Structural Dynamics of 15-Lipoxygenase‑2 via Hydrogen–Deuterium Exchange

Eicosanoids are inflammatory signaling lipids that are biosynthesized in response to cellular injury or threat. They were originally thought to be pro-inflammatory molecules, but members of at least one subclass, the lipoxins, are able to resolve inflammation. One step in lipoxin synthesis is the oxygenation of arachidonic acid by 15-lipoxygenase (15-LOX). 15-LOX contains two domains: a Ca²⁺ binding PLAT domain and a catalytic domain. 15-LOX is a soluble cytosolic protein until binding of Ca²⁺ to the PLAT domain promotes translocation to the membrane surface. The role of 15-LOX structural dynamics in this translocation has remained unclear. We investigated the dynamics of 15-LOX isoform B (15-LOX-2) upon binding of Ca²⁺ and ligands, as well as upon membrane association using hydrogen–deuterium exchange mass spectrometry (HDX-MS). We used HDX-MS to probe the solvent accessibility and backbone flexibility of 15-LOX-2, revealing significant differences in deuterium incorporation between the PLAT and catalytic domains, with the PLAT domain demonstrating higher flexibility. Comparison of HDX for 15-LOX-2 in the presence and absence of Ca²⁺ indicates there are few differences in structural dynamics. Furthermore, our HDX results involving nanodisc-associated 15-LOX-2 suggest that significant structural and dynamic changes in 15-LOX-2 are not required for membrane association. Our results also show that a substrate lipid binding to the active site in the catalytic domain does induce changes in incorporation of deuterium into the PLAT domain. Overall, our results challenge the previous hypothesis that Ca²⁺ binding induces major structural changes in the PLAT domain and support the hypothesis that is interdomain communication in 15-LOX-2

Myristoylation Exerts Direct and Allosteric Effects on Gα Conformation and Dynamics in Solution

Coupling of heterotrimeric G proteins to activated G protein-coupled receptors results in nucleotide exchange on the Gα subunit, which in turn decreases its affinity for both Gβγ and activated receptors. N-Terminal myristoylation of Gα subunits aids in membrane localization of inactive G proteins. Despite the presence of the covalently attached myristoyl group, Gα proteins are highly soluble after GTP binding. This study investigated factors facilitating the solubility of the activated, myristoylated protein. In doing so, we also identified myristoylation-dependent differences in regions of Gα known to play important roles in interactions with receptors, effectors, and nucleotide binding. Amide hydrogen–deuterium exchange and site-directed fluorescence of activated proteins revealed a solvent-protected amino terminus that was enhanced by myristoylation. Furthermore, fluorescence quenching confirmed that the myristoylated amino terminus is in proximity to the Switch II region in the activated protein. Myristoylation also stabilized the interaction between the guanine ring and the base of the α5 helix that contacts the bound nucleotide. The allosteric effects of myristoylation on protein structure, function, and localization indicate that the myristoylated amino terminus of Gα_i functions as a myristoyl switch, with implications for myristoylation in the stabilization of nucleotide binding and in the spatial regulation of G protein signaling

Observation of Two Modes of Inhibition of Human Microsomal Prostaglandin E Synthase 1 by the Cyclopentenone 15-Deoxy-Δ^12,14-prostaglandin J₂

Microsomal prostaglandin E synthase 1 (MPGES1) is an enzyme that produces the pro-inflammatory molecule prostaglandin E₂ (PGE₂). Effective inhibitors of MPGES1 are of considerable pharmacological interest for the selective control of pain, fever, and inflammation. The isoprostane, 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂), a naturally occurring degradation product of prostaglandin D₂, is known to have anti-inflammatory properties. In this paper, we demonstrate that 15d-PGJ₂ can inhibit MPGES1 by covalent modification of residue C59 and by noncovalent inhibition through binding at the substrate (PGH₂) binding site. The mechanism of inhibition is dissected by analysis of the native enzyme and the MPGES1 C59A mutant in the presence of glutathione (GSH) and glutathione sulfonate. The location of inhibitor adduction and noncovalent binding was determined by triple mass spectrometry sequencing and with backbone amide H/D exchange mass spectrometry. The kinetics, regiochemistry, and stereochemistry of the spontaneous reaction of GSH with 15d-PGJ₂ were determined. The question of whether the anti-inflammatory properties of 15d-PGJ₂ are due to inhibition of MPGES1 is discussed

Structural and Chemical Aspects of Resistance to the Antibiotic Fosfomycin Conferred by FosB from <i>Bacillus cereus</i>

