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At least 1,207 records · Page 67
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

A protein phosphatase 1 specific phos phatase ta rgeting p eptide (PhosTAP) to identify the PP1 phosphatome

Phosphoprotein phosphatases (PPPs) are the key serine/threonine phosphatases that regulate all essential signaling cascades. In particular, Protein Phosphatase 1 (PP1) dephosphorylates ~80% of all ser/thr phosphorylation sites. Here, we developed a phosphatase targeting peptide (PhosTAP) that binds all PP1 isoforms and does so with a stronger affinity than any other known PP1 regulator. This PhosTAP can be used as a PP1 recruitment tool for Phosphorylation Targeting Chimera (PhosTAC)-type recruitment in in vitro and cellular experiments, as well as in phosphoproteomics experiments to identify PP1-specific substrates and phosphosites. The latter is especially important to further our understanding of cellular signaling, as the identification of substrates and especially phosphosites that are targeted by specific phosphatases lags behind that of their kinase counterparts. Using PhosTAP-based proteomics, we show that, counter to our current understanding, many PP1 regulators are also substrates, that the number of residues between regulator PP1-binding and phosphosites vary significantly, and that PP1 counteracts the activities of mitotic kinases. Finally, we also found that Haspin kinase is a direct substrate of PP1 and that its PP1-dependent dephosphorylation modulates its activity during anaphase. Together, we show that PP1-specific PhosTAPs are a powerful tool for +studying PP1 activity in vitro and in cells.

Science & Technology - Other Topics↗
DOE OSTI2025

Thermophilic site-specific recombination system for rapid insertion of heterologous DNA into the Clostridium thermocellum chromosome

Clostridium thermocellum is an anaerobic thermophile capable of producing ethanol and other commodity chemicals from lignocellulosic biomass. The insertion of heterologous DNA into the C. thermocellum chromosome is currently achieved via a time-consuming homologous recombination process, where a single stable insertion can take 2–4 weeks or more to construct. In this work, we developed a thermostable version of the Serine recombinase Assisted Genome Engineering (tSAGE) approach for gene insertion in C. thermocellum utilizing a site-specific recombinase from Geobacillus sp. Y412MC61, enabling quick and easy insertion of DNA into the chromosome for accelerated genetic tool screening and heterologous gene expression. Using tSAGE, chromosomal insertion of plasmid DNA occurred at a maximum transformation efficiency of 5 × 10 3 CFU/µg, which is comparable to the transformation efficiency of a replicating control plasmid in C. thermocellum. Using tSAGE, we chromosomally integrated and characterized 17 reporter genes, 15 homologous and 31 heterologous constitutive promoters of varying strengths, 4 inducible promoters, and 5 riboswitches in C. thermocellum. We also determined that a 6–7 nucleotide gap between the ribosome binding site (RBS) and the start codon is optimal for high expression by employing a library of superfolder green fluorescent protein expression constructs driven by our strongest tested promoter (P clo1313_1194 ) with different distances between the RBS and start codon. The tools developed here will aid in accelerating C. thermocellum strain engineering for producing sustainable fuels and chemicals directly from plant biomass.

Biofuels↗
DOE OSTI2023

Follistatin Forms a Stable Complex With Inhibin A That Does Not Interfere With Activin A Antagonism

Abstract Inhibins are transforming growth factor-β family heterodimers that suppress follicle-stimulating hormone (FSH) secretion by antagonizing activin class ligands. Inhibins share a common β chain with activin ligands. Follistatin is another activin antagonist, known to bind the common β chain of both activins and inhibins. In this study, we characterized the antagonist-antagonist complex of inhibin A and follistatin to determine if their interaction impacted activin A antagonism. We isolated the inhibin A:follistatin 288 complex, showing that it forms in a 1:1 stoichiometric ratio, different from previously reported homodimeric ligand:follistatin complexes, which bind in a 1:2 ratio. Small angle X-ray scattering coupled with modeling provided a low-resolution structure of inhibin A in complex with follistatin 288. Inhibin binds follistatin via the shared activin β chain, leaving the α chain free and flexible. The inhibin A:follistatin 288 complex was also shown to bind heparin with lower affinity than follistatin 288 alone or in complex with activin A. Characterizing the inhibin A:follistatin 288 complex in an activin-responsive luciferase assay and by surface plasmon resonance indicated that the inhibitor complex readily dissociated upon binding type II receptor activin receptor type IIb, allowing both antagonists to inhibit activin signaling. Additionally, injection of the complex in ovariectomized female mice did not alter inhibin A suppression of FSH. Taken together, this study shows that while follistatin binds to inhibin A with a substochiometric ratio relative to the activin homodimer, the complex can dissociate readily, allowing both proteins to effectively antagonize activin signaling.

