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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 1,801 records · Page 100
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

The regulatory and transcriptional landscape associated with carbon utilization in a filamentous fungus

Filamentous fungi, such as Neurospora crassa , are very efficient in deconstructing plant biomass by the secretion of an arsenal of plant cell wall-degrading enzymes, by remodeling metabolism to accommodate production of secreted enzymes, and by enabling transport and intracellular utilization of plant biomass components. Although a number of enzymes and transcriptional regulators involved in plant biomass utilization have been identified, how filamentous fungi sense and integrate nutritional information encoded in the plant cell wall into a regulatory hierarchy for optimal utilization of complex carbon sources is not understood. Here, we performed transcriptional profiling of N. crassa on 40 different carbon sources, including plant biomass, to provide data on how fungi sense simple to complex carbohydrates. From these data, we identified regulatory factors in N. crassa and characterized one (PDR-2) associated with pectin utilization and one with pectin/hemicellulose utilization (ARA-1). Using in vitro DNA affinity purification sequencing (DAP-seq), we identified direct targets of transcription factors involved in regulating genes encoding plant cell wall-degrading enzymes. In particular, our data clarified the role of the transcription factor VIB-1 in the regulation of genes encoding plant cell wall-degrading enzymes and nutrient scavenging and revealed a major role of the carbon catabolite repressor CRE-1 in regulating the expression of major facilitator transporter genes. These data contribute to a more complete understanding of cross talk between transcription factors and their target genes, which are involved in regulating nutrient sensing and plant biomass utilization on a global level.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Molecular recognition of a host protein by NS1 of pandemic and seasonal influenza A viruses

The 1918 influenza A virus (IAV) caused the most severe flu pandemic in recorded human history. Nonstructural protein 1 (NS1) is an important virulence factor of the 1918 IAV. NS1 antagonizes host defense mechanisms through interactions with multiple host factors. One pathway by which NS1 increases virulence is through the activation of phosphoinositide 3-kinase (PI3K) by binding to its p85β subunit. Here we present the mechanism underlying the molecular recognition of the p85β subunit by 1918 NS1. Using X-ray crystallography, we determine the structure of 1918 NS1 complexed with p85β of human PI3K. We find that the 1918 NS1 effector domain (1918 NS1 ED ) undergoes a conformational change to bind p85β. Using NMR relaxation dispersion and molecular dynamics simulation, we identify that free 1918 NS1 ED exists in a dynamic equilibrium between p85β-binding–competent and –incompetent conformations in the submillisecond timescale. Moreover, we discover that NS1 ED proteins of 1918 (H1N1) and Udorn (H3N2) strains exhibit drastically different conformational dynamics and binding kinetics to p85β. These results provide evidence of strain-dependent conformational dynamics of NS1. Using kinetic modeling based on the experimental data, we demonstrate that 1918 NS1 ED can result in the faster hijacking of p85β compared to Ud NS1 ED , although the former has a lower affinity to p85β than the latter. Our results suggest that the difference in binding kinetics may impact the competition with cellular antiviral responses for the activation of PI3K. We anticipate that our findings will increase the understanding of the strain-dependent behaviors of influenza NS1 proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Genomic discovery of an evolutionarily programmed modality for small-molecule targeting of an intractable protein surface

The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional therapeutic modalities are needed to overcome this deficiency. Here, the identification and characterization of a natural product, WDB002, reveals a therapeutic modality that dramatically expands the currently accepted limits of druggability. WDB002, in complex with the FK506-binding protein (FKBP12), potently and selectively binds the human centrosomal protein 250 (CEP250), resulting in disruption of CEP250 function in cells. The recognition mode is unprecedented in that the targeted domain of CEP250 is a coiled coil and is topologically featureless, embodying both a structural motif and surface topology previously considered on the extreme limits of “undruggability” for an intracellular target. Structural studies reveal extensive protein–WDB002 and protein–protein contacts, with the latter being distinct from those seen in FKBP12 ternary complexes formed by FK506 and rapamycin. Outward-facing structural changes in a bound small molecule can thus reprogram FKBP12 to engage diverse, otherwise “undruggable” targets. The flat-targeting modality demonstrated here has the potential to expand the druggable target range of small-molecule therapeutics. As CEP250 was recently found to be an interaction partner with the Nsp13 protein of the SARS-CoV-2 virus that causes COVID-19 disease, it is possible that WDB002 or an analog may exert useful antiviral activity through its ability to form high-affinity ternary complexes containing CEP250 and FKBP12.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

An isolated water droplet in the aqueous solution of a supramolecular tetrahedral cage

