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Results for “DNA crystallography”

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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68 records · Page 4
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Molecular dissection of the glutamine synthetase-GlnR nitrogen regulatory circuitry in Gram-positive bacteria

How bacteria sense and respond to nitrogen levels are central questions in microbial physiology. In Gram-positive bacteria, nitrogen homeostasis is controlled by an operon encoding glutamine synthetase (GS), a dodecameric machine that assimilates ammonium into glutamine, and the GlnR repressor. GlnR detects nitrogen excess indirectly by binding glutamine-feedback-inhibited-GS (FBI-GS), which activates its transcription-repression function. The molecular mechanisms behind this regulatory circuitry, however, are unknown. Here we describe biochemical and structural analyses of GS and FBI-GS-GlnR complexes from pathogenic and non-pathogenic Gram-positive bacteria. The structures show FBI-GS binds the GlnR C-terminal domain within its active-site cavity, juxtaposing two GlnR monomers to form a DNA-binding-competent GlnR dimer. The FBI-GS-GlnR interaction stabilizes the inactive GS conformation. Strikingly, this interaction also favors a remarkable dodecamer to tetradecamer transition in some GS, breaking the paradigm that all bacterial GS are dodecamers. These data thus unveil unique structural mechanisms of transcription and enzymatic regulation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Deciphering the crystal structure of a novel nanobody against the NEIL1 DNA glycosylase

Nanobodies (VHHs) are single-domain antibodies with three antigenic CDR regions and are used in diverse scientific applications. Here, an ∼14 kDa nanobody (A5) specific for the endonuclease VIII (Nei)-like 1 or NEIL1 DNA glycosylase involved in the first step of the base-excision repair pathway was crystallized and its structure was determined to 2.1 Å resolution. The crystals posed challenges due to potential twinning and anisotropic diffraction. Despite inconclusive twinning indicators, reprocessing in an orthorhombic setting and molecular replacement in space groupP2 1 2 1 2 enabled the successful modeling of 96% of residues in the asymmetric unit, with finalR work andR free values of 0.199 and 0.229, respectively.

Biochemistry & Molecular Biology↗
DOE OSTI2022

Structural basis of R-loop recognition by the S9.6 monoclonal antibody

R-loops are ubiquitous, dynamic nucleic-acid structures that play fundamental roles in DNA replication and repair, chromatin and transcription regulation, as well as telomere maintenance. The DNA-RNA hybrid–specific S9.6 monoclonal antibody is widely used to map R-loops. Here, we report crystal structures of a S9.6 antigen-binding fragment (Fab) free and bound to a 13-bp hybrid duplex. We demonstrate that S9.6 exhibits robust selectivity in binding hybrids over double-stranded (ds) RNA and in categorically rejecting dsDNA. S9.6 asymmetrically recognizes a compact epitope of two consecutive RNA nucleotides via their 2'-hydroxyl groups and six consecutive DNA nucleotides via their backbone phosphate and deoxyribose groups. Recognition is mediated principally by aromatic and basic residues of the S9.6 heavy chain, which closely track the curvature of the hybrid minor groove. These findings reveal the molecular basis for S9.6 recognition of R-loops, detail its binding specificity, identify a new hybrid-recognition strategy, and provide a framework for S9.6 protein engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Base pairing, structural and functional insights into N 4 -methylcytidine (m 4 C) and N 4 ,N 4 -dimethylcytidine (m 4 2 C) modified RNA

The N 4 -methylation of cytidine (m 4 C and m 4 2 C) in RNA plays important roles in both bacterial and eukaryotic cells. In this work, we synthesized a series of m 4 C and m 4 2 C modified RNA oligonucleotides, conducted their base pairing and bioactivity studies, and solved three new crystal structures of the RNA duplexes containing these two modifications. Our thermostability and X-ray crystallography studies, together with the molecular dynamic simulation studies, demonstrated that m 4 C retains a regular C:G base pairing pattern in RNA duplex and has a relatively small effect on its base pairing stability and specificity. By contrast, the m 4 2 C modification disrupts the C:G pair and significantly decreases the duplex stability through a conformational shift of native Watson-Crick pair to a wobble-like pattern with the formation of two hydrogen bonds. This double-methylated m 4 2 C also results in the loss of base pairing discrimination between C:G and other mismatched pairs like C:A, C:T and C:C. The biochemical investigation of these two modified residues in the reverse transcription model shows that both mono- or di-methylated cytosine bases could specify the C:T pair and induce the G to T mutation using HIV-1 RT. In the presence of other reverse transcriptases with higher fidelity like AMV-RT, the methylation could either retain the normal nucleotide incorporation or completely inhibit the DNA synthesis. These results indicate the methylation at N 4 -position of cytidine is a molecular mechanism to fine tune base pairing specificity and affect the coding efficiency and fidelity during gene replication.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structural understanding of non-nucleoside inhibition in an elongating herpesvirus polymerase

