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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 1,009 records · Page 56
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

The intrinsically disordered protein TgIST from Toxoplasma gondii inhibits STAT1 signaling by blocking cofactor recruitment

Signal transducer and activator of transcription (STAT) proteins communicate from cell-surface receptors to drive transcription of immune response genes. The parasite Toxoplasma gondii blocks STAT1-mediated gene expression by secreting the intrinsically disordered protein TgIST that traffics to the host nucleus, binds phosphorylated STAT1 dimers, and occupies nascent transcription sites that unexpectedly remain silenced. Here we define a core region within internal repeats of TgIST that is necessary and sufficient to block STAT1-mediated gene expression. Cellular, biochemical, mutational, and structural data demonstrate that the repeat region of TgIST adopts a helical conformation upon binding to STAT1 dimers. The binding interface is defined by a groove formed from two loops in the STAT1 SH2 domains that reorient during dimerization. TgIST binding to this newly exposed site at the STAT1 dimer interface alters its conformation and prevents the recruitment of co-transcriptional activators, thus defining the mechanism of blocked transcription.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Muconic acid production from glucose and xylose in Pseudomonas putida via evolution and metabolic engineering

Muconic acid is a bioprivileged molecule that can be converted into direct replacement chemicals for incumbent petrochemicals and performance-advantaged bioproducts. In this study, Pseudomonas putida KT2440 is engineered to convert glucose and xylose, the primary carbohydrates in lignocellulosic hydrolysates, to muconic acid using a model-guided strategy to maximize the theoretical yield. Using adaptive laboratory evolution (ALE) and metabolic engineering in a strain engineered to express the D-xylose isomerase pathway, we demonstrate that mutations in the heterologous D-xylose:H + symporter (XylE), increased expression of a major facilitator superfamily transporter (PP_2569), and overexpression of aroB encoding the native 3-dehydroquinate synthase, enable efficient muconic acid production from glucose and xylose simultaneously. Using the rationally engineered strain, we produce 33.7 g L -1 muconate at 0.18 g L -1 h -1 and a 46% molar yield (92% of the maximum theoretical yield). This engineering strategy is promising for the production of other shikimate pathway-derived compounds from lignocellulosic sugars.

09 BIOMASS FUELS↗
DOE OSTI2022

Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform

Abstract Noncanonical redox cofactors are attractive low-cost alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P) + ) in biotransformation. However, engineering enzymes to utilize them is challenging. Here, we present a high-throughput directed evolution platform which couples cell growth to the in vivo cycling of a noncanonical cofactor, nicotinamide mononucleotide (NMN + ). We achieve this by engineering the life-essential glutathione reductase in Escherichia coli to exclusively rely on the reduced NMN + (NMNH). Using this system, we develop a phosphite dehydrogenase (PTDH) to cycle NMN + with ~147-fold improved catalytic efficiency, which translates to an industrially viable total turnover number of ~45,000 in cell-free biotransformation without requiring high cofactor concentrations. Moreover, the PTDH variants also exhibit improved activity with another structurally deviant noncanonical cofactor, 1-benzylnicotinamide (BNA + ), showcasing their broad applications. Structural modeling prediction reveals a general design principle where the mutations and the smaller, noncanonical cofactors together mimic the steric interactions of the larger, natural cofactors NAD(P) + .

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis

Enzymes with high activity are readily produced through protein engineering, but intentionally and efficiently engineering enzymes for an expanded substrate scope is a contemporary challenge. One approach to address this challenge is Substrate Multiplexed Screening (SUMS), where enzyme activity is measured on competing substrates. SUMS has long been used to rigorously quantitate native enzyme specificity, primarily for in vivo settings. SUMS has more recently found sporadic use as a protein engineering approach but has not been widely adopted by the field, despite its potential utility. Here, we develop principles of how to design and interpret SUMS assays to guide protein engineering. This rich information enables improving activity with multiple substrates simultaneously, identifies enzyme variants with altered scope, and indicates potential mutational hot-spots as sites for further engineering. These advances leverage common laboratory equipment and represent a highly accessible and customizable method for enzyme engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