Water under nanoconfinement at ambient conditions has exhibited low-dimensional ice formation and liquid–solid phase transitions, but with structural and dynamical signatures that map onto known regions of water’s phase diagram. Using terahertz (THz) absorption spectroscopy and ab initio molecular dynamics, we have investigated the ambient water confined in a supramolecular tetrahedral assembly, and determined that a dynamically distinct network of 9 ± 1 water molecules is present within the nanocavity of the host. The low-frequency absorption spectrum and theoretical analysis of the water in the Ga 4 L 6 12– host demonstrate that the structure and dynamics of the encapsulated droplet is distinct from any known phase of water. A further inference is that the release of the highly unusual encapsulated water droplet creates a strong thermodynamic driver for the high-affinity binding of guests in aqueous solution for the Ga 4 L 6 12– supramolecular construct.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Computationally designed peptide macrocycle inhibitors of New Delhi metallo-β-lactamase 1

The rise of antibiotic resistance calls for new therapeutics targeting resistance factors such as the New Delhi metallo-β-lactamase 1 (NDM-1), a bacterial enzyme that degrades β-lactam antibiotics. We present structure-guided computational methods for designing peptide macrocycles built from mixtures of l - and d -amino acids that are able to bind to and inhibit targets of therapeutic interest. Our methods explicitly consider the propensity of a peptide to favor a binding-competent conformation, which we found to predict rank order of experimentally observed IC 50 values across seven designed NDM-1- inhibiting peptides. We were able to determine X-ray crystal structures of three of the designed inhibitors in complex with NDM-1, and in all three the conformation of the peptide is very close to the computationally designed model. In two of the three structures, the binding mode with NDM-1 is also very similar to the design model, while in the third, we observed an alternative binding mode likely arising from internal symmetry in the shape of the design combined with flexibility of the target. Although challenges remain in robustly predicting target backbone changes, binding mode, and the effects of mutations on binding affinity, our methods for designing ordered, binding-competent macrocycles should have broad applicability to a wide range of therapeutic targets.

42 ENGINEERING↗
DOE OSTI2021

Structurally silent peptide anchor modifications allosterically modulate T cell recognition in a receptor-dependent manner

Presentation of peptides by class I MHC proteins underlies T cell immune responses to pathogens and cancer. The association between peptide binding affinity and immunogenicity has led to the engineering of modified peptides with improved MHC binding, with the hope that these peptides would be useful for eliciting cross-reactive immune responses directed toward their weak binding, unmodified counterparts. Increasing evidence, however, indicates that T cell receptors (TCRs) can perceive such anchor-modified peptides differently than wild-type (WT) peptides, although the scope of discrimination is unclear. We show here that even modifications at primary anchors that have no discernible structural impact can lead to substantially stronger or weaker T cell recognition depending on the TCR. Surprisingly, the effect of peptide anchor modification can be sensed by a TCR at regions distant from the site of modification, indicating a through-protein mechanism in which the anchor residue serves as an allosteric modulator for TCR binding. Our findings emphasize caution in the use and interpretation of results from anchor-modified peptides and have implications for how anchor modifications are accounted for in other circumstances, such as predicting the immunogenicity of tumor neoantigens. Our data also highlight an important need to better understand the highly tunable dynamic nature of class I MHC proteins and the impact this has on various forms of immune recognition.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Enhancing ion transport in charged block copolymers by stabilizing low symmetry morphology: Electrostatic control of interfaces

Recently, the interest in charged polymers has been rapidly growing due to their uses in energy storage and transfer devices. Yet, polymer electrolyte-based devices are not on the immediate horizon because of the low ionic conductivity. In the present study, we developed a methodology to enhance the ionic conductivity of charged block copolymers comprising ionic liquids through the electrostatic control of the interfacial layers. Unprecedented reentrant phase transitions between lamellar and A15 structures were seen, which cannot be explained by well-established thermodynamic factors. X-ray scattering experiments and molecular dynamics simulations revealed the formation of fascinating, thin ionic shell layers composed of ionic complexes. The ionic liquid cations of these complexes predominantly presented near the micellar interfaces if they had strong binding affinity with the charged polymer chains. Therefore, the interfacial properties and concentration fluctuations of the A15 structures were crucially dependent on the type of tethered acid groups in the polymers. Overall, the stabilization energies of the A15 structures were greater when enriched, attractive electrostatic interactions were present at the micellar interfaces. Contrary to the conventional wisdom that block copolymer interfaces act as “dead zone” to significantly deteriorate ion transport, this study establishes a prospective avenue for advanced polymer electrolyte having tailor-made interfaces.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

DNA-encoded chemistry technology yields expedient access to SARS-CoV-2 M pro inhibitors

