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At least 1,243 records · Page 69
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Improved galactic foreground removal for B-mode detection with clustering methods

ABSTRACT Characterizing the sub-mm Galactic emission has become increasingly critical especially in identifying and removing its polarized contribution from the one emitted by the cosmic microwave background (CMB). In this work, we present a parametric foreground removal performed on to sub-patches identified in the celestial sphere by means of spectral clustering. Our approach takes into account efficiently both the geometrical affinity and the similarity induced by the measurements and the accompanying errors. The optimal partition is then used to parametrically separate the Galactic emission encoding thermal dust and synchrotron from the CMB one applied on two nominal observations of forthcoming experiments from the ground and from the space. Moreover, the clustering is performed on tracers that are different from the data used for component separation, e.g. the spectral index maps of dust and synchrotron. Performing the parametric fit singularly on each of the clustering derived regions results in an overall improvement: both controlling the bias and the uncertainties in the CMB B-mode recovered maps. We finally apply this technique using the map of the number of clouds along the line of sight, $\mathcal {N}_c$, as estimated from H i emission data and perform parametric fitting on to patches derived by clustering on this map. We show that adopting the $\mathcal {N}_c$ map as a tracer for the patches related to the thermal dust emission, results in reducing the B-mode residuals post-component separation. The code is made publicly available https://github.com/giuspugl/fgcluster.

79 ASTRONOMY AND ASTROPHYSICS↗
DOE OSTI2024

Substitution Models of Protein Evolution with Selection on Enzymatic Activity

Abstract Substitution models of evolution are necessary for diverse evolutionary analyses including phylogenetic tree and ancestral sequence reconstructions. At the protein level, empirical substitution models are traditionally used due to their simplicity, but they ignore the variability of substitution patterns among protein sites. Next, in order to improve the realism of the modeling of protein evolution, a series of structurally constrained substitution models were presented, but still they usually ignore constraints on the protein activity. Here, we present a substitution model of protein evolution with selection on both protein structure and enzymatic activity, and that can be applied to phylogenetics. In particular, the model considers the binding affinity of the enzyme–substrate complex as well as structural constraints that include the flexibility of structural flaps, hydrogen bonds, amino acids backbone radius of gyration, and solvent-accessible surface area that are quantified through molecular dynamics simulations. We applied the model to the HIV-1 protease and evaluated it by phylogenetic likelihood in comparison with the best-fitting empirical substitution model and a structurally constrained substitution model that ignores the enzymatic activity. We found that accounting for selection on the protein activity improves the fitting of the modeled functional regions with the real observations, especially in data with high molecular identity, which recommends considering constraints on the protein activity in the development of substitution models of evolution.

Ferreiro, David↗
DOE OSTI2022

The development and use of metal-based probes for X-ray fluorescence microscopy

Abstract X-ray fluorescence microscopy (XFM) has become a widely used technique for imaging the concentration and distribution of metal ions in cells and tissues. Recent advances in synchrotron sources, optics, and detectors have improved the spatial resolution of the technique to <10 nm with attogram detection sensitivity. However, to make XFM most beneficial for bioimaging—especially at the nanoscale—the metal ion distribution must be visualized within the subcellular context of the cell. Over the years, a number of approaches have been taken to develop X-ray-sensitive tags that permit the visualization of specific organelles or proteins using XFM. In this review, we examine the types of X-ray fluorophore used, including nanomaterials and metal ions, and the approaches used to incorporate the metal into their target binding site via antibodies, genetically encoded metal-binding peptides, affinity labeling, or cell-specific peptides. We evaluate their advantages and disadvantages, review the scientific findings, and discuss the needs for future development.

