Cryo-EM structures of CusA reveal a mechanism of metal-ion export

Gram-negative bacteria utilize the resistance-nodulation-cell division (RND) superfamily of efflux pumps to expel a variety of toxic compounds from the cell. The CusA membrane protein, which recognizes and extrudes biocidal Cu(I) and Ag(I) ions, belongs to the heavy-metal efflux (HME) subfamily of RND efflux pumps. We here report four structures of the trimeric CusA heavy-metal efflux pump in the presence of Cu(I) using single-particle cryo-electron microscopy (cryo-EM). We discover that different CusA protomers within the trimer are able to bind Cu(I) ions simultaneously. Our structural data combined with molecular dynamics (MD) simulations allow us to propose a mechanism for ion transport where each CusA protomer functions independently within the trimer. The bacterial RND superfamily of efflux pumps mediate resistance to a variety of biocides, including Cu(I) and Ag(I) ions. Here we report four cryo-EM structures of the trimeric CusA pump in the presence of Cu(I). Combined with MD simulations, our data indicate that each CusA protomer within the trimer recognizes and extrudes Cu(I) independently. [Abstract copyright: Copyright © 2021 Moseng et al.

HIV-1 vaccines and adaptive trial designs.

Developing a vaccine against the human immunodeficiency virus (HIV) poses an exceptional challenge. There are no documented cases of immune-mediated clearance of HIV from an infected individual, and no known correlates of immune protection. Although nonhuman primate models of lentivirus infection have provided valuable data about HIV pathogenesis, such models do not predict HIV vaccine efficacy in humans. The combined lack of a predictive animal model and undefined biomarkers of immune protection against HIV necessitate that vaccines to this pathogen be tested directly in clinical trials. Adaptive clinical trial designs can accelerate vaccine development by rapidly screening out poor vaccines while extending the evaluation of efficacious ones, improving the characterization of promising vaccine candidates and the identification of correlates of immune protection

Cryo-Electron Microscopy Structure of an Acinetobacter baumannii Multidrug Efflux Pump

Acinetobacter baumannii is a successful human pathogen which has emerged as one of the most problematic and highly antibiotic-resistant Gram-negative bacteria worldwide. Multidrug efflux is a major mechanism that A. baumannii uses to counteract the action of multiple classes of antibiotics, such as β-lactams, tetracyclines, fluoroquinolones, and aminoglycosides. Here, we report a cryo-electron microscopy (cryo-EM) structure of the prevalent A. baumannii AdeB multidrug efflux pump, which indicates a plausible pathway for multidrug extrusion. Overall, our data suggest a mechanism for energy coupling that powers up this membrane protein to export antibiotics from bacterial cells. Our studies will ultimately inform an era in structure-guided drug design to combat multidrug resistance in these Gram-negative pathogens.Resistance-nodulation-cell division multidrug efflux pumps are membrane proteins that catalyze the export of drugs and toxic compounds out of bacterial cells. Within the hydrophobe-amphiphile subfamily, these multidrug-resistant proteins form trimeric efflux pumps. The drug efflux process is energized by the influx of protons. Here, we use single-particle cryo-electron microscopy to elucidate the structure of the Acinetobacter baumannii AdeB multidrug efflux pump embedded in lipidic nanodiscs to a resolution of 2.98 Å. We found that each AdeB molecule within the trimer preferentially takes the resting conformational state in the absence of substrates. We propose that proton influx and drug efflux are synchronized and coordinated within the transport cycle

Novel chloroacetamido compound CWR-J02 is an anti-inflammatory glutaredoxin-1 inhibitor

<div><p>Glutaredoxin (Grx1) is a ubiquitously expressed thiol-disulfide oxidoreductase that specifically catalyzes reduction of S-glutathionylated substrates. Grx1 is known to be a key regulator of pro-inflammatory signaling, and Grx1 silencing inhibits inflammation in inflammatory disease models. Therefore, we anticipate that inhibition of Grx1 could be an anti-inflammatory therapeutic strategy. We used a rapid screening approach to test 504 novel electrophilic compounds for inhibition of Grx1, which has a highly reactive active-site cysteine residue (pKa 3.5). From this chemical library a chloroacetamido compound, CWR-J02, was identified as a potential lead compound to be characterized. CWR-J02 inhibited isolated Grx1 with an IC₅₀ value of 32 μM in the presence of 1 mM glutathione. Mass spectrometric analysis documented preferential adduction of CWR-J02 to the active site Cys-22 of Grx1, and molecular dynamics simulation identified a potential non-covalent binding site. Treatment of the BV2 microglial cell line with CWR-J02 led to inhibition of intracellular Grx1 activity with an IC₅₀ value (37 μM). CWR-J02 treatment decreased lipopolysaccharide-induced inflammatory gene transcription in the microglial cells in a parallel concentration-dependent manner, documenting the anti-inflammatory potential of CWR-J02. Exploiting the alkyne moiety of CWR-J02, we used click chemistry to link biotin azide to CWR-J02-adducted proteins, isolating them with streptavidin beads. Tandem mass spectrometric analysis identified many CWR-J02-reactive proteins, including Grx1 and several mediators of inflammatory activation. Taken together, these data identify CWR-J02 as an intracellularly effective Grx1 inhibitor that may elicit its anti-inflammatory action in a synergistic manner by also disabling other pro-inflammatory mediators. The CWR-J02 molecule provides a starting point for developing more selective Grx1 inhibitors and anti-inflammatory agents for therapeutic development.</p></div

J02 inhibits cytokine expression in BV2 cells, not due to induction of cytotoxicity.