Endocrinology & Metabolism↗
DOE OSTI2024

Automated AI-driven Molecular Design for Therapeutic Discovery

In recent years, artificial intelligence and machine learning (AI/ML) approaches have revolutionized the process of designing new therapeutics, enabling scientists to rapidly respond to emerging threats from various pathogens. A prime example is the SARS-CoV-2 main protease, a key target for the development of antiviral inhibitors. In this study, we employed a novel, integrated approach that combines AI-driven iterative design of inhibitor candidates, screening based on physio-chemical properties and toxicity, physics-based computational modeling of protein-inhibitor interactions, and AI-assisted analysis of Native MS biophysical assay and characterization of designed candidates. Our deep learning 3D-scaffold model, which uses an input scaffold as a starting point, generated tens of thousands of compounds while preserving the key scaffold. To optimize these candidates, we calculated a comprehensive set of 136 descriptors, including both 2D and 3D molecular features, for compounds targeting the SARS-CoV-2 Main protease (Mpro) and a neurodegenerative disease-associated protein, cyclophilin (Cyp). The generated compounds were initially filtered based on their properties and then ranked according to their predicted binding affinity using our automated modeling and ML methods. Experimental validation of the Mpro candidates showing inhibitory activity demonstrates that our workflow can expedite the therapeutic discovery.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2019

Structure Based Prediction of Neoantigen Immunogenicity

The development of immunological therapies that incorporate peptide antigens presented to T cells by MHC proteins is a long sought-after goal, particularly for cancer, where mutated neoantigens are being explored as personalized cancer vaccines. Although neoantigens can be identified through sequencing, bioinformatics and mass spectrometry, identifying those which are immunogenic and able to promote tumor rejection remains a significant challenge. Here we examined the potential of high-resolution structural modeling followed by energetic scoring of structural features for predicting neoantigen immunogenicity. After developing a strategy to rapidly and accurately model nonameric peptides bound to the common class I MHC protein HLA-A2, we trained a neural network on structural features that influence T cell receptor (TCR) and peptide binding energies. The resulting structurally-parameterized neural network outperformed methods that do not incorporate explicit structural or energetic properties in predicting CD8 + T cell responses of HLA-A2 presented nonameric peptides, while also providing insight into the underlying structural and biophysical mechanisms governing immunogenicity. Our proof-of-concept study demonstrates the potential for structure-based immunogenicity predictions in the development of personalized peptide-based vaccines.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Pseudomonas aeruginosa C-Terminal Processing Protease CtpA Assembles into a Hexameric Structure That Requires Activation by a Spiral-Shaped Lipoprotein-Binding Partner

Pseudomonas aeruginosa CtpA is a carboxyl-terminal processing protease that partners with the outer membrane lipoprotein LbcA to degrade at least five cell wall-associated proteins, four of which are cell wall hydrolases. This activity plays an important role in supporting P. aeruginosa virulence in a mouse model of acute pneumonia. However, almost nothing is known about the molecular mechanisms underlying CtpA and LbcA function. Here, we used structural analysis to show that CtpA alone assembles into an inactive hexamer comprising a trimer of dimers, which limits its substrate access and prevents nonspecific degradation. The adaptor protein LbcA is a right-handed open spiral with 11 tetratricopeptide repeats, which might wrap around a substrate to deliver it to CtpA for degradation. By structure-guided mutagenesis and functional assays, we also showed that the interfaces of the CtpA trimer of dimers and an N-terminal helix of LbcA are important for LbcA-mediated substrate degradation by CtpA both in vitro and in vivo. This work improves our understanding of the molecular mechanism of the LbcA-CtpA proteolytic system and reveals some striking differences from the arrangements found in some other bacterial CTPs.

structural biology↗
DOE OSTI2024

Biomimetic mineralization of positively charged silica nanoparticles templated by thermoresponsive protein micelles: applications to electrostatic assembly of hierarchical and composite superstructures

