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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 181 records · Page 10
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Application of Cell Painting for chemical hazard evaluation in support of screening-level chemical assessments

‘Cell Painting’ is an imaging-based high-throughput phenotypic profiling (HTPP) method in which cultured cells are fluorescently labeled to visualize subcellular structures (i.e., nucleus, nucleoli, endoplasmic reticulum, cytoskeleton, Golgi apparatus / plasma membrane and mitochondria) and to quantify morphological changes in response to chemicals or other perturbagens. HTPP is a high-throughput and cost-effective bioactivity screening method that detects effects associated with many different molecular mechanisms in an untargeted manner, enabling rapid in vitro hazard assessment for thousands of chemicals. Here, 1201 chemicals from the ToxCast library were screened in concentration-response up to ~100 μM in human U-2 OS cells using HTPP. A phenotype altering concentration (PAC) was estimated for chemicals active in the tested range. PACs tended to be higher than lower bound potency values estimated from a broad collection of targeted high-throughput assays, but lower than the threshold for cytotoxicity. In vitro to in vivo extrapolation (IVIVE) was used to estimate administered equivalent doses (AEDs) based on PACs for comparison to human exposure predictions. AEDs for 18/412 chemicals overlapped with predicted human exposures. Phenotypic profile information was also leveraged to identify putative mechanisms of action and group chemicals. Of 58 known nuclear receptor modulators, only glucocorticoids and retinoids produced characteristic profiles; and both receptor types are expressed in U-2 OS cells. Thirteen chemicals with profile similarity to glucocorticoids were tested in a secondary screen and one chemical, pyrene, was confirmed by an orthogonal gene expression assay as a novel putative GR modulating chemical. Most active chemicals demonstrated profiles not associated with a known mechanism-of-action. However, many structurally related chemicals produced similar profiles, with exceptions such as diniconazole, whose profile differed from other active conazoles. Overall, the present study demonstrates how HTPP can be applied in screening-level chemical assessments through a series of examples and brief case studies.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Analysis of 26S Proteasome Activity across Arabidopsis Tissues

Plants utilize the ubiquitin proteasome system (UPS) to orchestrate numerous essential cellular processes, including the rapid responses required to cope with abiotic and biotic stresses. The 26S proteasome serves as the central catalytic component of the UPS that allows for the proteolytic degradation of ubiquitin-conjugated proteins in a highly specific manner. Despite the increasing number of studies employing cell-free degradation assays to dissect the pathways and target substrates of the UPS, the precise extraction methods of highly potent tissues remain unexplored. Here, we utilize a fluorogenic reporting assay using two extraction methods to survey proteasomal activity in different Arabidopsis thaliana tissues. This study provides new insights into the enrichment of activity and varied presence of proteasomes in specific plant tissues.

26S proteasome↗
DOE OSTI2023

Development of the Safe and Broad‐Spectrum Aldehyde and Ketoamide Mpro inhibitors Derived from the Constrained α, γ‐AA Peptide Scaffold

Abstract SARS‐CoV‐2 is still wreaking havoc all over the world with surging morbidity and high mortality. The main protease (M pro ) is essential in the replication of SARS‐CoV‐2, enabling itself an active target for antiviral development. Herein, we reported the design and synthesis of a new class of peptidomimetics‐constrained α, γ‐AA peptides, based on which a series of aldehyde and ketoamide inhibitors of the M pro of SARS‐CoV‐2 were prepared. The lead compounds showed excellent inhibitory activity in the FRET‐based M pro enzymatic assay not only for the M pro of SARS‐CoV‐2 but also for SARS‐CoV and MERS‐CoV, along with HCoVs like HCoV‐OC43, HCoV‐229E, HCoV‐NL63 and HKU1. The X‐ray crystallographic results demonstrated that our compounds form a covalent bond with the catalytic Cys145. They also demonstrated effective antiviral activity against live SARS‐CoV‐2. Overall, the results suggest that α, γ‐AA peptide could be a promising molecular scaffold in designing novel M pro inhibitors of SARS‐CoV‐2 and other coronaviruses.