36 MATERIALS SCIENCE↗
DOE OSTI2020

Mechanistic insights from structure of Mycobacterium smegmatis topoisomerase I with ssDNA bound to both N- and C-terminal domains

Abstract Type IA topoisomerases interact with G-strand and T-strand ssDNA to regulate DNA topology. However, simultaneous binding of two ssDNA segments to a type IA topoisomerase has not been observed previously. We report here the crystal structure of a type IA topoisomerase with ssDNA segments bound in opposite polarity to the N- and C-terminal domains. Titration of small ssDNA oligonucleotides to Mycobacterium smegmatis topoisomerase I with progressive C-terminal deletions showed that the C-terminal region has higher affinity for ssDNA than the N-terminal active site. This allows the C-terminal domains to capture one strand of underwound negatively supercoiled DNA substrate first and position the N-terminal domains to bind and cleave the opposite strand in the relaxation reaction. Efficiency of negative supercoiling relaxation increases with the number of domains that bind ssDNA primarily with conserved aromatic residues and possibly with assistance from polar/basic residues. A comparison of bacterial topoisomerase I structures showed that a conserved transesterification unit (N-terminal toroid structure) for cutting and rejoining of a ssDNA strand can be combined with two different types of C-terminal ssDNA binding domains to form diverse bacterial topoisomerase I enzymes that are highly efficient in their physiological role of preventing excess negative supercoiling in the genome.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Crystal structure of an atypical cobalamin riboswitch reveals RNA structural adaptability as basis for promiscuous ligand binding

Cobalamin riboswitches encompass a structurally diverse group of cis-acting, gene regulatory elements found mostly in bacterial messenger RNA and are classified into subtypes based on secondary and tertiary characteristics. An unusual variant of the cobalamin riboswitch with predicted structural features was identified in Bacillus subtilis over a decade ago, but its structure and mechanisms of cobalamin selectivity and translational control have remained unsolved. We present the crystal structure of the aptamer domain of this atypical cobalamin riboswitch and a model for the complete riboswitch, including its expression platform domain. We demonstrate that this riboswitch binds to multiple cobalamin derivatives and correlate its promiscuous behavior to its structure and unique arrangement of peripheral elements. Comparative structural analyses between conventional cobalamin riboswitches and the B. subtilis cobalamin riboswitch reveal that the likely basis for this promiscuous ligand binding is intrinsic structural adaptability encoded in the RNA structure. It suggests that cobalamin selectivity might ultimately be viewed as existing on a spectrum of affinity for each derivative rather than as belonging to distinct types based on ligand specificities. Our work provides an interesting and notable example of functional coupling of ligand-sensing and adaptive folding by a structured RNA molecule.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Allosteric autoregulation of DNA binding via a DNA-mimicking protein domain: a biophysical study of ZNF410–DNA interaction using small angle X-ray scattering

ZNF410 is a highly-conserved transcription factor, remarkable in that it recognizes a 15-base pair DNA element but has just a single responsive target gene in mammalian erythroid cells. ZNF410 includes a tandem array of five zinc-fingers (ZFs), surrounded by uncharacterized N- and C-terminal regions. Unexpectedly, full-length ZNF410 has reduced DNA binding affinity, compared to that of the isolated DNA binding ZF array, both in vitro and in cells. AlphaFold predicts a partially-folded N-terminal subdomain that includes a 30-residue long helix, preceded by a hairpin loop rich in acidic (aspartate/glutamate) and serine/threonine residues. This hairpin loop is predicted by AlphaFold to lie against the DNA binding interface of the ZF array. In solution, ZNF410 is a monomer and binds to DNA with 1:1 stoichiometry. Surprisingly, the single best-fit model for the experimental small angle X-ray scattering profile, in the absence of DNA, is the original AlphaFold model with the N-terminal long-helix and the hairpin loop occupying the ZF DNA binding surface. For DNA binding, the hairpin loop presumably must be displaced. After combining biophysical, biochemical, bioinformatic and artificial intelligence-based AlphaFold analyses, we suggest that the hairpin loop mimics the structure and electrostatics of DNA, and provides an additional mechanism, supplementary to sequence specificity, of regulating ZNF410 DNA binding.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structures of CTCF–DNA complexes including all 11 zinc fingers

