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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 433 records · Page 24
All sourcesNASA NTRSDOE OSTI
DOE OSTI2014

Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

Highlights: • Crystal structure of AtQR has been determined at 1.72 Å. • NADH binding induces the formation of substrate binding site. • AtQR possesses a conserved hydrophobic wall for stereospecific binding of substrate. • Additional Glu197 residue is critical to the high binding affinity. - Abstract: (R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior

Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bioinspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. We designed sequences with significantly increased phase separation propensity by shuffling the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that despite these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance our fundamental understanding of key biophysical principles and sequence features important to phase separation.

36 MATERIALS SCIENCE↗
DOE OSTI2010

Oligosaccharide Binding in Escherichia coli Glycogen Synthase

Glycogen/starch synthase elongates glucan chains and is the key enzyme in the synthesis of glycogen in bacteria and starch in plants. Cocrystallization of Escherichia coli wild-type glycogen synthase (GS) with substrate ADPGlc and the glucan acceptor mimic HEPPSO produced a closed form of GS and suggests that domain-domain closure accompanies glycogen synthesis. Cocrystallization of the inactive GS mutant E377A with substrate ADPGlc and oligosaccharide results in the first oligosaccharide-bound glycogen synthase structure. Four bound oligosaccharides are observed, one in the interdomain cleft (G6a) and three on the N-terminal domain surface (G6b, G6c, and G6d). Extending from the center of the enzyme to the interdomain cleft opening, G6a mostly interacts with the highly conserved N-terminal domain residues lining the cleft of GS. The surface-bound oligosaccharides G6c and G6d have less interaction with enzyme and exhibit a more curled, helixlike structural arrangement. The observation that oligosaccharides bind only to the N-terminal domain of GS suggests that glycogen in vivo probably binds to only one side of the enzyme to ensure unencumbered interdomain movement, which is required for efficient, continuous glucan-chain synthesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2010

Preferential selection of isomer binding from chiral mixtures: alternate binding modes observed for the E and Z isomers of a series of 5-substituted 2,4-diaminofuro[2,3-d]pyrimidines as ternary complexes with NADPH and human dihydrofolate reductase

The structures of six chirally mixed E/Z-isomers of 5-substituted 2,4-diaminofuro[2,3-d]pyrimidines reveals only one isomer is bound in the active site of human DHFR. The configuration of all but one C9-analogue is observed as the E-isomer. The crystal structures of six human dihydrofolate reductase (hDHFR) ternary complexes with NADPH and a series of mixed E/Z isomers of 5-substituted 5-[2-(2-methoxyphenyl)-prop-1-en-1-yl]furo[2,3-d]pyrimidine-2,4-diamines substituted at the C9 position with propyl, isopropyl, cyclopropyl, butyl, isobutyl and sec-butyl (E2–E7, Z3) were determined and the results were compared with the resolved E and Z isomers of the C9-methyl parent compound. The configuration of all of the inhibitors, save one, was observed as the E isomer, in which the binding of the furopyrimidine ring is flipped such that the 4-amino group binds in the 4-oxo site of folate. The Z3 isomer of the C9-isopropyl analog has the normal 2,4-diaminopyrimidine ring binding geometry, with the furo oxygen near Glu30 and the 4-amino group interacting near the cofactor nicotinamide ring. Electron-density maps for these structures revealed the binding of only one isomer to hDHFR, despite the fact that chiral mixtures (E:Z ratios of 2:1, 3:1 and 3:2) of the inhibitors were incubated with hDHFR prior to crystallization. Superposition of the hDHFR complexes with E2 and Z3 shows that the 2′-methoxyphenyl ring of E2 is perpendicular to that of Z3. The most potent inhibitor in this series is the isopropyl analog Z3 and the least potent is the isobutyl analog E6, consistent with data that show that the Z isomer makes the most favorable interactions with the active-site residues. The isobutyl moiety of E6 is observed in two orientations and the resultant steric crowding of the E6 analog is consistent with its weaker activity. The alternative binding modes observed for the furopyrimidine ring in these E/Z isomers suggest that new templates can be designed to probe these binding regions of the DHFR active site.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
DOE OSTI2019

