12 Search Results

Mycobacterium abscessus (Mab), a nontuberculous mycobacterial (NTM) species, is an emerging pathogen with high intrinsic drug resistance. Current standard-of-care therapy results in poor outcomes, demonstrating the urgent need to develop effective antimycobacterial regimens. Through synthetic modification of spectinomycin (SPC), we have identified a distinct structural subclass of N-ethylene linked aminomethyl SPCs (eAmSPCs) that are up to 64-fold more potent against Mab over the parent SPC. Mechanism of action and crystallography studies demonstrate that the eAmSPCs display a mode of ribosomal inhibition consistent with SPC. However, they exert their increased antimicrobial activity through enhanced accumulation, largely by circumventing efflux mechanisms. Themore » N-ethylene linkage within this series plays a critical role in avoiding TetV-mediated efflux, as lead eAmSPC 2593 displays a mere fourfold susceptibility improvement against Mab ΔtetV, in contrast to the 64-fold increase for SPC. Even a minor shortening of the linkage by a single carbon, akin to 1st generation AmSPC 1950, results in a substantial increase in MICs and a 16-fold rise in susceptibility against Mab ΔtetV. These shifts suggest that longer linkages might modify the kinetics of drug expulsion by TetV, ultimately shifting the equilibrium towards heightened intracellular concentrations and enhanced antimicrobial efficacy. Furthermore, lead eAmSPCs were also shown to synergize with various classes of anti-Mab antibiotics and retain activity against clinical isolates and other mycobacterial strains. Encouraging pharmacokinetic profiles coupled with robust efficacy in Mab murine infection models suggest that eAmSPCs hold the potential to be developed into treatments for Mab and other NTM infections.« less

Na⁺ is the most abundant ion in living organisms and plays essential roles in regulating nutrient uptake, muscle contraction, and neurotransmission. The identification of Na⁺ in protein structures is crucial for gaining a deeper understanding of protein function in a physiological context. LeuT, a bacterial homolog of the neurotransmitter:sodium symporter family, uses the Na⁺ gradient to power the uptake of amino acids into cells and has been used as a paradigm for the study of Na⁺-dependent transport systems. We have devised a low-energy multi-crystal approach for characterizing low-Z (Z ≤ 20) anomalous scattering ions such as Na⁺, Mg²⁺, K⁺, andmore » Ca²⁺ by combining Bijvoet-difference Fourier syntheses for ion detection and f” refinements for ion speciation. Using the approach, we experimentally identify two Na⁺ bound near the central leucine binding site in LeuT. Using LeuT microcrystals, we also demonstrate that Na⁺ may be depleted to study conformational changes in the LeuT transport cycle.« less

Abstract Glycosylinositolphosphorylceramides (GIPCs) are the predominant lipid in the outer leaflet of the plasma membrane. Characterized GIPC glycosylation mutants have severe or lethal plant phenotypes. However, the function of the glycosylation is unclear. Previously, we characterized Arabidopsis thaliana GONST1 and showed that it was a nucleotide sugar transporter which provides GDP‐mannose for GIPC glycosylation. gonst1 has a severe growth phenotype, as well as a constitutive defense response. Here, we characterize a mutant in GONST1’s closest homolog, GONST2. The gonst2‐ 1 allele has a minor change to GIPC headgroup glycosylation. Like other reported GIPC glycosylation mutants, gonst1‐1gonst2‐1 has reduced cellulose, amore » cell wall polymer that is synthesized at the plasma membrane. The gonst2‐1 allele has increased resistance to a biotrophic pathogen Golovinomyces orontii but not the necrotrophic pathogen Botrytis cinerea . Expression of GONST2 under the GONST1 promoter can rescue the gonst1 phenotype, indicating that GONST2 has a similar function to GONST1 in providing GDP‐D‐Man for GIPC mannosylation.« less