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed more than 4 million humans globally, but there is no bona fide Food and Drug Administration–approved drug-like molecule to impede the COVID-19 pandemic. The sluggish pace of traditional therapeutic discovery is poorly suited to producing targeted treatments against rapidly evolving viruses. Here, we used an affinity-based screen of 4 billion DNA-encoded molecules en masse to identify a potent class of virus-specific inhibitors of the SARS-CoV-2 main protease (M pro ) without extensive and time-consuming medicinal chemistry. CDD-1714, the initial three-building-block screening hit (molecular weight [MW] = 542.5 g/mol), was a potent inhibitor (inhibition constant [K i ] = 20 nM). CDD-1713, a smaller two-building-block analog (MW = 353.3 g/mol) of CDD-1714, is a reversible covalent inhibitor of M pro (K i = 45 nM) that binds in the protease pocket, has specificity over human proteases, and shows in vitro efficacy in a SARS-CoV-2 infectivity model. Subsequently, key regions of CDD-1713 that were necessary for inhibitory activity were identified and a potent (K i = 37 nM), smaller (MW = 323.4 g/mol), and metabolically more stable analog (CDD-1976) was generated. Thus, screening of DNA-encoded chemical libraries can accelerate the discovery of efficacious drug-like inhibitors of emerging viral disease targets.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Interleukin-2 superkines by computational design

Significance While computational engineering of therapeutic proteins is a desirable goal, in practice the optimization of protein–protein interactions requires substantial experimental intervention. We present here a computational approach that focuses on stabilizing core protein structures rather than engineering the protein–protein interface. Using this approach, we designed thermostabilized interleukin-2 (IL-2) variants that bind tightly to their receptor without experimental optimization, mimicking the properties of the yeast-display engineered IL-2 variant “super-2.” Our results suggest that structure-guided stabilization may be a general method for in silico affinity maturation of protein–protein interactions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

HP1-driven phase separation recapitulates the thermodynamics and kinetics of heterochromatin condensate formation

The spatial segregation of pericentromeric heterochromatin (PCH) into distinct, membrane-less nuclear compartments involves the binding of Heterochromatin Protein 1 (HP1) to H3K9me2/3-rich genomic regions. While HP1 exhibits liquid–liquid phase separation properties in vitro, its mechanistic impact on the structure and dynamics of PCH condensate formation in vivo remains largely unresolved. Here, using a minimal theoretical framework, we systematically investigate the mutual coupling between self-interacting HP1-like molecules and the chromatin polymer. We reveal that the specific affinity of HP1 for H3K9me2/3 loci facilitates coacervation in nucleo and promotes the formation of stable PCH condensates at HP1 levels far below the concentration required to observe phase separation in purified protein assays in vitro. These heterotypic HP1–chromatin interactions give rise to a strong dependence of the nucleoplasmic HP1 density on HP1-H3K9me2/3 stoichiometry, consistent with the thermodynamics of multicomponent phase separation. The dynamical cross talk between HP1 and the viscoelastic chromatin scaffold also leads to anomalously slow equilibration kinetics, which strongly depend on the genomic distribution of H3K9me2/3 domains and result in the coexistence of multiple long-lived, microphase-separated PCH compartments. The morphology of these complex coacervates is further found to be governed by the dynamic establishment of the underlying H3K9me2/3 landscape, which may drive their increasingly abnormal, aspherical shapes during cell development. These findings compare favorably to 4D microscopy measurements of HP1 condensate formation in live Drosophila embryos and suggest a general quantitative model of PCH formation based on the interplay between HP1-based phase separation and chromatin polymer mechanics.

3D genomics↗
DOE OSTI2023

Iterative computational design and crystallographic screening identifies potent inhibitors targeting the Nsp3 macrodomain of SARS-CoV-2

The nonstructural protein 3 (NSP3) of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) contains a conserved macrodomain enzyme (Mac1) that is critical for pathogenesis and lethality. While small-molecule inhibitors of Mac1 have great therapeutic potential, at the outset of the COVID-19 pandemic, there were no well-validated inhibitors for this protein nor, indeed, the macrodomain enzyme family, making this target a pharmacological orphan. Here, we report the structure-based discovery and development of several different chemical scaffolds exhibiting low- to sub-micromolar affinity for Mac1 through iterations of computer-aided design, structural characterization by ultra-high-resolution protein crystallography, and binding evaluation. Potent scaffolds were designed with in silico fragment linkage and by ultra-large library docking of over 450 million molecules. Both techniques leverage the computational exploration of tangible chemical space and are applicable to other pharmacological orphans. Overall, 160 ligands in 119 different scaffolds were discovered, and 153 Mac1-ligand complex crystal structures were determined, typically to 1 Å resolution or better. Our analyses discovered selective and cell-permeable molecules, unexpected ligand-mediated conformational changes within the active site, and key inhibitor motifs that will template future drug development against Mac1.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Investigation of the yielding transition in concentrated colloidal systems via rheo-XPCS