Feature Design for Protein Interface Hotspots Using KFC2 and Rosetta

Protein–protein interactions regulate many essential biological processes and play an important role in health and disease. The process of experimentally characterizing protein residues that contribute the most to protein–protein interaction affinity and specificity is laborious. Thus, developing models that accurately characterize hotspots at protein–protein interfaces provides important information about how to inhibit therapeutically relevant protein–protein interactions. During the course of the ICERM WiSDM workshop 2017, we combined the KFC2a protein–protein interaction hotspot prediction features with Rosetta scoring function terms and interface filter metrics. A two-way and three-way forward selection strategy was employed to train support vector machine classifiers, as was a reverse feature elimination strategy. From these results, we identified subsets of KFC2a and Rosetta combined features that show improved performance over KFC2a features alone.

Seeger, Franziska↗
DOE OSTI2021

Structural analysis of carbohydrate binding by the macrophage mannose receptor CD206

The human mannose receptor expressed on macrophages and hepatic endothelial cells scavenges released lysosomal enzymes, glycopeptide fragments of collagen, and pathogenic microorganisms and thus reduces damage following tissue injury. The receptor binds mannose, fucose, or N-acetylglucosamine (GlcNAc) residues on these targets. C-type carbohydrate recognition domain 4 (CRD4) of the receptor contains the site for Ca 2+ -dependent interaction with sugars. To investigate the details of CRD4 binding, glycan array screening was used to identify oligosaccharide ligands. The strongest signals were for glycans that contain either Manα1-2Man constituents or fucosein various linkages. The mechanisms of binding to monosaccharides and oligosaccharide substructures present in many of these ligands were examined in multiple crystal structures of CRD4. Binding of mannose residues to CRD4 results primarily from interaction of the equatorial 3- and 4-OH groups with a conserved principal Ca 2+ common to almost all sugar-binding C-type CRDs. In the Manα1-2Man complex, supplementary interactions with the reducing mannose residue explain the enhanced affinity for this disaccharide. Bound GlcNAc also interacts with the principal Ca 2+ through equatorial 3- and 4-OH groups, whereas fucose residues can bind in several orientations, through either the 2- and 3-OH groups or the 3- and 4-OH groups. Secondary contacts with additional sugars in fucose-containing oligosaccharides, such as the Lewis-a trisaccharide, provide enhanced affinity for these glycans. These results explain many of the biologically important interactions of the mannose receptor with both mammalian glycoproteins and microbes such as yeast and suggest additional classes of ligands that have not been previously identified

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2011

Phase Constituents and Microstructure of Interaction Layer Formed in U-Mo Alloys vs Al Diffusion Couples Annealed at 873 K (600°C)

U-Mo dispersion and monolithic fuels are being developed to fulfill the requirements for research reactors, under the Reduced Enrichment for Research and Test Reactors program. In dispersion fuels, particles of U-Mo alloys are embedded in the Al-alloy matrix, while in monolithic fuels, U-Mo monoliths are roll bonded to the Al-alloy matrix. In this study, interdiffusion and microstructural development in the solid-to-solid diffusion couples, namely, U-15.7 at. pct Mo (7 wt pct Mo) vs pure Al, U-21.6 at. pct Mo (10 wt pct Mo) vs pure Al, and U-25.3 at. pct Mo (12 wt pct Mo) vs pure Al, annealed at 873 K (600 degrees C) for 24 hours, were examined in detail. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and electron probe microanalysis (EPMA) were employed to examine the development of a very fine multiphase interaction layer with an approximately constant average composition of 80 at. pct Al. Extensive TEM was carried out to identify the constituent phases across the interaction layer based on selected area electron diffraction and convergent beam electron diffraction (CBED). The cubic-UAl3, orthorhombic-UAl4, hexagonal-U6Mo4Al43, and cubic-UMo2Al20 phases were identified within the interaction layer that included two- and three-phase layers. Residual stress from large differences in molar volume, evidenced by vertical cracks within the interaction layer, high Al mobility, Mo supersaturation, and partitioning toward equilibrium in the interdiffusion zone were employed to describe the complex microstructure and phase constituents observed. A mechanism by compositional modification of the Al alloy is explored to mitigate the development of the U6Mo4Al43 phase, which exhibits poor irradiation behavior that includes void formation and swelling.