<p><b>A</b>, Cytokine mRNA levels in lysates from BV2 cells pre-treated with indicated concentrations of J02 for 30 min and stimulated with 1μg/ml LPS for 60 min. n = 3 ± SEM. **p<0.01. RQ—relative quantity. <b>B</b>, ATP content in lysates from BV2 cells pre-treated with indicated concentrations of J02 for 30 min, allowed to recover for indicated amount of time. ATP levels were normalized to those from control (DMSO treated) cells. n = 3 ± SEM. Arrow indicates J02 concentration used in cell-based assays. <b>C</b>, mRNA levels in BV2 treated with non-targeting (NT) scrambled siRNA (control) or Grx1-targted (GLRX) siRNA for 24 hours, treated with J02 (32 μM) or DMSO for 30 min, and stimulated with 100 ng/ml LPS for 24 hours. n ≥ 3 ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. RQ—relative quantity.</p

J02 interactome for BV2 cells–proteins involved in inflammatory responses.

<p>BV2 cells were treated with 40 μM J02 or DMSO. Resulting cell pellets were lysed, linked to a biotin azide probe, and run over a streptavidin column. <b>A</b>, Pulled down proteins were identified using mass spectrometry (see SI Materials and Methods section for further details). A, Inflammatory proteins shown to be regulated <i>via</i> S-glutathionylation and identified in the mass spectrometry dataset (supplemental <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187991#pone.0187991.s001" target="_blank">S1 Table</a></b>), including glutaredoxin-1. <b>B</b>, Grx1 and p65 are detected in J02-adducted samples. BV2 cells were treated with 40 μM J02 or equivalent volume DMSO for 30 min. Medium was changed, and cells were allowed to recover for 60 min. Resulting cell pellets were lysed, adducted with azide fluorescent fluorophore, and run over streptavidin beads to precipitate J02-adducted proteins. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187991#sec015" target="_blank">Materials and Methods</a> for further details. Eluted proteins were separated on SDS-PAGE gel, transferred to PVDF membrane, and probed with antibodies against Grx1 (left) and p65 (right).</p

J02 inhibits Grx1 in BV2 murine microglia cells.

<p><b>A</b>, Grx1 specific activity in BV2 cell lysates. Cells were treated with 32 μM J02 or DMSO for 30 min, medium changed, and cells were allowed to recover for 1 hour before lysing and assaying activity. <b>B</b>, <i>glrx1</i> mRNA levels in BV2 cells treated as in A, or BV2 cells pre-incubated with 32 μM J02 or DMSO for 30 min and then treated with 1 μg/ml LPS for 1 hour. <b>C</b>, Immunoblot of BV2 murine microglial cells treated as in <b>A</b>. Densitometric quantification on right. <b>D</b>, Immunoblot of purified Grx1 incubated with 96 μM J02 or DMSO. Incubation was performed in phosphate buffer pH 7.4 for 30 min at room temperature. Densitometric quantification is shown on right. E, Grx1 activity in BV2 cells treated with 40 μM gliotoxin (sporidesmin analog) or DMSO as in A. n = 3 ± SEM. *p<0.05, **p<0.01, ***p<0.001. RQ—relative quantity.</p

Novel chloroacetamide J02 inhibits Grx1 as isolated enzyme.

<p><b>A</b>, J02 chemical structure. <b>B</b>, % enzyme inhibition by J02 of Grx1 or GR as isolated enzymes. Grx1 or GR were pre-incubated with indicated concentrations of J02 in complete assay mix for 30 min. Enzyme activity was then measured using standard spectrophotometric assays. n≥3±SEM. <b>C</b>, Identification of J02 adducted to the active site cysteine of Grx1 by mass spectrometry. The tandem spectrum was collected for the m/z 654.3 [M+H]⁺³ ion that corresponds to the peptide </p><p>¹⁴VVVFIKPTCPYCR²⁶</p> modified by J02 adduction at Cys-22 and carbamidomethylation at Cys-25. Fragmentation of the parent ion revealed the presence of the cysteinyl-J02 moiety identified by a series of unique and subsequent “y” ions (y₄ –y₁₂). These fragments unambiguously confirm Cys-22 as the site of J02 adduction. The inset on the upper left of panel C of <b>Fig 1</b> indicates the observed fragment ions of the peptide containing modified Cys-22, labeled according to Biemann nomenclature. Of the five cysteine residues on Grx1, only cysteine-22 was found to be adducted under these conditions. <b>D</b>, J02 inhibition of Grx1 isolated enzyme activity in a concentration- and time-dependent manner. Grx1 (40 milliunits (nmol substrate/min)) was incubated with indicated concentrations of J02 in 0.33 M sodium potassium phosphate buffer pH 7.4 at 30°C for indicated time. The mixture was then diluted 20-fold into complete assay mix, and standard spectrophotometric assay was performed. <b>E</b>, modified Kitz-Wilson plot for Grx1 inactivation by J02. Grx1 (4 milliunits) was pre-incubated with various (10–90 μM) concentrations of J02 in 0.33 M sodium potassium phosphate buffer pH 7.4 for 5 min at 30°C. A separate experiment verified the log linear relationship between J02 concentration and % Grx1 inhibition for the range of experimental conditions (see <b>Fig 1D</b>). K_<i>I</i> and k_<i>inact</i> were determined according to the relationship ln(E₀/E_t)/t = k_i<i>nact</i>[I]/(K_<i>I</i> + [I]), where E₀ refers to Grx1 activity at time zero, and Et refers to Grx1 activity after 5 min pre-incubation, [I] refers to J02 concentration, K_<i>I</i> is the concentration of J02 that gives half the maximal rate of inactivation, and k_<i>inact</i> is the net rate constant for inactivation. The K_<i>I</i> for J02 is 40 μM and k_<i>inact</i> is 0.5 min^-1. n = 3 ± SEM.<p></p