Analyses of publicly available structural data reveal interesting insights into the impact of the three-dimensional (3D) structures of protein targets important for discovery of new drugs (e.g., G-protein-coupled receptors, voltage-gated ion channels, ligand-gated ion channels, transporters, and E3 ubiquitin ligases). The Protein Data Bank (PDB) archive currently holds > 155,000 atomic-level 3D structures of biomolecules experimentally determined using crystallography, nuclear magnetic resonance spectroscopy, and electron microscopy. The PDB was established in 1971 as the first open-access, digital-data resource in biology, and is now managed by the Worldwide PDB partnership (wwPDB; wwPDB.org). US PDB operations are the responsibility of the Researchmore » Collaboratory for Structural Bioinformatics PDB (RCSB PDB). The RCSB PDB serves millions of RCSB.org users worldwide by delivering PDB data integrated with ~40 external biodata resources, providing rich structural views of fundamental biology, biomedicine, and energy sciences. Recently published work showed that the PDB archival holdings facilitated discovery of ~90% of the 210 new drugs approved by the US Food and Drug Administration 2010–2016. Here, we review user-driven development of RCSB PDB services, examine growth of the PDB archive in terms of size and complexity, and present examples and opportunities for structure-guided drug discovery for challenging targets (e.g., integral membrane proteins).« less

Here, cytokinins are a group of mobile adenine derivatives that act as chemical signals regulating a variety of biological processes implicated in plant development and stress responses. Their synthesis, homeostasis, and signaling perception evoke complicated intracellular traffic, intercellular movement, and in short- and long-distance translocation. Over nearly two decades, subsets of membrane transporters have been recognized and implicated in the transport of cytokinins as well as the related adenylates. In this review, we aim to recapitulate the key progresses in the exploration of the transporter proteins involved in cytokinin traffic and translocation, discuss their functional implications in the cytokinin-mediated paracrinemore » and long-distance communication, and highlight some knowledge gaps and open issues toward comprehensively understanding the molecular mechanism of membrane transporters in controlling spatiotemporal distribution of cytokinin species.« less

Yarrowia lipolytica has been used to produce both citric acid and lipid-based bioproducts at high titers. In this study, we found that pH differentially affects citric acid and lipid production in Y. lipolytica W29, with citric acid production enhanced at more neutral pH’s and lipid production enhanced at more acid pH’s. To determine the mechanism governing this pH-dependent switch between citric acid and lipid production, we profiled gene expression at different pH’s and found that the relative expression of multiple transporters is increased at neutral pH. These results suggest that this pH-dependent switch is mediated at the level of citricmore » acid transport rather than changes in the expression of the enzymes involved in citric acid and lipid metabolism. In further support of this mechanism, thermodynamic calculations suggest that citric acid secretion is more energetically favorable at neutral pH’s, assuming the fully protonated acid is the substrate for secretion. Finally, collectively, these results provide new insights regarding citric acid and lipid production in Y. lipolytica and may offer new strategies for metabolic engineering and process design.« less

Plant health in natural environments depends on interactions with complex and dynamic communities comprising macro- and microorganisms. While many studies have provided insights into the composition of rhizosphere microbiomes (rhizobiomes), little is known about whether plants shape their rhizobiomes. In this work, we discuss physiological factors of plants that may govern plant–microbe interactions, focusing on root physiology and the role of root exudates. Given that only a few plant transport proteins are known to be involved in root metabolite export, we suggest novel families putatively involved in this process. Finally, building off of the features discussed in this review, andmore » in analogy to well-known symbioses, we elaborate on a possible sequence of events governing rhizobiome assembly. Recent advances in sequencing technology have been enabling high-throughput characterization of highly complex plant-associated microbial communities, which are relevant for plant health. Metagenomic approaches have been identifying the metabolic potential of microbes, and are starting to reveal functional groups of microbes, reducing the overall complexity of rhizobiomes. Metabolomic studies are uncovering differential root exudation in various environments and plant developmental stages, as well as consumption of specific exometabolites by microbes. Genome-wide association studies and other comparative genomic approaches have been revealing a link between plant genetic factors, exudation profiles, and microbiome compositions. Finally, molecular studies have been characterizing plant transport proteins necessary for interactions with specific microbes, although the transport of most nutrients and signaling molecules remains unexplored.« less