The CCCTC-binding factor (CTCF) binds tens of thousands of enhancers and promoters on mammalian chromosomes by means of its 11 tandem zinc finger (ZF) DNA-binding domain. In addition to the 12–15-bp CORE sequence, some of the CTCF binding sites contain 5' upstream and/or 3' downstream motifs. Here, we describe two structures for overlapping portions of human CTCF, respectively, including ZF1–ZF7 and ZF3–ZF11 in complex with DNA that incorporates the CORE sequence together with either 3' downstream or 5' upstream motifs. Like conventional tandem ZF array proteins, ZF1–ZF7 follow the right-handed twist of the DNA, with each finger occupying and recognizing one triplet of three base pairs in the DNA major groove. ZF8 plays a unique role, acting as a spacer across the DNA minor groove and positioning ZF9–ZF11 to make cross-strand contacts with DNA. We ascribe the difference between the two subgroups of ZF1–ZF7 and ZF8–ZF11 to residues at the two positions -6 and -5 within each finger, with small residues for ZF1–ZF7 and bulkier and polar/charged residues for ZF8–ZF11. ZF8 is also uniquely rich in basic amino acids, which allows salt bridges to DNA phosphates in the minor groove. Highly specific arginine–guanine and glutamine–adenine interactions, used to recognize G:C or A:T base pairs at conventional base-interacting positions of ZFs, also apply to the cross-strand interactions adopted by ZF9–ZF11. The differences between ZF1–ZF7 and ZF8–ZF11 can be rationalized structurally and may contribute to recognition of high-affinity CTCF binding sites.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural basis for cloverleaf RNA-initiated viral genome replication

The genomes of positive-strand RNA viruses serve as a template for both protein translation and genome replication. In enteroviruses, a cloverleaf RNA structure at the 5' end of the genome functions as a switch to transition from viral translation to replication by interacting with host poly(C)-binding protein 2 (PCBP2) and the viral 3CD pro protein. We determined the structures of cloverleaf RNA from coxsackievirus and poliovirus. Cloverleaf RNA folds into an H-type four-way junction and is stabilized by a unique adenosine-cytidine-uridine (A•C-U) base triple involving the conserved pyrimidine mismatch region. The two PCBP2 binding sites are spatially proximal and are located on the opposite end from the 3CD pro binding site on cloverleaf. We determined that the A•C-U base triple restricts the flexibility of the cloverleaf stem–loops resulting in partial occlusion of the PCBP2 binding site, and elimination of the A•C-U base triple increases the binding affinity of PCBP2 to the cloverleaf RNA. Based on the cloverleaf structures and biophysical assays, we propose a new mechanistic model by which enteroviruses use the cloverleaf structure as a molecular switch to transition from viral protein translation to genome replication.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

BindingDB in 2024: a FAIR knowledgebase of protein-small molecule binding data

Abstract BindingDB (bindingdb.org) is a public, web-accessible database of experimentally measured binding affinities between small molecules and proteins, which supports diverse applications including medicinal chemistry, biochemical pathway annotation, training of artificial intelligence models and computational chemistry methods development. This update reports significant growth and enhancements since our last review in 2016. Of note, the database now contains 2.9 million binding measurements spanning 1.3 million compounds and thousands of protein targets. This growth is largely attributable to our unique focus on curating data from US patents, which has yielded a substantial influx of novel binding data. Recent improvements include a remake of the website following responsive web design principles, enhanced search and filtering capabilities, new data download options and webservices and establishment of a long-term data archive replicated across dispersed sites. We also discuss BindingDB’s positioning relative to related resources, its open data sharing policies, insights gleaned from the dataset and plans for future growth and development.