Chemistry↗
DOE OSTI2021

Screening and Validation of Molecular Targeted Radiosensitizers

The development of molecular targeted drugs with radiation and chemotherapy is critically important for improving the outcomes of patients with hard-to-treat, potentially curable cancers. However, too many preclinical studies have not translated into successful radiation oncology trials. Major contributing factors to this insufficiency include poor reproducibility of preclinical data, inadequate preclinical modeling of intertumoral genomic heterogeneity that influences treatment sensitivity in the clinic, and a reliance on tumor growth delay instead of local control (TCD50) endpoints. There exists an urgent need to overcome these barriers to facilitate successful clinical translation of targeted radiosensitizers. To this end, we have used 3-dimensional (3D) cell culture assays to better model tumor behavior in vivo. Examples of successful prediction of in vivo effects with these 3D assays include radiosensitization of head and neck cancers by inhibiting epidermal growth factor receptor or focal adhesion kinase signaling, and radioresistance associated with oncogenic mutation of KRAS. To address the issue of tumor heterogeneity, we leveraged institutional resources that allow high-throughput 3D screening of radiation combinations with small-molecule inhibitors across genomically characterized cell lines from lung, head and neck, and pancreatic cancers. This high-throughput screen is expected to uncover genomic biomarkers that will inform the successful clinical translation of targeted agents from the National Cancer Institute Cancer Therapy Evaluation Program portfolio and other sources. Screening “hits” need to be subjected to refinement studies that include clonogenic assays, addition of disease-specific chemotherapeutics, target/biomarker validation, and integration of patient-derived tumor models. The chemoradiosensitizing activities of the most promising drugs should be confirmed in TCD50 assays in xenograft models with or without relevant biomarker and using clinically relevant radiation fractionation. We predict that appropriately validated and biomarker-directed targeted therapies will have a higher likelihood than past efforts of being successfully incorporated into the standard management of hard-to-treat tumors.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2021

LOXG473A induces the formation of osteoclasts in RAW264.7 cells via IL-6/JAK2/STAT3 signaling

Formation of osteoclasts is known to be closely associated with osteoporosis progression. LOX is a key enzyme that catalyzes the synthesis of collagen, which is the new mediator in osteoclast formation. However, the effect of LOXG473A on of osteoclast formation needs to be explored. Thereby, we sought to explore the effect of LOX{sub G473A} on formation of osteoclasts and its underlying mechanism. To investigate the function of LOX{sub G473A} in osteoclast formation, Raw264.7 cells were stably transfected with LOX-WT or LOX-MUT (LOX{sub G473A}). Real-time PCR and western blotting were used to detect the relative levels of osteoclast formation related genes and proteins. TRAP staining and immunofluorescence staining were used to test the ability of Raw264.7 cells to form osteoclasts and the ability of cells to form rings, respectively. Bone erosion assay was used to test bone resorptive activity. The data indicated that LOX{sub G473A} significantly enhanced the ability of osteoclasts forming, ring-forming and bone resorpting in Raw264.7 cells. Mechanically, LOX{sub G473A} upregulated the expressions of NFATC1, ACP5, CTSK, IL-6, and the proportion of p-JAK2/JAK2 and p-STAT3/STAT3, thereby promoting the formation of osteoclasts. In conclusion, we have verified that LOX{sub G473A} induces the proliferation of osteoclasts in Raw264.7 cells via IL-6/JAK2/STAT3 signaling, suggesting a novel strategy for studying osteoporosis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Identification of conserved skeletal enhancers associated with craniosynostosis risk genes

Abstract Craniosynostosis, defined by premature fusion of one or multiple cranial sutures, is a common congenital defect affecting more than 1/2000 infants and results in restricted brain expansion. Single gene mutations account for 15%–20% of cases, largely as part of a syndrome, but the majority are nonsyndromic with complex underlying genetics. We hypothesized that the two noncoding genomic regions identified by a GWAS for craniosynostosis contain distal regulatory elements for the risk genes BMPER and BMP2. To identify such regulatory elements, we surveyed conserved noncoding sequences from both risk loci for enhancer activity in transgenic Danio rerio. We identified enhancers from both regions that direct expression to skeletal tissues, consistent with the endogenous expression of bmper and bmp2. For each locus, we also found a skeletal enhancer that also contains a sequence variant associated with craniosynostosis risk. We examined the activity of each enhancer during craniofacial development and found that the BMPER-associated enhancer is active in the restricted region of cartilage closely associated with frontal bone initiation. The same enhancer is active in mouse skeletal tissues, demonstrating evolutionarily conserved activity. Using enhanced yeast one-hybrid assays, we identified transcription factors that bind each enhancer and observed differential binding between alleles, implicating multiple signaling pathways. Our findings help unveil the genetic mechanism of the two craniosynostosis risk loci. More broadly, our combined in vivo approach is applicable to many complex genetic diseases to build a link between association studies and specific genetic mechanisms.