11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗
DOE OSTI2014

Subfamily-specific adaptations in the structures of two penicillin-binding proteins from Mycobacterium tuberculosis

Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis , we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows that Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis . Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2010

NMR solution structure of the N-terminal domain of hERG and its interaction with the S4-S5 linker

Research highlights: {yields} The N-terminal domain (NTD, eag domain) containing 135 residues of hERG was expressed and purified from E. coli cells. {yields} Solution structure of NTD was determined with NMR spectroscopy. {yields} The alpha-helical region (residues 13-23) was demonstrated to possess the characteristics of an amphipathic helix. {yields} NMR titration confirmed the interaction between NTD and the peptide from the S4-S5 linker. -- Abstract: The human Ether-a-go-go Related Gene (hERG) potassium channel mediates the rapid delayed rectifier current (IKr) in the cardiac action potential. Mutations in the 135 amino acid residue N-terminal domain (NTD) cause channel dysfunction or mis-translocation. To study the structure of NTD, it was overexpressed and purified from Escherichia coli cells using affinity purification and gel filtration chromatography. The purified protein behaved as a monomer under purification conditions. Far- and near-UV, circular dichroism (CD) and solution nuclear magnetic resonance (NMR) studies showed that the purified protein was well-folded. The solution structure of NTD was obtained and the N-terminal residues 13-23 forming an amphipathic helix which may be important for the protein-protein or protein-membrane interactions. NMR titration experiment also demonstrated that residues from 88 to 94 in NTD are important for the molecular interaction with the peptide derived from the S4-S5 linker.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

The structure of importin α and the nuclear localization peptide of ChREBP, and small compound inhibitors of ChREBP–importin α interactions

The carbohydrate response element binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in glucose-mediated induction of genes involved in hepatic glycolysis and lipogenesis. In response to fluctuating blood glucose levels ChREBP activity is regulated mainly by nucleocytoplasmic shuttling of ChREBP. Under high glucose ChREBP binds to importin α and importin β and translocates into the nucleus to initiate transcription. We have previously shown that the nuclear localization signal site (NLS) for ChREBP is bipartite with the NLS extending from Arg158 to Lys190. Here, we report the 2.5 Å crystal structure of the ChREBP-NLS peptide bound to importin α. The structure revealed that the NLS binding is monopartite, with the amino acid residues K171RRI174 from the ChREBP-NLS interacting with ARM2–ARM5 on importin α. We discovered that importin α also binds to the primary binding site of the 14-3-3 proteins with high affinity, which suggests that both importin α and 14-3-3 are each competing with the other for this broad-binding region (residues 117–196) on ChREBP. We screened a small compound library and identified two novel compounds that inhibit the ChREBP-NLS/importin α interaction, nuclear localization, and transcription activities of ChREBP. These candidate molecules support developing inhibitors of ChREBP that may be useful in treatment of obesity and the associated diseases.

