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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 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Proteolytic processing induces a conformational switch required for antibacterial toxin delivery

Many Gram-negative bacteria use CdiA effector proteins to inhibit the growth of neighboring competitors. CdiA transfers its toxic CdiA-CT region into the periplasm of target cells, where it is released through proteolytic cleavage. The N-terminal cytoplasm-entry domain of the CdiA-CT then mediates translocation across the inner membrane to deliver the C-terminal toxin domain into the cytosol. Here, we show that proteolysis not only liberates the CdiA-CT for delivery, but is also required to activate the entry domain for membrane translocation. Translocation function depends on precise cleavage after a conserved VENN peptide sequence, and the processed ΔVENN entry domain exhibits distinct biophysical and thermodynamic properties. By contrast, imprecisely processed CdiA-CT fragments do not undergo this transition and fail to translocate to the cytoplasm. These findings suggest that CdiA-CT processing induces a critical structural switch that converts the entry domain into a membrane-translocation competent conformation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Highly Basic Clusters in the Herpes Simplex Virus 1 Nuclear Egress Complex Drive Membrane Budding by Inducing Lipid Ordering

During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC-membrane interactions and inhibit NEC-mediated budding in vitro, providing a biophysical explanation for the in vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an off switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding.

membrane curvature↗
DOE OSTI2020

Controlling protein adsorption modes electrostatically

Protein adsorption on surfaces is ubiquitous in biology and in biotechnology. There are various forces required for controlling protein adsorption. Here, we introduce an explicit ion coarse-grained molecular dynamics simulation approach for studying the effects of electrostatics on protein adsorption, and 2D protein assembly on charged surfaces. Our model accounts for the spatial distribution of protein charges. We use catalase as our model protein. We find that the preferential adsorption mode of proteins at low protein concentration on a charged surface is “standing up”. When the protein concentration in a solution increases to reach a critical density on the surface, the adsorption mode switches from “standing up” to a mixed state “flat on” and “standing up”, which increases the lateral correlations among the adsorbed proteins. As such, the changes in the adsorption mode arise from the protein adsorption that cancel the surface charge and the protein–protein repulsion. This correlated surface structure melts as the salt concentration increases because the charged surface is cancelled by the salt ions and the proteins de-adsorb. For the case of strongly charged surfaces the “standing up” conformation remains more favorable even at high protein adsorption at low salt concentrations since in that conformation the surface charge is cancelled more effectively, generating an even more laterally correlated structure. We elucidate the effects of parameters such as surface charge density, salt concentration, and protein charges on the different adsorption modes and the structure and organization of proteins on the charged surfaces. Finally, this study provides a guide for controlling protein assembly on surfaces.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

The crystal structure of bacteriophage λ RexA provides novel insights into the DNA binding properties of Rex-like phage exclusion proteins

Abstract RexA and RexB function as an exclusion system that prevents bacteriophage T4rII mutants from growing on Escherichia coli λ phage lysogens. Recent data established that RexA is a non-specific DNA binding protein that can act independently of RexB to bias the λ bistable switch toward the lytic state, preventing conversion back to lysogeny. The molecular interactions underlying these activities are unknown, owing in part to a dearth of structural information. Here, we present the 2.05-Å crystal structure of the λ RexA dimer, which reveals a two-domain architecture with unexpected structural homology to the recombination-associated protein RdgC. Modelling suggests that our structure adopts a closed conformation and would require significant domain rearrangements to facilitate DNA binding. Mutagenesis coupled with electromobility shift assays, limited proteolysis, and double electron–electron spin resonance spectroscopy support a DNA-dependent conformational change. In vivo phenotypes of RexA mutants suggest that DNA binding is not a strict requirement for phage exclusion but may directly contribute to modulation of the bistable switch. We further demonstrate that RexA homologs from other temperate phages also dimerize and bind DNA in vitro. Collectively, these findings advance our mechanistic understanding of Rex functions and provide new evolutionary insights into different aspects of phage biology.