He(何璇), Xuan Anita (ORCID:0000000313221972)↗
DOE OSTI2023

Secreted Effector Proteins of Poplar Leaf Spot and Stem Canker Pathogen Sphaerulina musiva Manipulate Plant Immunity and Contribute to Virulence in Diverse Ways

We report fungal effectors play critical roles in manipulating plant immune responses and promoting colonization. Sphaerulina musiva is a heterothallic ascomycete fungus that causes Septoria leaf spot and stem canker disease in poplar (Populus spp.) plantations. This disease can result in premature defoliation, branch and stem breakage, increased mortality, and plantation failure. However, little is known about the interaction between S. musiva and poplar. Previous work predicted 142 candidate secreted effector proteins in S. musiva (SmCSEPs), 19 of which were selected for further functional characterization in this study. SmCSEP3 induced plant cell death in Nicotiana benthamiana, while 8 out of 19 tested SmCSEPs suppressed cell death. The signal peptides of these eight SmCSEPs exhibited secretory activity in a yeast signal sequence trap assay. Confocal microscopy revealed that four of these eight SmCSEPs target both the cytoplasm and the nucleus, whereas four predominantly localize to discrete punctate structures. Pathogen challenge assays in N. benthamiana demonstrated that the transient expression of six SmCSEPs promoted Fusarium proliferatum infection. The expression of these six SmCSEP genes were induced during infection. SmCSEP2, SmCSEP13, and SmCSEP25 suppressed chitin-triggered reactive oxygen species burst and callose deposition in N. benthamiana. The candidate secreted effector proteins of S. musiva target multiple compartments in the plant cell and modulate different pattern-triggered immunity pathways.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Optimizing Cell-based Antimicrobials through Pooled Genomic Libraries

DNA synthesis and assembly technologies ushered in through synthetic biology have great promise for biomanufacturing, bioremediation, and the development of living therapeutics. Unfortunately, predicting sequence to function relationships, including for biosynthetic pathways expressed in a new host organism, is difficult and often requires many iterative cycles of design, construction, and testing. We are working to develop data-driven approaches to identify the genetic determinants of growth defects and productivity for the expression of a cell-based antimicrobial. We assayed the growth, pigment production, and antimicrobial activity of a collection of over 10,000 genetic mutants of the violacein biosynthetic pathway and sequenced the genetic variation of these mutants. Through this project, we have developed an innovative codebase to automate the determination of pigmentation and antimicrobial clearing diameter for tens of thousands of genetic mutants cultivated on agar dishes. Further, we have written DNA sequence analysis code to demultiplex & provide consensus sequences from high-throughput PacBio long-read circular consensus sequencing (CCS) datasets. From this foundation, we plan to map DNA sequence to function to predict an optimal genetic design to maximize antimicrobial activity while minimizing deleterious growth effects. The workflows and algorithms developed through this project can be broadly applied to other engineered functions in microbes, uncovering sequence to function relationships for complex phenotypes where function impacts fitness.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

A cell type-aware framework for nominating non-coding variants in Mendelian regulatory disorders