Liu, Tiqing↗
DOE OSTI2024

A phage nucleus-associated RNA-binding protein is required for jumbo phage infection

Abstract Large-genome bacteriophages (jumbo phages) of the proposed family Chimalliviridae assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and DNA-targeting CRISPR-Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here, we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d results in accumulation of phage-encoded mRNAs in the phage nucleus, reduces phage protein production, and compromises virion assembly. Taken together, our data show that the conserved ChmC protein plays crucial roles in the viral life cycle, potentially by facilitating phage mRNA translocation through the nuclear shell to promote protein production and virion development.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Symmetry breaking of fluorophore binding to a G-quadruplex generates an RNA aptamer with picomolar K D

Fluorogenic RNA aptamer tags with high affinity enable RNA purification and imaging. The G-quadruplex (G4) based Mango (M) series of aptamers were selected to bind a thiazole orange based (TO1-Biotin) ligand. Using a chemical biology and reselection approach, we have produced a MII.2 aptamer–ligand complex with a remarkable set of properties: Its unprecedented K D of 45 pM, formaldehyde resistance (8% v/v), temperature stability and ligand photo-recycling properties are all unusual to find simultaneously within a small RNA tag. Crystal structures demonstrate how MII.2, which differs from MII by a single A23U mutation, and modification of the TO1-Biotin ligand to TO1-6A-Biotin achieves these results. MII binds TO1-Biotin heterogeneously via a G4 surface that is surrounded by a stadium of five adenosines. Breaking this pseudo-rotational symmetry results in a highly cooperative and homogeneous ligand binding pocket: A22 of the G4 stadium stacks on the G4 binding surface while the TO1-6A-Biotin ligand completely fills the remaining three quadrants of the G4 ligand binding face. Similar optimization attempts with MIII.1, which already binds TO1-Biotin in a homogeneous manner, did not produce such marked improvements. We use the novel features of the MII.2 complex to demonstrate a powerful optically-based RNA purification system.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structural basis for a highly conserved RNA-mediated enteroviral genome replication

Abstract Enteroviruses contain conserved RNA structures at the extreme 5′ end of their genomes that recruit essential proteins 3CD and PCBP2 to promote genome replication. However, the high-resolution structures and mechanisms of these replication-linked RNAs (REPLRs) are limited. Here, we determined the crystal structures of the coxsackievirus B3 and rhinoviruses B14 and C15 REPLRs at 1.54, 2.2 and 2.54 Å resolution, revealing a highly conserved H-type four-way junction fold with co-axially stacked sA-sD and sB-sC helices that are stabilized by a long-range A•C•U base-triple. Such conserved features observed in the crystal structures also allowed us to predict the models of several other enteroviral REPLRs using homology modeling, which generated models almost identical to the experimentally determined structures. Moreover, our structure-guided binding studies with recombinantly purified full-length human PCBP2 showed that two previously proposed binding sites, the sB-loop and 3′ spacer, reside proximally and bind a single PCBP2. Additionally, the DNA oligos complementary to the 3′ spacer, the high-affinity PCBP2 binding site, abrogated its interactions with enteroviral REPLRs, suggesting the critical roles of this single-stranded region in recruiting PCBP2 for enteroviral genome replication and illuminating the promising prospects of developing therapeutics against enteroviral infections targeting this replication platform.

Biochemistry & Molecular Biology↗
DOE OSTI2024

A compact stem-loop DNA aptamer targets a uracil-binding pocket in the SARS-CoV-2 nucleocapsid RNA-binding domain

Abstract SARS-CoV-2 nucleocapsid (N) protein is a structural component of the virus with essential roles in the replication and packaging of the viral RNA genome. The N protein is also an important target of COVID-19 antigen tests and a promising vaccine candidate along with the spike protein. Here, we report a compact stem-loop DNA aptamer that binds tightly to the N-terminal RNA-binding domain of SARS-CoV-2 N protein. Crystallographic analysis shows that a hexanucleotide DNA motif (5′-TCGGAT-3′) of the aptamer fits into a positively charged concave surface of N-NTD and engages essential RNA-binding residues including Tyr109, which mediates a sequence-specific interaction in a uracil-binding pocket. Avid binding of the DNA aptamer allows isolation and sensitive detection of full-length N protein from crude cell lysates, demonstrating its selectivity and utility in biochemical applications. We further designed a chemically modified DNA aptamer and used it as a probe to examine the interaction of N-NTD with various RNA motifs, which revealed a strong preference for uridine-rich sequences. Our studies provide a high-affinity chemical probe for the SARS-CoV-2 N protein RNA-binding domain, which may be useful for diagnostic applications and investigating novel antiviral agents.

