin. Dashed line indicates sigmoidal fitting. <b>(c)</b> In the absence of cations, the anisotropy of PIP₂-F (0.5 μM) increased and reached steady-state at ~ 5 μM gelsolin, indicating saturation of binding. Dashed line shows the fit to the data (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201826#pone.0201826.e002" target="_blank">Eq 2</a>). <b>(d)</b> The anisotropy of PIP₂-F (0.5 μM) in the presence of gelsolin (5 μM) was not changed on titration with magnesium (M, orange circles), but inclusion of 0.5 mM ATP (MA, white circles) lowered the anisotropy to the value characteristic to free PIP₂-F, indicating complete dissociation of the gelsolin/PIP₂-F complex by ATP. This effect was diminished by magnesium concentrations above 7 mM (pMg 2.15). In the absence of ATP, PIP₂-F (0.5 μM) binds to gelsolin (5 μM), as revealed by the increase in anisotropy across a wide range of calcium concentrations (C, white triangles). Inclusion of ATP (0.2 mM, CA, purple triangles) lowered the anisotropy to the value characteristic to free PIP₂-F, indicating complete dissociation of the gelsolin/PIP₂-F complex by ATP.</p

ATP dependence of gelsolin binding to the surface of PIP₂-containing membrane vesicles.

<p><b>(a)</b> Gelsolin-Alexa488 (5 μM, red) was incubated with rhodamine590-filled, PIP₂-containing membrane vesicles (blue) in the absence of divalent cations and visualized by confocal microscopy. The merged image indicates that gelsolin and vesicles colocalized (top panel). After ATP (0.5 mM) was added, the majority of gelsolin-Alexa488 was released from the vesicles (middle panel). Following removal of ATP via buffer exchange and addition of fresh gelsolin-Alexa488 (15 μM), gelsolin re-associated with the vesicles (bottom panel). Scale bar = 10 μm. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201826#pone.0201826.s007" target="_blank">S1 Movie</a> details the time course of these changes. <b>(b)</b> Representative image of gelsolin-Alexa488 (15 μM, red) localized to the surface of a large rhodamine590-filled PIP₂-containing vesicle (blue) (top panel). After the addition of ATP (0.5 mM) the vesicle changed morphology concurrently with the release of gelsolin (red) from the vesicle surface (blue) (bottom panel). Scale bar = 10 μm. Panel <i>a</i> and <i>b</i> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201826#pone.0201826.s006" target="_blank">S6 Fig</a> show line scans across the images as indicated by the arrows and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201826#pone.0201826.s008" target="_blank">S2 Movie</a> details the time course. The confocal slice thickness was ~ 3 μm. There are two vesicles in focus, a small (d = 7 μm) and a large (d = 22.5 μm) one, the outer layer of the bigger vesicle is attached to the coverslip.</p

Stick models of small peptide fibril crystals from the PDB.

<p>Stick models of small peptide fibril crystals showing a similar pattern to Ac-YLD of alternating hydrophobic zipper regions with hydrophilic, water-stabilized regions. Hydrophobic regions are highlighted in green. Water stabilized regions are highlighted in red. Note that these regions can greatly vary in size and shape across crystal structures. (A) PDB: 2OMM, GNNQQNY (parallel) (B) PDB∶3LOZ, LSFSKD (antiparallel) (C) PDB∶2Y29, KLVFFA (antiparallel) (D) PDB∶3SGS, GDVIEV (parallel).</p

Details of crystallization, data collection and refinement.

<p>Details of crystallization, data collection and refinement.</p

Comparison of the viscoelastic properties of tripeptide.

<p>Comparison of the viscoelastic properties of tripeptide hydrogels via frequency sweep studies (strain = 0.1%) at 25°C: Ac-MYD (20 mM, 13 mg/mL), Ac-VIE (20 mM, 12 mg/mL). Every data point represents the mean of 10 repetitions. The error bars reflect the standard deviation of 10 repetitions. The data show that at 20 mM, Ac-MYD formed the stiffer hydrogel compared to Ac-VIE. The inset illustrates the physical forms of the hydrogels (left: Ac-VIE, right: Ac-MYD) on prolonged standing under ambient conditions.</p

Summary of results for Run 1.

<p>Summary of results for Run 1.</p

Model of Ac-YLD crystal structure.

<p>Stick model illustrating the network of hydrogen bonding linking one molecule of water and three molecules of Ac-YLD together. The intermolecular hydrogen bonds are labeled accordingly. Residue identities are labeled for the first row of peptides only. Most of the hydrogen atoms have been omitted for clarity. The diagram illustrates that the aromatic rings of tyrosine engage in π-π stacking interactions.</p