Abstract Unsolved Mendelian cases often lack obvious pathogenic coding variants, suggesting potential non-coding etiologies. Here, we present a single cell multi-omic framework integrating embryonic mouse chromatin accessibility, histone modification, and gene expression assays to discover cranial motor neuron (cMN)cis-regulatory elements and subsequently nominate candidate non-coding variants in the congenital cranial dysinnervation disorders (CCDDs), a set of Mendelian disorders altering cMN development. We generate single cell epigenomic profiles for ~86,000 cMNs and related cell types, identifying ~250,000 accessible regulatory elements with cognate gene predictions for ~145,000 putative enhancers. We evaluate enhancer activity for 59 elements using an in vivo transgenic assay and validate 44 (75%), demonstrating that single cell accessibility can be a strong predictor of enhancer activity. Applying our cMN atlas to 899 whole genome sequences from 270 genetically unsolved CCDD pedigrees, we achieve significant reduction in our variant search space and nominate candidate variants predicted to regulate known CCDD disease genesMAFB, PHOX2A, CHN1, andEBF3– as well as candidates in recurrently mutated enhancers through peak- and gene-centric allelic aggregation. This work delivers non-coding variant discoveries of relevance to CCDDs and a generalizable framework for nominating non-coding variants of potentially high functional impact in other Mendelian disorders.

Science & Technology - Other Topics↗
DOE OSTI2024

Potential biological control agents of Geosmithia morbida restrict fungal pathogen growth via mycoparasitism and antibiosis

Abstract Thousand cankers disease of Juglans (walnut) and Pterocarya (wingnut) spp. (Fagales: Juglandaceae) is caused by the fungal pathogen Geosmithia morbida Kolarík, Freeland, Utley, and Tisserat (Hypocreales: Bionectriaceae) and bark beetle pest/vector, Pityophthorus juglandis Blackman (Coleoptera: Curculionidae). To further the development of biological management strategies for thousand cankers disease, we assessed the ability of 14 endophytic Trichoderma (Hypocreales: Hypocreaceae) isolates and the commercially available isolate T. afroharzianum strain KRL-AG2 to inhibit the in vitro growth of three different G. morbida isolates via mycoparasitism and antibiosis. To identify factors that may affect field success of candidate biological control agents, we quantified the growth responses of Trichoderma spp. and the commercially available entomopathogenic fungus, Beauveria bassiana (Bals.-Criv.) Vuill. (Hypocreales: Cordycipitaceae) strain GHA, to the plant secondary metabolite and antimicrobial compound, juglone in vitro. A total of 12 Trichoderma isolates (from six different Trichoderma species) demonstrated antagonistic activity towards G. morbida in dual-plate assays. Juglone consistently reduced the growth of B. bassiana strain GHA and 14 of the 15 screened Trichoderma isolates in vitro. Additionally, one metabolite-producing Trichoderma isolate, TN4-47, completely inhibited the growth of all three G. morbida isolates across all tested metabolite concentrations and had comparatively greater tolerance to juglone compared to other Trichoderma isolates. Future lines of research should focus on characterizing the active antagonistic compound present in the metabolite filtrates, determine the mode of action of the active component(s), and elucidate how abiotic and biotic factors may influence the growth, persistence, and antagonistic activity of candidate biological control agents in planta .

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Polydopamine Surface Coating Synergizes the Antimicrobial Activity of Silver Nanoparticles

Metal nanoparticles, especially silver nanoparticles (AgNPs), have drawn increasing attention for antimicrobial applications. Most studies have emphasized on the correlations between the antibacterial potency of AgNPs and the kinetics of metallic to ionic Ag conversion, while other antimicrobial mechanisms have been underestimated. In this work, we focused on the surface effects of polydopamine (PDA) coating on the antimicrobial activity of AgNPs. A method of fast deposition of PDA was used to synthesize the PDA-AgNPs with controllable coating thickness ranging from 3 to 25 nm. The antimicrobial activities of the PDA-AgNPs were analyzed by fluorescence-based growth curve assays on Escherichia coli. The results indicated that the PDA-AgNPs exhibited significantly higher antibacterial activities than poly(vinylpyrrolidone)-passivated AgNPs (PVP-AgNPs) and PDA themselves. It was found that the PDA coating synergized with the AgNPs to prominently enhance the potency of the PDA-AgNPs against bacteria. Additionally, the analysis of X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy elucidated that the synergistic effects could be originated from the interaction/coordination between Ag and catechol group on the PDA coating. The synergistic effects led to increased generation of reactive oxygen species and the consequent bacterial damage. Overall, these findings demonstrated the importance of the surface effects on the antimicrobial properties of AgNPs. The underlying molecular mechanisms have shined light on the future development of more potent metal nanoparticle-based antimicrobial agents.