We probe the microstructural yielding dynamics of a concentrated colloidal system by performing creep/recovery tests with simultaneous collection of coherent scattering data via X-ray Photon Correlation Spectroscopy (XPCS). This combination of rheology and scattering allows for time-resolved observations of the microstructural dynamics as yielding occurs, which can be linked back to the applied rheological deformation to form structure–property relations. Under sufficiently small applied creep stresses, examination of the correlation in the flow direction reveals that the scattering response recorrelates with its predeformed state, indicating nearly complete microstructural recovery, and the dynamics of the system under these conditions slows considerably. Conversely, larger creep stresses increase the speed of the dynamics under both applied creep and recovery. The data show a strong connection between the microstructural dynamics and the acquisition of unrecoverable strain. By comparing this relationship to that predicted from homogeneous, affine shearing, we find that the yielding transition in concentrated colloidal systems is highly heterogeneous on the microstructural level.

42 ENGINEERING↗
DOE OSTI2023

Structure of the Wnt–Frizzled–LRP6 initiation complex reveals the basis for coreceptor discrimination

Wnt morphogens are critical for embryonic development and tissue regeneration. Canonical Wnts form ternary receptor complexes composed of tissue-specific Frizzled (Fzd) receptors together with the shared LRP5/6 coreceptors to initiate β-catenin signaling. The cryo-EM structure of a ternary initiation complex of an affinity-matured XWnt8–Frizzled8–LRP6 complex elucidates the basis of coreceptor discrimination by canonical Wnts by means of their N termini and linker domains that engage the LRP6 E1E2 domain funnels. Chimeric Wnts bearing modular linker “grafts” were able to transfer LRP6 domain specificity between different Wnts and enable non-canonical Wnt5a to signal through the canonical pathway. Synthetic peptides comprising the linker domain serve as Wnt-specific antagonists. The structure of the ternary complex provides a topological blueprint for the orientation and proximity of Frizzled and LRP6 within the Wnt cell surface signalosome.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Learning to learn by using nonequilibrium training protocols for adaptable materials

Evolution in time-varying environments naturally leads to adaptable biological systems that can easily switch functionalities. Advances in the synthesis of environmentally responsive materials therefore open up the possibility of creating a wide range of synthetic materials which can also be trained for adaptability. We consider high-dimensional inverse problems for materials where any particular functionality can be realized by numerous equivalent choices of design parameters. By periodically switching targets in a given design algorithm, we can teach a material to perform incompatible functionalities with minimal changes in design parameters. We exhibit this learning strategy for adaptability in two simulated settings: elastic networks that are designed to switch deformation modes with minimal bond changes and heteropolymers whose folding pathway selections are controlled by a minimal set of monomer affinities. The resulting designs can reveal physical principles, such as nucleation-controlled folding, that enable such adaptability.

Science & Technology - Other Topics↗
DOE OSTI2023

Engineered molecular sensors for quantifying cell surface crowding

Cells mediate interactions with the extracellular environment through a crowded assembly of transmembrane proteins, glycoproteins and glycolipids on their plasma membrane. The extent to which surface crowding modulates the biophysical interactions of ligands, receptors, and other macromolecules is poorly understood due to the lack of methods to quantify surface crowding on native cell membranes. In this work, we demonstrate that physical crowding on reconstituted membranes and live cell surfaces attenuates the effective binding affinity of macromolecules such as IgG antibodies in a surface crowding-dependent manner. We combine experiment and simulation to design a crowding sensor based on this principle that provides a quantitative readout of cell surface crowding. Our measurements reveal that surface crowding decreases IgG antibody binding by 2 to 20 fold in live cells compared to a bare membrane surface. Our sensors show that sialic acid, a negatively charged monosaccharide, contributes disproportionately to red blood cell surface crowding via electrostatic repulsion, despite occupying only ~1% of the total cell membrane by mass. We also observe significant differences in surface crowding for different cell types and find that expression of single oncogenes can both increase and decrease crowding, suggesting that surface crowding may be an indicator of both cell type and state. Our high-throughput, single-cell measurement of cell surface crowding may be combined with functional assays to enable further biophysical dissection of the cell surfaceome.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Structure-based design of nanobodies that inhibit seeding of Alzheimer’s patient–extracted tau fibrils