Biochemistry & Molecular Biology↗
DOE OSTI2021

Mitogen-activated protein kinase 6 negatively regulates secondary wall biosynthesis by modulating MYB46 protein stability in Arabidopsis thaliana

The R2R3-MYB transcription factor MYB46 functions as a master switch for secondary cell wall biosynthesis, ensuring the exquisite expression of the secondary wall biosynthetic genes in the tissues where secondary walls are critical for growth and development. At the same time, suppression of its function is needed when/where formation of secondary walls is not desirable. Little is known about how this opposing control of secondary cell wall formation is achieved. We used both transient and transgenic expression of MYB46 and mitogen-activated protein kinase 6 (MPK6) to investigate the molecular mechanism of the post-translational regulation of MYB46. We show that MYB46 is phosphorylated by MPK6, leading to site specific phosphorylation-dependent degradation of MYB46 by the ubiquitin-mediated proteasome pathway. In addition, the MPK6-mediated MYB46 phosphorylation was found to regulate in planta secondary wall forming function of MYB46. Furthermore, we provide experimental evidences that MYB83, a paralog of MYB46, is not regulated by MPK6. The coupling of MPK signaling to MYB46 function provides insights into the tissue- and/or condition-specific activity of MYB46 for secondary wall biosynthesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Molecular Dynamics Free Energy Simulations Reveal the Mechanism for the Antiviral Resistance of the M66I HIV-1 Capsid Mutation

While drug resistance mutations can often be attributed to the loss of direct or solvent-mediated protein−ligand interactions in the drug-mutant complex, in this study we show that a resistance mutation for the picomolar HIV-1 capsid (CA)-targeting antiviral (GS-6207) is mainly due to the free energy cost of the drug-induced protein side chain reorganization in the mutant protein. Among several mutations, M66I causes the most suppression of the GS-6207 antiviral activity (up to ~84,000-fold), and only 83- and 68-fold reductions for PF74 and ZW-1261, respectively. To understand the molecular basis of this drug resistance, we conducted molecular dynamics free energy simulations to study the structures, energetics, and conformational free energy landscapes involved in the inhibitors binding at the interface of two CA monomers. To minimize the protein−ligand steric clash, the I66 side chain in the M66I−GS-6207 complex switches to a higher free energy conformation from the one adopted in the apo M66I. In contrast, the binding of GS-6207 to the wild-type CA does not lead to any significant M66 conformational change. Based on an analysis that decomposes the absolute binding free energy into contributions from two receptor conformational states, it appears that it is the free energy cost of side chain reorganization rather than the reduced protein−ligand interaction that is largely responsible for the drug resistance against GS-6207.

Sun, Qinfang (ORCID:0000000173621543)↗
DOE OSTI2021

Boost of the Bio-memristor Performance for Artificial Electronic Synapses by Surface Reconstruction

Biomaterial-based memristors (bio-memristors) are often adopted to emulate biological synapse functions and applied to construct neural computing networks in brain-inspired chip systems. However, the randomness of conductive filament formation in bio-memristors inhibits their switching performance by causing the dispersion of the device-switching parameters. In this case, a facile porous silk fibroin (p-SF) memristor was obtained through a protein surface reconstruction strategy, in which the size of the hole can be adjusted by the density of hybrid nanoseeds. Here, the porous SF memristors exhibit greatly enhanced electrical characteristics, including uniform I-V cycles, centralized distribution of the switching voltages, and both high and low resistances, compared to devices without pores. The results of three-dimensional (3D) simulations based on classical density functional theory (cDFT) suggest that the reconstructed pores in the SF layers guide the formation and fracture of Ag filaments under an electric field and enhance the overall conductivity by separating Ag + ion and electron diffusion pathways. Ag + ions are predicted to preferentially diffuse through pores, whereas electrons diffuse through the SF network. Interestingly, the device conductance can be bidirectionally modulated gradually by positive and negative voltages, can faithfully simulate short-term and long-term plasticity, and can even realize the triplet-spike-timing-dependent plasticity (triplet-STDP) rule, which can be used for pattern recognition in biological systems. The simulation results reveal that a memristor network of this type has an accuracy of ~95.78% in memory learning and the capability of pattern learning. This work provides a facile technology route to improve the performance of bionic-material memristors.