36 MATERIALS SCIENCE↗
DOE OSTI2022

Sourcing thermotolerant poly(ethylene terephthalate) hydrolase scaffolds from natural diversity

Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases. We successfully express, purify, and assay 51 enzymes from seven distinct phylogenetic groups; observing PET hydrolysis activity on amorphous PET film from 37 enzymes in reactions spanning pH from 4.5–9.0 and temperatures from 30–70 °C. We conduct PET hydrolysis time-course reactions with the best-performing enzymes, where we observe differences in substrate selectivity as function of PET morphology. We employed X-ray crystallography and AlphaFold to examine the enzyme architectures of all 74 candidates, revealing protein folds and accessory domains not previously associated with PET deconstruction. Overall, this study expands the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Phylogenomic Analyses of Non-Dikarya Fungi Supports Horizontal Gene Transfer Driving Diversification of Secondary Metabolism in the Amphibian Gastrointestinal Symbiont, Basidiobolus

Research into secondary metabolism (SM) production by fungi has resulted in the discovery of diverse, biologically active compounds with significant medicinal applications. The fungi rich in SM production are taxonomically concentrated in the subkingdom Dikarya, which comprises the phyla Ascomycota and Basidiomycota. Here, we explore the potential for SM production in Mucoromycota and Zoopagomycota, two phyla of nonflagellated fungi that are not members of Dikarya, by predicting and identifying core genes and gene clusters involved in SM. The majority of non-Dikarya have few genes and gene clusters involved in SM production except for the amphibian gut symbionts in the genus Basidiobolus. Basidiobolus genomes exhibit an enrichment of SM genes involved in siderophore, surfactin-like, and terpene cyclase production, all these with evidence of constitutive gene expression. Gene expression and chemical assays also confirm that Basidiobolus has significant siderophore activity. Finally, the expansion of SMs in Basidiobolus are partially due to horizontal gene transfer from bacteria, likely as a consequence of its ecology as an amphibian gut endosymbiont.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Multi-scale organization in communicating active matter

The emergence of collective motion among interacting, self-propelled agents is a central paradigm in non-equilibrium physics. Examples of such active matter range from swimming bacteria and cytoskeletal motility assays to synthetic self-propelled colloids and swarming microrobots. Remarkably, the aggregation capabilities of many of these systems rely on a theme as fundamental as it is ubiquitous in nature: communication. Despite its eminent importance, the role of communication in the collective organization of active systems is not yet fully understood. Here we report on the multi-scale self-organization of interacting self-propelled agents that locally process information transmitted by chemical signals. We show that this communication capacity dramatically expands their ability to form complex structures, allowing them to self-organize through a series of collective dynamical states at multiple hierarchical levels. Our findings provide insights into the role of self-sustained signal processing for self-organization in biological systems and open routes to applications using chemically driven colloids or microrobots.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Protein Structure Inspired Discovery of a Novel Inducer of Anoikis in Human Melanoma

Drug discovery historically starts with an established function, either that of compounds or proteins. This can hamper discovery of novel therapeutics. As structure determines function, we hypothesized that unique 3D protein structures constitute primary data that can inform novel discovery. Using a computationally intensive physics-based analytical platform operating at supercomputing speeds, we probed a high-resolution protein X-ray crystallographic library developed by us. For each of the eight identified novel 3D structures, we analyzed binding of sixty million compounds. Top-ranking compounds were acquired and screened for efficacy against breast, prostate, colon, or lung cancer, and for toxicity on normal human bone marrow stem cells, both using eight-day colony formation assays. Effective and non-toxic compounds segregated to two pockets. One compound, Dxr2-017, exhibited selective anti-melanoma activity in the NCI-60 cell line screen. In eight-day assays, Dxr2-017 had an IC50 of 12 nM against melanoma cells, while concentrations over 2100-fold higher had minimal stem cell toxicity. Dxr2-017 induced anoikis, a unique form of programmed cell death in need of targeted therapeutics. Our findings demonstrate proof-of-concept that protein structures represent high-value primary data to support the discovery of novel acting therapeutics. This approach is widely applicable.