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

Pairing instability on a Luttinger surface: A non-Fermi liquid to superconductor transition and its Sachdev-Ye-Kitaev dual

Superconductivity results from an instability of the Fermi surface—contour of poles of the single-particle propagator—to an infinitesimally small attraction between electrons. Here, we instead discuss the analogous problem on a model Luttinger surface, or contour of zeros of the Green function. At zero temperature ($β → \infty$) and a critical interaction strength ($u_{c∞}$) characterized by the residue of self-energy pole, we find that the pair susceptibility diverges leading to a superconducting instability. We evaluate the pair fluctuation partition function and find that the spectral density in the normal state has an interaction-driven, power-law $\frac{1}{\sqrt{ω}}$ type, van-Hove singularity (vHS), indicating non-Fermi liquid (NFL) physics. Crucially, in the strong coupling limit ($βu \gg 1$), the leading-order fluctuation free-energy terms in the normal state of this NFL-SC transition resembles the equivalent [$\textit{O}(1)$] terms of the Sachdev-Ye-Kitaev (SYK) model. This free-energy contribution takes a simple form $–βF = βu_{c∞} – γ \text{ln}(βu_{c∞}$), where $\gamma$ is a constant equal to $\frac{1}{2}$. Weak impurity scattering ($τ \gg β^{–1}$) leaves the low-energy spectral density unaffected but leads to an interaction-driven enhancement of superconductivity. Our results in this work shed light on the role played by order-parameter fluctuations in providing the key missing link between Mott physics and strongly coupled toy models exhibiting gravity duals.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
DOE OSTI2017

Inclusive muon and $B$ quark production cross-sections in $p \bar{p}$ collisions at $\sqrt{s}$ = 1.8-TeV

We have calculated the double and triple differential cross sections for electron ejection with energy of 14.6 eV in single ionization of H2 by 75 keV proton impact. A molecular version of the continuum distorted wave-eikonal initial state approach is applied, where the interaction between the projectile and the residual molecular ion is considered more properly than in previous applications of the method. For triple differential cross sections, the present results are in better agreement with the experimental data than those of other descriptions when large momentum transfer values are considered. For double differential cross sections the experimental data are reproduced quite well for both coherent and incoherent proton beams.

DIFFERENTIAL CROSS SECTIONS↗
DOE OSTI2017

Lysines in the RNA Polymerase II C-Terminal Domain Contribute to TAF15 Fibril Recruitment

Many cancer-causing chromosomal translocations result in transactivating protein products encoding FET family (FUS, EWSR1, TAF15) low-complexity (LC) domains fused to a DNA binding domain from one of several transcription factors. Recent work demonstrates that higher-order assemblies of FET LC domains bind the carboxy-terminal domain of the large subunit of RNA polymerase II (RNA pol II CTD), suggesting FET oncoproteins may mediate aberrant transcriptional activation by recruiting RNA polymerase II to promoters of target genes. Here we use nuclear magnetic resonance (NMR) spectroscopy and hydrogel fluorescence microscopy localization and fluorescence recovery after photobleaching to visualize atomic details of a model of this process, interactions of RNA pol II CTD with high-molecular weight TAF15 LC assemblies. Here we report NMR resonance assignments of the intact degenerate repeat half of human RNA pol II CTD alone and verify its predominant intrinsic disorder by molecular simulation. By measuring NMR spin relaxation and dark-state exchange saturation transfer, we characterize the interaction of RNA pol II CTD with amyloid-like hydrogel fibrils of TAF15 and hnRNP A2 LC domains and observe that heptads far from the acidic C-terminal tail of RNA pol II CTD bind TAF15 fibrils most avidly. Mutation of CTD lysines in heptad position 7 to consensus serines reduced the overall level of TAF15 fibril binding, suggesting that electrostatic interactions contribute to complex formation. Conversely, mutations of position 7 asparagine residues and truncation of the acidic tail had little effect. Thus, weak, multivalent interactions between TAF15 fibrils and heptads throughout RNA pol II CTD collectively mediate complex formation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Molecular dynamics simulation studies of the wild type and E92Q/N155H mutant of Elvitegravir-resistance HIV-1 integrase