36 MATERIALS SCIENCE↗
DOE OSTI2020

Structure of the Diphtheria Toxin at Acidic pH: Implications for the Conformational Switching of the Translocation Domain

Diphtheria toxin, an exotoxin secreted by Corynebacterium that causes disease in humans by inhibiting protein synthesis, enters the cell via receptor-mediated endocytosis. The subsequent endosomal acidification triggers a series of conformational changes, resulting in the refolding and membrane insertion of the translocation (T-)domain and ultimately leading to the translocation of the catalytic domain into the cytoplasm. Here, we use X-ray crystallography along with circular dichroism and fluorescence spectroscopy to gain insight into the mechanism of the early stages of pH-dependent conformational transition. For the first time, we present the high-resolution structure of the diphtheria toxin at a mildly acidic pH (5–6) and compare it to the structure at neutral pH (7). We demonstrate that neither catalytic nor receptor-binding domains change their structure upon this acidification, while the T-domain undergoes a conformational change that results in the unfolding of the TH2–3 helices. Surprisingly, the TH1 helix maintains its conformation in the crystal of the full-length toxin even at pH 5. This contrasts with the evidence from the new and previously published data, obtained by spectroscopic measurements and molecular dynamics computer simulations, which indicate the refolding of TH1 upon the acidification of the isolated T-domain. The overall results imply that the membrane interactions of the T-domain are critical in ensuring the proper conformational changes required for the preparation of the diphtheria toxin for the cellular entry.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Multi-layered heterochromatin interaction as a switch for DIM2-mediated DNA methylation

Functional crosstalk between DNA methylation, histone H3 lysine-9 trimethylation (H3K9me3) and heterochromatin protein 1 (HP1) is essential for proper heterochromatin assembly and genome stability. However, how repressive chromatin cues guide DNA methyltransferases for region-specific DNA methylation remains largely unknown. Here, we report structure-function characterizations of DNA methyltransferase Defective-In-Methylation-2 (DIM2) in Neurospora . The DNA methylation activity of DIM2 requires the presence of both H3K9me3 and HP1. Our structural study reveals a bipartite DIM2-HP1 interaction, leading to a disorder-to-order transition of the DIM2 target-recognition domain that is essential for substrate binding. Furthermore, the structure of DIM2-HP1-H3K9me3-DNA complex reveals a substrate-binding mechanism distinct from that for its mammalian orthologue DNMT1. In addition, the dual recognition of H3K9me3 peptide by the DIM2 RFTS and BAH1 domains allosterically impacts the DIM2-substrate binding, thereby controlling DIM2-mediated DNA methylation. Together, this study uncovers how multiple heterochromatin factors coordinately orchestrate an activity-switching mechanism for region-specific DNA methylation.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

SPARC: Structural properties associated with residue constraints

SPARC facilitates the generation of plausible hypotheses regarding underlying biochemical mechanisms by structurally characterizing protein sequence constraints. Such constraints appear as residues co-conserved in functionally related subgroups, as subtle pairwise correlations (i.e., direct couplings), and as correlations among these sequence features or with structural features. SPARC performs three types of analyses. First, based on pairwise sequence correlations, it estimates the biological relevance of alternative conformations and of homomeric contacts, as illustrated here for death domains. Second, it estimates the statistical significance of the correspondence between directly coupled residue pairs and interactions at heterodimeric interfaces. Third, given molecular dynamics simulated structures, it characterizes interactions among constrained residues or between such residues and ligands that: (a) are stably maintained during the simulation; (b) undergo correlated formation and/or disruption of interactions with other constrained residues; or (c) switch between alternative interactions. We illustrate this for two homohexameric complexes: the bacterial enhancer binding protein (bEBP) NtrC1, which activates transcription by remodeling RNA polymerase (RNAP) containing σ 54 , and for DnaB helicase, which opens DNA at the bacterial replication fork. Based on the NtrC1 analysis, we hypothesize possible mechanisms for inhibiting ATP hydrolysis until ADP is released from an adjacent subunit and for coupling ATP hydrolysis to restructuring of σ 54 binding loops. Based on the DnaB analysis, we hypothesize that DnaB ‘grabs’ ssDNA by flipping every fourth base and inserting it into cavities between subunits and that flipping of a DnaB-specific glutamine residue triggers ATP hydrolysis.