An active site loop toggles between conformations to control antibiotic hydrolysis and inhibition potency for CTX-M β-lactamase drug-resistance enzymes

Abstract β-lactamases inactivate β-lactam antibiotics leading to drug resistance. Consequently, inhibitors of β-lactamases can combat this resistance, and the β-lactamase inhibitory protein (BLIP) is a naturally occurring inhibitor. The widespread CTX-M-14 and CTX-M-15 β-lactamases have an 83% sequence identity. In this study, we show that BLIP weakly inhibits CTX-M-14 but potently inhibits CTX-M-15. The structure of the BLIP/CTX-M-15 complex reveals that binding is associated with a conformational change of an active site loop of β-lactamase. Surprisingly, the loop structure in the complex is similar to that in a drug-resistant variant (N106S) of CTX-M-14. We hypothesized that the pre-established favorable loop conformation of the N106S mutant would facilitate binding. The N106S substitution results in a ~100- and 10-fold increase in BLIP inhibition potency for CTX-M-14 and CTX-M-15, respectively. Thus, this indicates that an active site loop in β-lactamase toggles between conformations that control antibiotic hydrolysis and inhibitor susceptibility. These findings highlight the role of accessible active site conformations in controlling enzyme activity and inhibitor susceptibility as well as the influence of mutations in selectively stabilizing discrete conformations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Orthogonal glycolytic pathway enables directed evolution of noncanonical cofactor oxidase

Abstract Noncanonical cofactor biomimetics (NCBs) such as nicotinamide mononucleotide (NMN + ) provide enhanced scalability for biomanufacturing. However, engineering enzymes to accept NCBs is difficult. Here, we establish a growth selection platform to evolve enzymes to utilize NMN + -based reducing power. This is based on an orthogonal, NMN + -dependent glycolytic pathway in Escherichia coli which can be coupled to any reciprocal enzyme to recycle the ensuing reduced NMN + . With a throughput of >10 6 variants per iteration, the growth selection discovers a Lactobacillus pentosus NADH oxidase variant with ~10-fold increase in NMNH catalytic efficiency and enhanced activity for other NCBs. Molecular modeling and experimental validation suggest that instead of directly contacting NCBs, the mutations optimize the enzyme’s global conformational dynamics to resemble the WT with the native cofactor bound. Restoring the enzyme’s access to catalytically competent conformation states via deep navigation of protein sequence space with high-throughput evolution provides a universal route to engineer NCB-dependent enzymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

An in silico method to assess antibody fragment polyreactivity

Antibodies are essential biological research tools and important therapeutic agents, but some exhibit non-specific binding to off-target proteins and other biomolecules. Such polyreactive antibodies compromise screening pipelines, lead to incorrect and irreproducible experimental results, and are generally intractable for clinical development. Here, we design a set of experiments using a diverse naïve synthetic camelid antibody fragment (nanobody) library to enable machine learning models to accurately assess polyreactivity from protein sequence (AUC > 0.8). Moreover, our models provide quantitative scoring metrics that predict the effect of amino acid substitutions on polyreactivity. We experimentally test our models’ performance on three independent nanobody scaffolds, where over 90% of predicted substitutions successfully reduced polyreactivity. Importantly, the models allow us to diminish the polyreactivity of an angiotensin II type I receptor antagonist nanobody, without compromising its functional properties. We provide a companion web-server that offers a straightforward means of predicting polyreactivity and polyreactivity-reducing mutations for any given nanobody sequence.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Quantifying the local adaptive landscape of a nascent bacterial community