High information content building blocks offer a path toward the construction of precision materials by supporting the organization and reconfiguration of organic and inorganic components through engineered functions. Here, we combine thermoresponsiveness with biomimetic mineralization by fusing the Car9 silica-binding dodecapeptide to the C-terminus of the (VPGVG) 54 elastin-like polypeptide (ELP). Using small angle X-ray scattering, we show that the short Car9 cationic block is sufficient to promote the conversion of disordered unimers into 30 nm micelles comprising about 150 proteins, 5 °C above the transition temperature of the ELP. While both species catalyze self-limiting silica precipitation, micelles template the mineralization of highly monodisperse (62 nm) nanoparticles, while unimers yield larger polydisperse species. Strikingly, and unlike traditional synthetic silica, these particles exhibit a positive surface charge, likely due to cationic Car9 sidechains projecting from their surface. Capitalizing on the high monodispersity and positive charge of the micelle-templated products, we use smaller silica and gold particles bearing a native negative charge to create a variety of superstructures via electrostatic co-assembly. Furthermore, this simple biomimetic route to positively charged silica eliminates the need for multiple precursors or surface modifications and enables the rapid creation of single-material and composite architectures in which components of different sizes or compositions are well dispersed and integrated.

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

Thioester synthesis by a designed nickel enzyme models prebiotic energy conversion

The formation of carbon–carbon bonds from prebiotic precursors such as carbon dioxide represents the foundation of all primordial life processes. In extant organisms, this reaction is carried out by the carbon monoxide dehydrogenase (CODH)/acetyl coenzyme A synthase (ACS) enzyme, which performs the cornerstone reaction in the ancient Wood–Ljungdahl metabolic pathway to synthesize the key biological metabolite, acetyl-CoA. Despite its significance, a fundamental understanding of this transformation is lacking, hampering efforts to harness analogous chemistry. To address these knowledge gaps, we have designed an artificial metalloenzyme within the azurin protein scaffold as a structural, functional, and mechanistic model of ACS. We demonstrate the intermediacy of the Ni I species and requirement for ordered substrate binding in the bioorganometallic carbon–carbon bond-forming reaction from the one-carbon ACS substrates. The electronic and geometric structures of the nickel-acetyl intermediate have been characterized using time-resolved optical, electron paramagnetic resonance, and X-ray absorption spectroscopy in conjunction with quantum chemical calculations. Moreover, we demonstrate that the nickel-acetyl species is chemically competent for selective acyl transfer upon thiol addition to biosynthesize an activated thioester. Drawing an analogy to the native enzyme, a mechanism for thioester generation by this ACS model has been proposed. The fundamental insight into the enzymatic process provided by this rudimentary ACS model has implications for the evolution of primitive ACS-like proteins. Ultimately, these findings offer strategies for development of highly active catalysts for sustainable generation of liquid fuels from one-carbon substrates, with potential for broad applications across diverse fields ranging from energy storage to environmental remediation.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Serodominant SARS-CoV-2 Nucleocapsid Peptides Map to Unstructured Protein Regions

The SARS-CoV-2 nucleocapsid (N) protein is highly immunogenic, and anti-N antibodies are commonly used as markers for prior infection. While several studies have examined or predicted the antigenic regions of N, these have lacked consensus and structural context. Using COVID-19 patient sera to probe an overlapping peptide array, we identified six public and four private epitope regions across N, some of which are unique to this study. We further report the first deposited X-ray structure of the stable dimerization domain at 2.05 Å as similar to all other reported structures. Structural mapping revealed that most epitopes are derived from surface-exposed loops on the stable domains or from the unstructured linker regions. An antibody response to an epitope in the stable RNA binding domain was found more frequently in sera from patients requiring intensive care. Since emerging amino acid variations in N map to immunogenic peptides, N protein variation could impact detection of seroconversion for variants of concern.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

The SARS-CoV-2 Spike Variant D614G Favors an Open Conformational State

The COVID-19 pandemic underwent a rapid transition with the emergence of a dominant viral variant (from the “D-form” to the “G-form”) that carried an amino acid substitution D614G in its “Spike” protein. The G-form is more infectious in vitro and associated with increased viral loads. To gain insight into the molecular-level underpinnings of these characteristics, we employed microsecond all-atom simulations. Here we show that changes in the protein energetics favor a higher population of infection-capable (open) states through release of hydrogen bonds of an asymmetry present in the D-form but not the G-form. Thus, the increased infectivity of the G-form is likely due to a higher rate of profitable binding encounters with the host receptor. It is also predicted to be more neutralization sensitive due to enhanced exposure of the receptor binding domain, a key target region for neutralizing antibodies. These results are significant for vaccine design. The Molecular Dynamics Simulations datasets generated in this study, namely that of the soluble form SARS-CoV-2 Spike protein are made available here. Details of the trajectory and supporting files are given in the README.txt file.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Crystal structures and functional analysis of the ZnF5-WWE1-WWE2 region of PARP13/ZAP define a distinctive mode of engaging poly(ADP-ribose)