97 MATHEMATICS AND COMPUTING↗
DOE OSTI2025

Eucalyptus grandis MYB‐Like and RAN‐Like Zinc Finger Proteins Display Dual Roles in Regulating Plant Immunity and Symbiosis Pathways

Plant roots live in constant contact with diverse microbes in the soil. Plant fitness, therefore, relies on signaling pathways that mount an effective immune response against pathogens while fostering mutualistic symbioses. Plant pathways, and specifically immune genes that may act as "switches," discriminating between pathogenic or mutualistic fungi, remain largely unknown. Using Eucalyptus grandis as a model system, we investigate alterations to the root transcriptomic landscape during pre-symbiosis with either the pathogen Armillaria luteobubalina or the mutualistic fungus Pisolithus microcarpus. Comparative analyses identified three strongly counter-regulated genes that may act as immune switches to accommodate or to repress fungal colonization. We characterized two of these, a MYB-like and RAN-like zinc finger protein, using a transgenic approach and demonstrated that they have bifunctional roles in the regulation of cell death and a hypersensitive-like response, depending on the lifestyle of the associated fungus. Using co-expression network analysis, we identified hypothetical pathways correlated to these genes. We functionally validated these predictions using plants with transgenic roots with increased or decreased transcription of these genes, thereby showing the power of co-expression networks as an a priori approach to identify key immune response pathways in plants. Overall, our results demonstrate that prior to physical contact with microbes, MYB-like and RAN-like zinc finger proteins are key regulators of plant immune signaling that respond to fungal signals and enable or repress symbiotic establishment.

mycorrhizal fungi↗
DOE OSTI2023

Indefinite and bidirectional near-infrared nanocrystal photoswitching

Materials whose luminescence can be switched by optical stimulation drive technologies ranging from superresolution imaging, nanophotonics, and optical data storage, to targeted pharmacology, optogenetics, and chemical reactivity. Furthermore, these photoswitchable probes, including organic fluorophores and proteins, can be prone to photodegradation and often operate in the ultraviolet or visible spectral regions. Colloidal inorganic nanoparticles can offer improved stability, but the ability to switch emission bidirectionally, particularly with near-infrared (NIR) light, has not, to our knowledge, been reported in such systems. Here, we present two-way, NIR photoswitching of avalanching nanoparticles (ANPs), showing full optical control of upconverted emission using phototriggers in the NIR-I and NIR-II spectral regions useful for subsurface imaging. Employing single-step photodarkening and photobrightening, we demonstrate indefinite photoswitching of individual nanoparticles (more than 1,000 cycles over 7 h) in ambient or aqueous conditions without measurable photodegradation. Critical steps of the photoswitching mechanism are elucidated by modelling and by measuring the photon avalanche properties of single ANPs in both bright and dark states. Unlimited, reversible photoswitching of ANPs enables indefinitely rewritable two-dimensional and three-dimensional multilevel optical patterning of ANPs, as well as optical nanoscopy with sub-Å localization superresolution that allows us to distinguish individual ANPs within tightly packed clusters.

36 MATERIALS SCIENCE↗
DOE OSTI2021

A structured RNA motif locks Argonaute2:miR-122 onto the 5’ end of the HCV genome

microRNAs (miRNAs) form regulatory networks in metazoans. Viruses engage miRNA networks in numerous ways, with Flaviviridae members exploiting direct interactions of their RNA genomes with host miRNAs. For hepatitis C virus (HCV), binding of liver-abundant miR-122 stabilizes the viral RNA and regulates viral translation. Here, we investigate the structural basis for these activities, taking into consideration that miRNAs function in complex with Argonaute (Ago) proteins. The crystal structure of the Ago2:miR-122:HCV complex reveals a structured RNA motif that traps Ago2 on the viral RNA, masking its 5’ end from enzymatic attack. The trapped Ago2 can recruit host factor PCBP2, implicated in viral translation, while binding of a second Ago2:miR-122 competes with PCBP2, creating a potential molecular switch for translational control. Combined results reveal a viral RNA structure that modulates Ago2:miR-122 dynamics and repurposes host proteins to generate a functional analog of the mRNA cap-binding complex.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structure of metallochaperone in complex with the cobalamin-binding domain of its target mutase provides insight into cofactor delivery