Abstract The fitness effects of all possible mutations available to an organism largely shape the dynamics of evolutionary adaptation. Yet, whether and how this adaptive landscape changes over evolutionary times, especially upon ecological diversification and changes in community composition, remains poorly understood. We sought to fill this gap by analyzing a stable community of two closely related ecotypes (“L” and “S”) shortly after they emerged within the E. coli Long-Term Evolution Experiment (LTEE). We engineered genome-wide barcoded transposon libraries to measure the invasion fitness effects of all possible gene knockouts in the coexisting strains as well as their ancestor, for many different, ecologically relevant conditions. We find consistent statistical patterns of fitness effect variation across both genetic background and community composition, despite the idiosyncratic behavior of individual knockouts. Additionally, fitness effects are correlated with evolutionary outcomes for a number of conditions, possibly revealing shifting patterns of adaptation. Together, our results reveal how ecological and epistatic effects combine to shape the adaptive landscape in a nascent ecological community.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Rapid assembly of SARS-CoV-2 genomes reveals attenuation of the Omicron BA.1 variant through NSP6

Although the SARS-CoV-2 Omicron variant (BA.1) spread rapidly across the world and effectively evaded immune responses, its viral fitness in cell and animal models was reduced. The precise nature of this attenuation remains unknown as generating replication-competent viral genomes is challenging because of the length of the viral genome (~30 kb). Here, we present a plasmid-based viral genome assembly and rescue strategy (pGLUE) that constructs complete infectious viruses or noninfectious subgenomic replicons in a single ligation reaction with >80% efficiency. Fully sequenced replicons and infectious viral stocks can be generated in 1 and 3 weeks, respectively. By testing a series of naturally occurring viruses as well as Delta-Omicron chimeric replicons, we show that Omicron nonstructural protein 6 harbors critical attenuating mutations, which dampen viral RNA replication and reduce lipid droplet consumption. Thus, pGLUE overcomes remaining barriers to broadly study SARS-CoV-2 replication and reveals deficits in nonstructural protein function underlying Omicron attenuation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Ligand dependent interaction between PC-TP and PPARδ mitigates diet-induced hepatic steatosis in male mice

Phosphatidylcholine transfer protein (PC-TP; synonym StarD2) is a soluble lipid-binding protein that transports phosphatidylcholine (PC) between cellular membranes. To better understand the protective metabolic effects associated with hepatic PC-TP, we generated a hepatocyte-specific PC-TP knockdown (L-Pctp -/- ) in male mice, which gains less weight and accumulates less liver fat compared to wild-type mice when challenged with a high-fat diet. Hepatic deletion of PC-TP also reduced adipose tissue mass and decreases levels of triglycerides and phospholipids in skeletal muscle, liver and plasma. Gene expression analysis suggest that the observed metabolic changes are related to transcriptional activity of peroxisome proliferative activating receptor (PPAR) family members. An in-cell protein complementation screen between lipid transfer proteins and PPARs uncovered a direct interaction between PC-TP and PPARδ that was not observed for other PPARs. We confirmed the PC-TP– PPARδ interaction in Huh7 hepatocytes, where it was found to repress PPARδ-mediated transactivation. Mutations of PC-TP residues implicated in PC binding and transfer reduce the PC-TP-PPARδ interaction and relieve PC-TP-mediated PPARδ repression. Reduction of exogenously supplied methionine and choline reduces the interaction while serum starvation enhances the interaction in cultured hepatocytes. Together our data points to a ligand sensitive PC-TP– PPARδ interaction that suppresses PPAR activity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Dynamic conformational switching underlies TFIIH function in transcription and DNA repair and impacts genetic diseases