Biochemistry & Molecular Biology↗
DOE OSTI2025

Crystal structure of MutYX: a novel clusterless adenine DNA glycosylase with a distinct C-terminal domain and 8-oxoguanine recognition sphere

Abstract The [4Fe–4S] cluster is an important cofactor of the base excision repair (BER) adenine DNA glycosylase MutY to prevent mutations associated with 8-oxoguanine (OG). Several MutYs lacking the [4Fe–4S] cofactor have been identified. Phylogenetic analysis shows that clusterless MutYs are distributed in two clades suggesting cofactor loss has occurred in multiple independent evolutionary events. Herein, we determined the first crystal structure of a clusterless MutY complexed with DNA. On the basis of the dramatic structural divergence from canonical MutYs, we refer to this as representative of a clusterless MutY subgroup “MutYX.” Interestingly, MutYX compensates for the missing [4Fe–4S] cofactor to maintain positioning of catalytic residues by expanding a pre-existing α-helix and acquisition of a new α-helix. Surprisingly, MutYX also acquired a new C-terminal domain that uniquely recognizes OG using residues Gln201 and Arg209. Adenine glycosylase assays and binding affinity measurements indicate that Arg209 is the primary residue responsible for OG:A lesion specificity, while Gln201 assists by bridging OG and Arg209. Surprisingly, replacement of Arg209 and Gln201 with Ala increased activity toward G:A mismatches. The MutYX structure serves as an example of devolution, capturing structural features required to retain function in the absence of a metal cofactor considered indispensable.

Trasviña-Arenas, Carlos H [University of Californi↗
DOE OSTI2025

Structural basis for sensitivity and acquired resistance of fungal cap guanine-N7 methyltransferases to the antifungal antibiotic sinefungin

Abstract The essential enzyme messenger RNA (mRNA) (guanine-N7) methyltransferase catalyzes S-adenosylmethionine (SAM)-dependent conversion of GpppRNA ends to the m7GpppRNA cap structure characteristic of eukaryal mRNAs. The antibiotic sinefungin (SFG) is a SAM analog in which the S-CH3 sulfonium moiety of SAM is replaced by a C-NH2 amine. Available evidence indicates that the antifungal activity of SFG is exerted via inhibition of fungal cap methyltransferase Abd1. Here we report that recombinant Kluyveromyces lactis and Saccharomyces cerevisiae Abd1 are 240-fold and 485-fold more sensitive to inhibition by SFG than by the reaction product S-adenosylhomocysteine (SAH). Crystal structures of K. lactis and S. cerevisiae Abd1 as binary complexes with SAH or SFG and ternary complexes with GTP•SFG highlight how SFG makes two hydrogen bonds from its C-NH2 amine to the guanine-O6 and -N7 atoms of GTP that account for its higher affinity vis-à-vis SAH and SAM. Through a genetic screen to isolate SFG-resistant S. cerevisiae strains, a conserved tyrosine (Tyr416) that interacts with the cap guanine in Abd1 was identified as a key determinant of SFG potency. Tyr416 Abd1 variants confer SFG resistance in vitro by weakening cap-assisted SFG interactions with Abd1. Our study illuminates the basis for the exquisite SFG sensitivity of fungal cap methyltransferases.