G-protein metallochaperone MeaB in bacteria [methylmalonic aciduria type A (MMAA) in humans] is responsible for facilitating the delivery of adenosylcobalamin (AdoCbl) to methylmalonyl-CoA mutase (MCM), the only AdoCbl-dependent enzyme in humans. Genetic defects in the switch III region of MMAA lead to the genetic disorder methylmalonic aciduria in which the body is unable to process certain lipids. Here, we present a crystal structure of Methylobacterium extorquens MeaB bound to a nonhydrolyzable guanosine triphosphate (GTP) analog guanosine-5'-[(β,γ)-methyleno]triphosphate (GMPPCP) with the Cbl-binding domain of its target mutase enzyme (MeMCM cbl ). This structure provides an explanation for the stimulation of the GTP hydrolyase activity of MeaB afforded by target protein binding. We find that upon MCM cbl association, one protomer of the MeaB dimer rotates ~180°, such that the inactive state of MeaB is converted to an active state in which the nucleotide substrate is now surrounded by catalytic residues. Importantly, it is the switch III region that undergoes the largest change, rearranging to make direct contacts with the terminal phosphate of GMPPCP. These structural data additionally provide insights into the molecular basis by which this metallochaperone contributes to AdoCbl delivery without directly binding the cofactor. Our data suggest a model in which GTP-bound MeaB stabilizes a conformation of MCM that is open for AdoCbl insertion, and GTP hydrolysis, as signaled by switch III residues, allows MCM to close and trap its cofactor. Substitutions of switch III residues destabilize the active state of MeaB through loss of protein:nucleotide and protein:protein interactions at the dimer interface, thus uncoupling GTP hydrolysis from AdoCbl delivery.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Cryo-EM captures the coordination of asymmetric electron transfer through a di-copper site in DPOR

Enzymes that catalyze long-range electron transfer (ET) reactions often function as higher order complexes that possess two structurally symmetrical halves. The functional advantages for such an architecture remain a mystery. Using cryoelectron microscopy we capture snapshots of the nitrogenase-like dark-operative protochlorophyllide oxidoreductase (DPOR) during substrate binding and turnover. DPOR catalyzes reduction of the C17 = C18 double bond in protochlorophyllide during the dark chlorophyll biosynthetic pathway. DPOR is composed of electron donor (L-protein) and acceptor (NB-protein) component proteins that transiently form a complex in the presence of ATP to facilitate ET. NB-protein is an α 2 β 2 heterotetramer with two structurally identical halves. However, our structures reveal that NB-protein becomes functionally asymmetric upon substrate binding. Asymmetry results in allosteric inhibition of L-protein engagement and ET in one half. Residues that form a conduit for ET are aligned in one half while misaligned in the other. An ATP hydrolysis-coupled conformational switch is triggered once ET is accomplished in one half. These structural changes are then relayed to the other half through a di-nuclear copper center at the tetrameric interface of the NB-protein and leads to activation of ET and substrate reduction. These findings provide a mechanistic blueprint for regulation of long-range electron transfer reactions.

Computational biophysics↗
DOE OSTI2020

Structural evidence for a dynamic metallocofactor during N 2 reduction by Mo-nitrogenase

The enzyme nitrogenase uses a suite of complex metallocofactors to reduce dinitrogen (N 2 ) to ammonia. Mechanistic details of this reaction remain sparse. We report a 1.83-angstrom crystal structure of the nitrogenase molybdenum-iron (MoFe) protein captured under physiological N 2 turnover conditions. This structure reveals asymmetric displacements of the cofactor belt sulfurs (S2B or S3A and S5A) with distinct dinitrogen species in the two αβ dimers of the protein. The sulfur-displaced sites are distinct in the ability of protein ligands to donate protons to the bound dinitrogen species, as well as the elongation of either the Mo–O5 (carboxyl) or Mo–O7 (hydroxyl) distance that switches the Mo-homocitrate ligation from bidentate to monodentate. These results highlight the dynamic nature of the cofactor during catalysis and provide evidence for participation of all belt-sulfur sites in this process.

Science & Technology - Other Topics↗