Transcription factor IIH (TFIIH) is a protein assembly essential for transcription initiation and nucleotide excision repair (NER). Yet, understanding of the conformational switching underpinning these diverse TFIIH functions remains fragmentary. TFIIH mechanisms critically depend on two translocase subunits, XPB and XPD. To unravel their functions and regulation, we build cryo-EM based TFIIH models in transcription- and NER-competent states. Using simulations and graph-theoretical analysis methods, we reveal TFIIH’s global motions, define TFIIH partitioning into dynamic communities and show how TFIIH reshapes itself and self-regulates depending on functional context. Our study uncovers an internal regulatory mechanism that switches XPB and XPD activities making them mutually exclusive between NER and transcription initiation. By sequentially coordinating the XPB and XPD DNA-unwinding activities, the switch ensures precise DNA incision in NER. Mapping TFIIH disease mutations onto network models reveals clustering into distinct mechanistic classes, affecting translocase functions, protein interactions and interface dynamics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Genome analyses reveal population structure and a purple stigma color gene candidate in finger millet

Finger millet is a key food security crop widely grown in eastern Africa, India and Nepal. Long considered a ‘poor man’s crop’, finger millet has regained attention over the past decade for its climate resilience and the nutritional qualities of its grain. To bring finger millet breeding into the 21 st century, here we present the assembly and annotation of a chromosome-scale reference genome. We show that this ~1.3 million years old allotetraploid has a high level of homoeologous gene retention and lacks subgenome dominance. Population structure is mainly driven by the differential presence of large wild segments in the pericentromeric regions of several chromosomes. Trait mapping, followed by variant analysis of gene candidates, reveals that loss of purple coloration of anthers and stigma is associated with loss-of-function mutations in the finger millet orthologs of the maize R1/B1 and Arabidopsis GL3/EGL3 anthocyanin regulatory genes. Proanthocyanidin production in seed is not affected by these gene knockouts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Architecture of the human G-protein-methylmalonyl-CoA mutase nanoassembly for B 12 delivery and repair

G-proteins function as molecular switches to power cofactor translocation and confer fidelity in metal trafficking. The G-protein, MMAA, together with MMAB, an adenosyltransferase, orchestrate cofactor delivery and repair of B 12 -dependent human methylmalonyl-CoA mutase (MMUT). The mechanism by which the complex assembles and moves a >1300 Da cargo, or fails in disease, are poorly understood. Herein, we report the crystal structure of the human MMUT-MMAA nano-assembly, which reveals a dramatic 180° rotation of the B 12 domain, exposing it to solvent. The complex, stabilized by MMAA wedging between two MMUT domains, leads to ordering of the switch I and III loops, revealing the molecular basis of mutase-dependent GTPase activation. The structure explains the biochemical penalties incurred by methylmalonic aciduria-causing mutations that reside at the MMAA-MMUT interfaces we identify here.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Affinity-matured homotypic interactions induce spectrum of PfCSP structures that influence protection from malaria infection

The generation of high-quality antibody responses to Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP), the primary surface antigen of Pf sporozoites, is paramount to the development of an effective malaria vaccine. Here we present an in-depth structural and functional analysis of a panel of potent antibodies encoded by the immunoglobulin heavy chain variable (IGHV) gene IGHV3-33, which is among the most prevalent and potent antibody families induced in the anti-PfCSP immune response and targets the Asn-Ala-Asn-Pro (NANP) repeat region. Cryo-electron microscopy (cryo-EM) reveals a remarkable spectrum of helical antibody-PfCSP structures stabilized by homotypic interactions between tightly packed fragments antigen binding (Fabs), many of which correlate with somatic hypermutation. We demonstrate a key role of these mutated homotypic contacts for high avidity binding to PfCSP and in protection from Pf malaria infection. Together, these data emphasize the importance of anti-homotypic affinity maturation in the frequent selection of IGHV3–33 antibodies and highlight key features underlying the potent protection of this antibody family.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Hydrophobic interactions dominate the recognition of a KRAS G12V neoantigen