PARP13/ZAP (zinc-finger antiviral protein) acts against multiple viruses by promoting degradation of viral mRNA. PARP13 has four N-terminal zinc (Zn) fingers that bind CG-rich nucleotide sequences, a C-terminal ADP ribosyltransferase fold, and a central region with a fifth Zn finger and tandem WWE domains. The central PARP13 region, ZnF5-WWE1-WWE2, is implicated in binding poly(ADP-ribose); however, there are limited insights into its structure and function. We present crystal structures of ZnF5-WWE1-WWE2 from mouse PARP13 in complex with ADP-ribose and in complex with ATP. The crystal structures and binding studies demonstrate that WWE2 interacts with ADP-ribose and ATP, whereas WWE1 does not have a functional binding site. Binding studies with poly(ADP-ribose) ligands indicate that WWE2 serves as an anchor for preferential binding to the terminal end of poly(ADP-ribose) chains. The composite ZnF5-WWE1-WWE2 structure forms an extended surface to engage ADP-ribose chains, representing a distinctive mode of recognition that provides a framework for investigating the impact of poly(ADP-ribose) on PARP13 function.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Mycobacterial DNA polymerase I: activities and crystal structures of the POL domain as apoenzyme and in complex with a DNA primer-template and of the full-length FEN/EXO–POL enzyme

Mycobacterial Pol1 is a bifunctional enzyme composed of an N-terminal DNA flap endonuclease/5' exonuclease domain (FEN/EXO) and a C-terminal DNA polymerase domain (POL). Here we document additional functions of Pol1: FEN activity on the flap RNA strand of an RNA:DNA hybrid and reverse transcriptase activity on a DNA-primed RNA template. We report crystal structures of the POL domain, as apoenzyme and as ternary complex with 3'-dideoxy-terminated DNA primer-template and dNTP. The thumb, palm, and fingers subdomains of POL form an extensive interface with the primer-template and the triphosphate of the incoming dNTP. Progression from an open conformation of the apoenzyme to a nearly closed conformation of the ternary complex entails a disordered-to-ordered transition of several segments of the thumb and fingers modules and an inward motion of the fingers subdomain—especially the O helix—to engage the primer-template and dNTP triphosphate. Distinctive structural features of mycobacterial Pol1 POL include a manganese binding site in the vestigial 3' exonuclease subdomain and a non-catalytic water-bridged magnesium complex at the protein-DNA interface. We report a crystal structure of the bifunctional FEN/EXO–POL apoenzyme that reveals the positions of two active site metals in the FEN/EXO domain.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

CasDinG is a 5′-3′ dsDNA and RNA/DNA helicase with three accessory domains essential for type IV CRISPR immunity

Abstract CRISPR-associated DinG protein (CasDinG) is essential to type IV-A CRISPR function. Here, we demonstrate that CasDinG from Pseudomonas aeruginosa strain 83 is an ATP-dependent 5′-3′ DNA translocase that unwinds double-stranded (ds)DNA and RNA/DNA hybrids. The crystal structure of CasDinG reveals a superfamily 2 helicase core of two RecA-like domains with three accessory domains (N-terminal, arch, and vestigial FeS). To examine the in vivo function of these domains, we identified the preferred PAM sequence for the type IV-A system (5′-GNAWN-3′ on the 5′-side of the target) with a plasmid library and performed plasmid clearance assays with domain deletion mutants. Plasmid clearance assays demonstrated that all three domains are essential for type IV-A immunity. Protein expression and biochemical assays suggested the vFeS domain is needed for protein stability and the arch for helicase activity. However, deletion of the N-terminal domain did not impair ATPase, ssDNA binding, or helicase activities, indicating a role distinct from canonical helicase activities that structure prediction tools suggest involves interaction with dsDNA. This work demonstrates CasDinG helicase activity is essential for type IV-A CRISPR immunity as well as the yet undetermined activity of the CasDinG N-terminal domain.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