Oncology↗
DOE OSTI2025

N-terminal domain swapping: A new paradigm for spermidine/spermine N -acetyltransferase (SSAT) protein structures?

Enterococcus faecalis is a multi-drug-resistant human pathogen that is found in a variety of environments and is challenging to treat. Under stress conditions, some bacteria regulate intracellular polyamine concentrations via polyamine acetyltransferases to reduce their toxicity. The E. faecalis genome encodes two polyamine acetyltransferases: PmvE and BltD. Both of these proteins belong to the Gcn5-related N-acetyltransferase (GNAT) superfamily. It is unclear why there are two enzymes with similar substrate specificities in this organism. To better understand the structure/function relationship of the E. faecalis BltD enzyme, we determined its crystal structure and performed additional assays to explore its oligomeric state and enzymatic activity. The goal was to determine whether there were structural or catalytic differences between this enzyme and other polyamine acetyltransferases that could explain this redundancy and be exploited for future development of targeted inhibitors for this important human pathogen. We found the BltD enzyme was structurally unique due to its N-terminal domain swapped dimer. However, this enzyme adopts a catalytically active monomer rather than dimer in solution. This indicates the crystal structure we obtained may represent a state that forms at high protein and salt concentrations and at low pH used during crystallization. The BltD dimer found in the crystal may represent a unique view of how an inhibitory peptide or molecule could be designed to occupy its active site. Additionally, this structure shows the extensive flexibility of the N-terminal portion of the E. faecalis BltD enzyme.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Effect of docetaxel on mechanical properties of ovarian cancer cells

Docetaxel could inhibit the proliferation of tumor cells by targeting microtubules. The extension of cellular microtubules plays an important role in the invasion and metastasis of tumor cells. This paper aims to study the distribution and mechanical properties of cytoskeletal proteins with low concentration of docetaxel. MTT assay was used to detect the minimum drug activity concentration of docetaxel on SKOV-3 cells, fluorescence staining was used to analyze the distribution of cytoskeleton proteins, scanning electron microscopy(SEM) was used to observe the morphology of single cells, and atomic force microscopy(AFM) was used to determine the microstructure and mechanical properties of cells. The results showed that the IC{sub 10} of docetaxel was 1 ng/ml. Docetaxel can effectively inhibit the formation of cell pseudopodia, hinder the indirectness between cells, reduce the cell extension area, and make the cells malformed. In addition, when AFM analyzes the effects of drugs on cell microstructure and mechanical properties, the average cell surface roughness and cell height are positively correlated with the concentration of docetaxel. Especially when the concentration was 100 ng/ml, the adhesion decreased by 37.04% and Young's modulus increased by 1.57 times compared with the control group. This may be because docetaxel leads to microtubule remodeling and membrane protein aggregation, which affects cell microstructure and increases cell strength, leading to significant changes in the mechanical properties of ovarian cells. This is of great significance to the study of the formation mechanism of tumor cell invasion and migration activities mediated by actin.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Molecular basis for polysaccharide recognition and modulated ATP hydrolysis by the O antigen ABC transporter

O antigens are ubiquitous protective extensions of lipopolysaccharides in the extracellular leaflet of the Gram-negative outer membrane. Following biosynthesis in the cytosol, the lipid-linked polysaccharide is transported to the periplasm by the WzmWzt ABC transporter. Often, O antigen secretion requires the chemical modification of its elongating terminus, which the transporter recognizes via a carbohydrate-binding domain (CBD). Here, using components from A. aeolicus, we identify the O antigen structure with methylated mannose or rhamnose as its cap. Crystal and cryo electron microscopy structures reveal how WzmWzt recognizes this cap between its carbohydrate and nucleotide-binding domains in a nucleotide-free state. ATP binding induces drastic conformational changes of its CBD, terminating interactions with the O antigen. ATPase assays and site directed mutagenesis reveal reduced hydrolytic activity upon O antigen binding, likely to facilitate polymer loading into the ABC transporter. Our results elucidate critical steps in the recognition and translocation of polysaccharides by ABC transporters.

59 BASIC BIOLOGICAL SCIENCES↗