The fosfomycin resistance enzymes, FosB, from Gram-positive organisms, are M²⁺-dependent thiol tranferases that catalyze nucleophilic addition of either l-cysteine (l-Cys) or bacillithiol (BSH) to the antibiotic, resulting in a modified compound with no bacteriacidal properties. Here we report the structural and functional characterization of FosB from Bacillus cereus (FosB^<i>Bc</i>). The overall structure of FosB^<i>Bc</i>, at 1.27 Å resolution, reveals that the enzyme belongs to the vicinal oxygen chelate (VOC) superfamily. Crystal structures of FosB^<i>Bc</i> cocrystallized with fosfomycin and a variety of divalent metals, including Ni²⁺, Mn²⁺, Co²⁺, and Zn²⁺, indicate that the antibiotic coordinates to the active site metal center in an orientation similar to that found in the structurally homologous manganese-dependent fosfomycin resistance enzyme, FosA. Surface analysis of the FosB^<i>Bc</i> structures show a well-defined binding pocket and an access channel to C1 of fosfomycin, the carbon to which nucleophilic addition of the thiol occurs. The pocket and access channel are appropriate in size and shape to accommodate l-Cys or BSH. Further investigation of the structures revealed that the fosfomycin molecule, anchored by the metal, is surrounded by a cage of amino acids that hold the antibiotic in an orientation such that C1 is centered at the end of the solvent channel, positioning the compound for direct nucleophilic attack by the thiol substrate. In addition, the structures of FosB^<i>Bc</i> in complex with the l-Cys-fosfomycin product (1.55 Å resolution) and in complex with the bacillithiol-fosfomycin product (1.77 Å resolution) coordinated to a Mn²⁺ metal in the active site have been determined. The l-Cys moiety of either product is located in the solvent channel, where the thiol has added to the backside of fosfomycin C1 located at the end of the channel. Concomitant kinetic analyses of FosB^<i>Bc</i> indicated that the enzyme has a preference for BSH over l-Cys when activated by Mn²⁺ and is inhibited by Zn²⁺. The fact that Zn²⁺ is an inhibitor of FosB^<i>Bc</i> was used to obtain a ternary complex structure of the enzyme with both fosfomycin and l-Cys bound

Prostanoid profiles for WT MPGES1 and variants Asp49Ala, Arg73Ala, Arg73Leu, Arg126Ala, Arg126Leu, Ser127Ala, expressed in <i>E</i>. <i>Coli</i>.

<p>Prostanoid production was measured by LC-MS/MS after 60 seconds incubations of membrane fractions with PGH₂ (10μM final concentration) and GSH (2.5mM final concentration) at room temperature. Denatured enzymes by boiling were incorporated in the activity assay as controls. All prostanoid measurements of membrane fractions are expressed as mean ± SD from two independent experiment performed in duplicates.</p

Synthesis of Bacillithiol and the Catalytic Selectivity of FosB-Type Fosfomycin Resistance Proteins

Bacillithiol (BSH) has been prepared on the gram scale from the inexpensive starting material, d-glucosamine hydrochloride, in 11 steps and 8–9% overall yield. The BSH was used to survey the substrate and metal-ion selectivity of FosB enzymes from four Gram-positive microorganisms associated with the deactivation of the antibiotic fosfomycin. The <i>in vitro</i> results indicate that the preferred thiol substrate and metal ion for the FosB from <i>Staphylococcus aureus</i> are BSH and Ni(II), respectively. However, the metal-ion selectivity is less distinct with FosB from <i>Bacillus subtilis</i>, <i>Bacillus anthracis</i>, or <i>Bacillus cereus</i>

Suggested chemical mechanism of PGH₂ isomerization to PGE₂ by MPGES1 and its active site structure highlighting the amino acids altered.

<p>(A) The thiolate of glutathione (GSH) could be stabilized by Arg126 and attack the C9 oxygen of the PGH₂ endoperoxide forming a sulfenic acid ester. An unidentified proton donor protonates the developing C11 oxyanion. This is followed by proton abstraction at C9 via Asp49. A carbonyl forms and the oxygen sulfur bond is broken forming PGE₂. The leaving GSH thiolate could again be stabilized by Arg126. The unidentified proton donor could then take up the proton from Asp49. (B) The reaction starts by proton abstraction at C9 via Asp49. A carbonyl forms and the endoperoxide bridge is broken. The thiol of GSH functions as a proton donor to the developing C11 oxyanion. After that the proton taken up by Asp49 can reprotonate the GSH thiolate. (C) The interaction between MPGES1 and GSH highlighting the positions of Asp49, Arg126 as well as Ser127 that was proposed to stabilize the GSH thiolate.</p

Glutathione transferase (GST) activity assay of purified MPGES1, expressed in <i>Baculovirus</i> infected <i>SF9 cells</i>.

<p>The enzymatic activity of purified MPGES1 was measured by the conjugation of GSH to the substrate 1-chloro-2,4-dinitrobenzene (CDNB). The spontaneous conjugation of CDNB to GSH was subtracted from each enzymatic reaction and the specific activity values were calculated based on the slope of the initial linear portion of the absorbance curve. The GST activity is expressed as mean ± SD from two independent experiment performed in triplicates.</p

Western Blot analysis of WT MPGES1 and variants Asp49Ala, Arg73Ala, Arg73Leu, Arg126Ala, Arg126Leu, Ser127Ala, expressed in <i>E</i>. <i>Coli</i>.

<p>40μg of membrane fraction was loaded into each well. The exposure time was 5 minutes. As a positive control purified MPGES1 was loaded at different concentrations, yielding: 75ng, 100ng, 150ng, 200ng and 450ng.</p

Specific activity of WT MPGES1 and variants Asp49Ala, Arg73Ala, Arg73Leu, Arg126Ala, Arg126Leu and Ser127Ala, expressed and measured in <i>E</i>. <i>Coli</i> membrane fractions.

<p>Specific activity of WT MPGES1 and variants Asp49Ala, Arg73Ala, Arg73Leu, Arg126Ala, Arg126Leu and Ser127Ala, expressed and measured in <i>E</i>. <i>Coli</i> membrane fractions.</p