Specificity remains a major challenge to current therapeutic strategies for cancer. Mutation associated neoantigens (MANAs) are products of genetic alterations, making them highly specific therapeutic targets. MANAs are HLA-presented (pHLA) peptides derived from intracellular mutant proteins that are otherwise inaccessible to antibody-based therapeutics. Here, we describe the cryo-EM structure of an antibody-MANA pHLA complex. Specifically, we determine a TCR mimic (TCRm) antibody bound to its MANA target, the KRAS G12V peptide presented by HLA-A*03:01. Hydrophobic residues appear to account for the specificity of the mutant G12V residue. We also determine the structure of the wild-type G12 peptide bound to HLA-A*03:01, using X-ray crystallography. Based on these structures, we perform screens to validate the key residues required for peptide specificity. These experiments led us to a model for discrimination between the mutant and the wild-type peptides presented on HLA-A*03:01 based exclusively on hydrophobic interactions.

36 MATERIALS SCIENCE↗
DOE OSTI2023

Targeted mutagenesis of the herpesvirus fusogen central helix captures transition states

Herpesviruses remain a burden for animal and human health, including the medically important varicella-zoster virus (VZV). Membrane fusion mediated by conserved core glycoproteins, the fusogen gB and the heterodimer gH-gL, enables herpesvirus cell entry. The ectodomain of gB orthologs has five domains and is proposed to transition from a prefusion to postfusion conformation but the functional relevance of the domains for this transition remains poorly defined. Here we describe structure-function studies of the VZV gB DIII central helix targeting residues 526 EHV 528 . Critically, a H527P mutation captures gB in a prefusion conformation as determined by cryo-EM, a loss of membrane fusion in a virus free assay, and failure of recombinant VZV to spread in cell monolayers. Importantly, two predominant cryo-EM structures of gB[H527P] are identified by 3D classification and focused refinement, suggesting they represented gB conformations in transition. These studies reveal gB DIII as a critical element for herpesvirus gB fusion function.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural and dynamic mechanisms for coupled folding and tRNA recognition of a translational T-box riboswitch

T-box riboswitches are unique riboregulators where gene regulation is mediated through interactions between two highly structured RNAs. Despite extensive structural insights, how RNA-RNA interactions drive the folding and structural transitions of T-box to achieve functional conformations remains unclear. Here, by combining SAXS, single-molecule FRET and computational modeling, we elaborate the folding energy landscape of a translational T-box aptamer consisting of stems I, II and IIA/B, which Mg 2+ -induced global folding and tRNA binding are cooperatively coupled. smFRET measurements reveal that high Mg 2+ stabilizes IIA/B and its stacking on II, which drives the pre-docking of I and II into a competent conformation, subsequent tRNA binding promotes docking of I and II to form a high-affinity tRNA binding groove, of which the essentiality of IIA/B and S-turn in II is substantiated with mutational analysis. We highlight a delicate balance among Mg 2+ , the intra- and intermolecular RNA-RNA interactions in modulating RNA folding and function.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Engineered immunogens to elicit antibodies against conserved coronavirus epitopes

Immune responses to SARS-CoV-2 primarily target the receptor binding domain of the spike protein, which continually mutates to escape acquired immunity. Other regions in the spike S2 subunit, such as the stem helix and the segment encompassing residues 815-823 adjacent to the fusion peptide, are highly conserved across sarbecoviruses and are recognized by broadly reactive antibodies, providing hope that vaccines targeting these epitopes could offer protection against both current and emergent viruses. Here we employ computational modeling to design scaffolded immunogens that display the spike 815-823 peptide and the stem helix epitopes without the distracting and immunodominant receptor binding domain. These engineered proteins bind with high affinity and specificity to the mature and germline versions of previously identified broadly protective human antibodies. Epitope scaffolds interact with both sera and isolated monoclonal antibodies with broadly reactivity from individuals with pre-existing SARS-CoV-2 immunity. When used as immunogens, epitope scaffolds elicit sera with broad betacoronavirus reactivity and protect as “boosts” against live virus challenge in mice, illustrating their potential as components of a future pancoronavirus vaccine.

60 APPLIED LIFE SCIENCES↗