Although Elvitegravir (EVG) is a newly developed antiretrovirals drug to treat the acquired immunodeficiency syndrome (AIDS), drug resistance has already been found in clinic, such as E92Q/N155H and Q148H/G140S. Several structural investigations have already been reported to reveal the molecular mechanism of the drug resistance. As full length crystal structure for HIV-1 integrase is still unsolved, we use in this paper the crystal structure of the full length prototype foamy virus (PFV) in complex with virus DNA and inhibitor Elvitegravir as a template to construct the wild type and E92Q/N155H mutant system of HIV-1 integrase. Molecular dynamic simulations was used to revel the binding mode and the drug resistance of the EVG ligand in E92Q/N155H. Several important interactions were discovered between the mutated residues and the residues in the active site of the E92Q/N155H double mutant pattern, and cross correlation and clustering methods were used for detailed analysis. The results from the MD simulation studies will be used to guide the experimental efforts of developing novel inhibitors against drug-resistant HIV integrase mutants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Characterization of alternate encounter assemblies of SARS-CoV-2 main protease

The assembly of two monomeric constructs spanning segments 1-199 (MPro 1-199 ) and 10-306 (MPro 10-306 ) of SARS-CoV-2 main protease (MPro) was examined to assess the existence of a transient heterodimer intermediate in the N-terminal autoprocessing pathway of MPro model precursor. Together, they form a heterodimer population accompanied by a 13-fold increase in catalytic activity. Addition of inhibitor GC373 to the proteins increases the activity further by ~7-fold with a 1:1 complex and higher order assemblies approaching 1:2 and 2:2 molecules of MPro 1-199 and MPro 10-306 detectable by analytical ultracentrifugation and native mass estimation by light scattering. Assemblies larger than a heterodimer (1:1) are discussed in terms of alternate pathways of domain III association, either through switching the location of helix 201 to 214 onto a second helical domain of MPro 10-306 and vice versa or direct interdomain III contacts like that of the native dimer, based on known structures and AlphaFold 3 prediction, respectively. At a constant concentration of MPro 1-199 with molar excess of GC373, the rate of substrate hydrolysis displays first order dependency on the MPro 10-306 concentration and vice versa. An equimolar composition of the two proteins with excess GC373 exhibits half-maximal activity at ~6 μM MPro 1-199 . Catalytic activity arises primarily from MPro 1-199 and is dependent on the interface interactions involving the N-finger residues 1 to 9 of MPro 1-199 and E290 of MPro 10-306 . Importantly, our results confirm that a single N-finger region with its associated intersubunit contacts is sufficient to form a heterodimeric MPro intermediate with enhanced catalytic activity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Self-inhibited State of Venezuelan Equine Encephalitis Virus (VEEV) nsP2 Cysteine Protease: A Crystallographic and Molecular Dynamics Analysis

The Venezuelan equine encephalitis virus (VEEV) belongs to the Togaviridae family and is pathogenic to both humans and equines. The VEEV non-structural protein 2 (nsP2) is a cysteine protease (nsP2pro) that processes the polyprotein and thus it is a drug target for inhibitor discovery. The atomic structure of the VEEV nsP2 catalytic domain was previously characterized by both X-ray crystallography and computational studies. A modified nsP2pro harboring a N475A mutation in the N terminus was observed to exhibit an unexpected conformation: the N-terminal residues bind to the active site, mimicking binding of a substrate. The large conformational change of the N terminus was assumed to be induced by the N475A mutation, as N475 has an important role in stabilization of the N terminus and the active site. This conformation was first observed in the N475A mutant, but we also found it while determining a crystal structure of the catalytically active nsP2pro containing the wild-type N475 active site residue and K741A/K767A surface entropy reduction mutations. This suggests that the N475A mutation is not a prerequisite for self-inhibition. Here, we describe a high resolution (1.46 Å) crystal structure of a truncated nsP2pro (residues 463–785, K741A/K767A) and analyze the structure further by molecular dynamics to study the active and self-inhibited conformations of nsP2pro and its N475A mutant. A comparison of the different conformations of the N-terminal residues sheds a light on the interactions that play an important role in the stabilization of the enzyme.

59 BASIC BIOLOGICAL SCIENCES↗