3D_MolGNN_RL

3D-MolGNNRL, couples reinforcement learning (RL) to a deep generative model based on 3D-Scaffold to generate target candidates specific to a protein pocket building up atom by atom from the core scaffold. 3D-MolGNNRL provides an efficient way to optimize key features within a protein pocket using a parallel graph neural network model. The agent learns to build molecules in 3D space while optimizing the binding affinity, potency, and synthetic accessibility of the candidates generated for the SARS-CoV-2 Main protease

Kumar, Neeraj↗
DOE OSTI2022

Classifying Metal-Binding Sites with Neural Networks

A pre-trained convolutional neural network was fine-tuned for three separate classification tasks, distinguishing 2D images of: 1) single amino acids, 2) protein structural ball and stick images of metalloproteins, and 3) protein structural ball and stick images of metalloenzymes with the metal cofactors removed. Images used in the training, testing and validation are shared here.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Structure-Based Identification of Novel Histone Deacetylase 4 (HDAC4) Inhibitors

Histone deacetylases (HDACs) are important cancer drug targets. Existing FDA-approved drugs target the catalytic pocket of HDACs, which is conserved across subfamilies (classes) of HDAC. However, engineering specificity is an important goal. Herein, we use molecular modeling approaches to identify and target potential novel pockets specific to Class IIA HDAC-HDAC4 at the interface between HDAC4 and the transcriptional corepressor component protein NCoR. These pockets were screened using an ensemble docking approach combined with consensus scoring to identify compounds with a different binding mechanism than the currently known HDAC modulators. Binding was compared in experimental assays between HDAC4 and HDAC3, which belong to a different family of HDACs. HDAC4 was significantly inhibited by compound 88402 but not HDAC3. Two other compounds (67436 and 134199) had IC50 values in the low micromolar range for both HDACs, which is comparable to the known inhibitor of HDAC4, SAHA (Vorinostat). However, both of these compounds were significantly weaker inhibitors of HDAC3 than SAHA and thus more selective, albeit to a limited extent. Five compounds exhibited activity on human breast carcinoma and/or urothelial carcinoma cell lines. The present result suggests potential mechanistic and chemical approaches for developing selective HDAC4 modulators.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

A dynamic charge-charge interaction modulates PP2A:B56 substrate recruitment

The recruitment of substrates by the ser/thr protein phosphatase 2A (PP2A) is poorly understood, limiting our understanding of PP2A-regulated signaling. Recently, the first PP2A:B56 consensus binding motif, LxxIxE, was identified. However, most validated LxxIxE motifs bind PP2A:B56 with micromolar affinities, suggesting that additional motifs exist to enhance PP2A:B56 binding. Here, we report the requirement of a positively charged motif in a subset of PP2A:B56 interactors, including KIF4A, to facilitate B56 binding via dynamic, electrostatic interactions. Using molecular and cellular experiments, we show that a conserved, negatively charged groove on B56 mediates dynamic binding. We also discovered that this positively charged motif, in addition to facilitating KIF4A dephosphorylation, is essential for condensin I binding, a function distinct and exclusive from PP2A-B56 binding. Together, these results reveal how dynamic, charge-charge interactions fine-tune the interactions mediated by specific motifs, providing a new framework for understanding how PP2A regulation drives cellular signaling.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A synthetic cell density signal can drive proliferation in chick embryonic tendon cells and tendon cells from a full size rooster can produce high levels of procollagen in cell culture

Cell density signaling drives tendon morphogenesis by regulating both procollagen production and cell proliferation. The signal is composed of a small, highly conserved protein (SNZR P) tightly bound to a tissue-specific, unique lipid (SNZR L). This allows the complex (SNZR PL) to bind to the membrane of the cell and locally diffuse over a radius of ~1 mm. The cell produces low levels of this signal but the binding to the membrane increases with the number of tendon cells in the local environment. In this article SNZR P was produced in E.coli and SNZR L was chemically synthesized. The two bind together when heated to 60 °C in the presence of Ca ++ and Mg ++ and the synthesized SNZR PL at ng/ml levels can replace serum. Adding SNZR PL to the medium was also tested on primary tendon cells from adult roosters. The older cells were in a maintenance state in vivo and in cell culture they proliferate more slowly than embryonic cells. Nevertheless, after reaching a moderately high cell density, they produced high levels of procollagen similar to the embryonic cells. This data was not expected from older cells but suggests that adult tendon cells can regenerate the tissue after injury when given the correct signals.

59 BASIC BIOLOGICAL SCIENCES↗