Biochemistry & Molecular Biology↗
DOE OSTI2025

Mechanistic studies of small molecule ligands selective to RNA single G bulges

Abstract Small-molecule RNA binders have emerged as an important pharmacological modality. A profound understanding of the ligand selectivity, binding mode, and influential factors governing ligand engagement with RNA targets is the foundation for rational ligand design. Here, we report a novel class of coumarin derivatives exhibiting selective binding affinity towards single G RNA bulges. Harnessing the computational power of all-atom Gaussian accelerated molecular dynamics simulations, we unveiled a rare minor groove binding mode of the ligand with a key interaction between the coumarin moiety and the G bulge. This predicted binding mode is consistent with results obtained from structure-activity relationship studies and transverse relaxation measurements by nuclear magnetic resonance spectroscopy. We further generated 444 molecular descriptors from 69 coumarin derivatives and identified key contributors to the binding events, such as charge state and planarity, by lasso (least absolute shrinkage and selection operator) regression. Our work deepened the understanding of RNA-small molecule interactions and integrated a new framework for the rational design of selective small-molecule RNA binders.

Biochemistry & Molecular Biology↗
DOE OSTI2024

Biochemical Characterization of Rice Xylan Biosynthetic Enzymes in Determining Xylan Chain Elongation and Substitutions

Abstract Grass xylan consists of a linear chain of β-1,4-linked xylosyl residues that often form domains substituted only with either arabinofuranose (Araf) or glucuronic acid (GlcA)/methylglucuronic acid (MeGlcA) residues, and it lacks the unique reducing end tetrasaccharide sequence found in dicot xylan. The mechanism of how grass xylan backbone elongation is initiated and how its distinctive substitution pattern is determined remains elusive. Here, we performed biochemical characterization of rice xylan biosynthetic enzymes, including xylan synthases, glucuronyltransferases and methyltransferases. Activity assays of rice xylan synthases demonstrated that they required short xylooligomers as acceptors for their activities. While rice xylan glucuronyltransferases effectively glucuronidated unsubstituted xylohexaose acceptors, they transferred little GlcA residues onto (Araf)-substituted xylohexaoses and rice xylan 3-O-arabinosyltransferase could not arabinosylate GlcA-substituted xylohexaoses, indicating that their intrinsic biochemical properties may contribute to the distinctive substitution patterns of rice xylan. In addition, we found that rice xylan methyltransferase exhibited a low substrate binding affinity, which may explain the partial GlcA methylation in rice xylan. Furthermore, immunolocalization of xylan in xylem cells of both rice and Arabidopsis showed that it was deposited together with cellulose in secondary walls without forming xylan-rich nanodomains. Together, our findings provide new insights into the biochemical mechanisms underlying xylan backbone elongation and substitutions in grass species.

Cell Biology↗
DOE OSTI2024

Proteomic Insights into Trichome Responses to Elevated Elemental Stress in Cation Exchanger (CAX) Mutants

Abstract Research on elemental distribution in plants is crucial for understanding nutrient uptake, environmental adaptation and optimizing agricultural practices for sustainable food production. Plant trichomes, with their self-contained structures and easy accessibility, offer a robust model system for investigating elemental repartitioning. Transport proteins, such as the four functional cation exchangers (CAXs) in Arabidopsis, are low-affinity, high-capacity transporters primarily located on the vacuole. Mutants in these transporters have been partially characterized, one of the phenotypes of the CAX1 mutant being altered with tolerance to low-oxygen conditions. A simple visual screen demonstrated trichome density and morphology in cax1, and quadruple CAX (cax1-4: qKO) mutants remained unaltered. Here, we used synchrotron X-ray fluorescence (SXRF) to show that trichomes in CAX-deficient lines accumulated high levels of chlorine, potassium, calcium and manganese. Proteomic analysis on isolated Arabidopsis trichomes showed changes in protein abundance in response to changes in element accumulation. The CAX mutants showed an increased abundance of plasma membrane ATPase and vacuolar H-pumping proteins, and proteins associated with water movement and endocytosis, while also showing changes in proteins associated with the regulation of plasmodesmata. These findings advance our understanding of the integration of CAX transport with elemental homeostasis within trichomes and shed light on how plants modulate protein abundance under conditions of altered elemental levels.

59 BASIC BIOLOGICAL SCIENCES↗