Despite much effort, antibody therapies for Alzheimer’s disease (AD) have shown limited efficacy. Challenges to the rational design of effective antibodies include the difficulty of achieving specific affinity to critical targets, poor expression, and antibody aggregation caused by buried charges and unstructured loops. To overcome these challenges, we grafted previously determined sequences of fibril-capping amyloid inhibitors onto a camel heavy chain antibody scaffold. These sequences were designed to cap fibrils of tau, known to form the neurofibrillary tangles of AD, thereby preventing fibril elongation. The nanobodies grafted with capping inhibitors blocked tau aggregation in biosensor cells seeded with postmortem brain extracts from AD and progressive supranuclear palsy (PSP) patients. The tau capping nanobody inhibitors also blocked seeding by recombinant tau oligomers. Another challenge to the design of effective antibodies is their poor blood–brain barrier (BBB) penetration. In this study, we also designed a bispecific nanobody composed of a nanobody that targets a receptor on the BBB and a tau capping nanobody inhibitor, conjoined by a flexible linker. We provide evidence that the bispecific nanobody improved BBB penetration over the tau capping inhibitor alone after intravenous administration in mice. Our results suggest that the design of synthetic antibodies that target sequences that drive protein aggregation may be a promising approach to inhibit the prion-like seeding of tau and other proteins involved in AD and related proteinopathies.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Exploring switch II pocket conformation of KRAS(G12D) with mutant-selective monobody inhibitors

The G12D mutation is among the most common KRAS mutations associated with cancer, in particular, pancreatic cancer. Here, we have developed monobodies, small synthetic binding proteins, that are selective to KRAS(G12D) over KRAS(wild type) and other oncogenic KRAS mutations, as well as over the G12D mutation in HRAS and NRAS. Crystallographic studies revealed that, similar to other KRAS mutant-selective inhibitors, the initial monobody bound to the S-II pocket, the groove between switch II and α3 helix, and captured this pocket in the most widely open form reported to date. Unlike other G12D-selective polypeptides reported to date, the monobody used its backbone NH group to directly recognize the side chain of KRAS Asp12, a feature that closely resembles that of a small-molecule inhibitor, MTRX1133. The monobody also directly interacted with H95, a residue not conserved in RAS isoforms. These features rationalize the high selectivity toward the G12D mutant and the KRAS isoform. Structure-guided affinity maturation resulted in monobodies with low nM KD values. Deep mutational scanning of a monobody generated hundreds of functional and nonfunctional single-point mutants, which identified crucial residues for binding and those that contributed to the selectivity toward the GTP- and GDP-bound states. When expressed in cells as genetically encoded reagents, these monobodies engaged selectively with KRAS(G12D) and inhibited KRAS(G12D)-mediated signaling and tumorigenesis. These results further illustrate the plasticity of the S-II pocket, which may be exploited for the design of next-generation KRAS(G12D)-selective inhibitors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Polyubiquitin ligand-induced phase transitions are optimized by spacing between ubiquitin units

Biomolecular condensates form via multivalent interactions among key macromolecules and are regulated through ligand binding and/or posttranslational modifications. One such modification is ubiquitination, the covalent addition of ubiquitin (Ub) or polyubiquitin chains to target macromolecules. Specific interactions between polyubiquitin chains and partner proteins, including hHR23B, NEMO, and UBQLN2, regulate condensate assembly or disassembly. Here, we used a library of designed polyubiquitin hubs and UBQLN2 as model systems for determining the driving forces of ligand-mediated phase transitions. Perturbations to either the UBQLN2-binding surface of Ub or the spacing between Ub units reduce the ability of hubs to modulate UBQLN2 phase behavior. By developing an analytical model based on polyphasic linkage principles that accurately described the effects of different hubs on UBQLN2 phase separation, we determined that introduction of Ub to UBQLN2 condensates incurs a significant inclusion energetic penalty. This penalty antagonizes the ability of polyUb hubs to scaffold multiple UBQLN2 molecules and cooperatively amplify phase separation. The extent to which polyubiquitin hubs promote UBQLN2 phase separation is encoded in the spacings between Ub units. This spacing is modulated by chains of different linkages and designed chains of different architectures, thus illustrating how the ubiquitin code regulates functionality via the emergent properties of the condensate. The spacing in naturally occurring linear polyubiquitin chains is already optimized to promote phase separation with UBQLN2. We expect our findings to extend to other condensates, emphasizing the importance of ligand properties, including concentration, valency, affinity, and spacing between binding sites in studies and designs of condensates.

97 MATHEMATICS AND COMPUTING↗

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