All herpesviruses encode a conserved DNA polymerase that is required for viral genome replication and serves as an important therapeutic target. Currently available herpesvirus therapies include nucleoside and non-nucleoside inhibitors (NNI) that target the DNA-bound state of herpesvirus polymerase and block replication. Here we report the ternary complex crystal structure of Herpes Simplex Virus 1 DNA polymerase bound to DNA and a 4-oxo-dihydroquinoline NNI, PNU-183792 (PNU), at 3.5 Å resolution. PNU bound at the polymerase active site, displacing the template strand and inducing a conformational shift of the fingers domain into an open state. These results demonstrate that PNU inhibits replication by blocking association of dNTP and stalling the enzyme in a catalytically incompetent conformation, ultimately acting as a nucleotide competing inhibitor (NCI). Sequence conservation of the NCI binding pocket further explains broad-spectrum activity while a direct interaction between PNU and residue V823 rationalizes why mutations at this position result in loss of inhibition.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

The structural basis for 2′−5′/3′−5′-cGAMP synthesis by cGAS

Abstract cGAS activates innate immune responses against cytosolic double-stranded DNA. Here, by determining crystal structures of cGAS at various reaction stages, we report a unifying catalytic mechanism. apo-cGAS assumes an array of inactive conformations and binds NTPs nonproductively. Dimerization-coupled double-stranded DNA-binding then affixes the active site into a rigid lock for productive metal•substrate binding. A web-like network of protein•NTP, intra-NTP, and inter-NTP interactions ensures the stepwise synthesis of 2′−5′/3′−5′-linked cGAMP while discriminating against noncognate NTPs and off-pathway intermediates. One divalent metal is sufficient for productive substrate binding, and capturing the second divalent metal is tightly coupled to nucleotide and linkage specificities, a process which manganese is preferred over magnesium by 100-fold. Additionally, we elucidate how mouse cGAS achieves more stringent NTP and linkage specificities than human cGAS. Together, our results reveal that an adaptable, yet precise lock-and-key-like mechanism underpins cGAS catalysis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Tousled-like kinase 2 targets ASF1 histone chaperones through client mimicry

Tousled-like kinases (TLKs) are nuclear serine-threonine kinases essential for genome maintenance and proper cell division in animals and plants. A major function of TLKs is to phosphorylate the histone chaperone proteins ASF1a and ASF1b to facilitate DNA replication-coupled nucleosome assembly, but how TLKs selectively target these critical substrates is unknown. Here, we show that TLK2 selectivity towards ASF1 substrates is achieved in two ways. First, the TLK2 catalytic domain recognizes consensus phosphorylation site motifs in the ASF1 C-terminal tail. Second, a short sequence at the TLK2 N-terminus docks onto the ASF1a globular N-terminal domain in a manner that mimics its histone H3 client. Disrupting either catalytic or non-catalytic interactions through mutagenesis hampers ASF1 phosphorylation by TLK2 and cell growth. Our results suggest that the stringent selectivity of TLKs for ASF1 is enforced by an unusual interaction mode involving mutual recognition of a short sequence motifs by both kinase and substrate.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural basis of the amidase ClbL central to the biosynthesis of the genotoxin colibactin

Colibactin is a genotoxic natural product produced by select commensal bacteria in the human gut microbiota. The compound is a bis-electrophile that is predicted to form interstrand DNA cross-links in target cells, leading to double-strand DNA breaks. The biosynthesis of colibactin is carried out by a mixed NRPS–PKS assembly line with several noncanonical features. An amidase, ClbL, plays a key role in the pathway, catalyzing the final step in the formation of the pseudodimeric scaffold. ClbL couples α-aminoketone and β-ketothioester intermediates attached to separate carrier domains on the NRPS–PKS assembly. Here, the 1.9 Å resolution structure of ClbL is reported, providing a structural basis for this key step in the colibactin biosynthetic pathway. The structure reveals an open hydrophobic active site surrounded by flexible loops, and comparison with homologous amidases supports its unusual function and predicts macromolecular interactions with pathway carrier-protein substrates. Modeling protein–protein interactions supports a predicted molecular basis for enzyme–carrier domain interactions. Overall, the work provides structural insight into this unique enzyme that is central to the biosynthesis of colibactin.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structural and mechanistic basis for redox sensing by the cyanobacterial transcription regulator RexT

Abstract Organisms have a myriad of strategies for sensing, responding to, and combating reactive oxygen species, which are unavoidable consequences of aerobic life. In the heterocystous cyanobacterium Nostoc sp. PCC 7120, one such strategy is the use of an ArsR-SmtB transcriptional regulator RexT that senses H 2 O 2 and upregulates expression of thioredoxin to maintain cellular redox homeostasis. Different from many other members of the ArsR-SmtB family which bind metal ions, RexT has been proposed to use disulfide bond formation as a trigger to bind and release DNA. Here, we present high-resolution crystal structures of RexT in the reduced and H 2 O 2 -treated states. These structures reveal that RexT showcases the ArsR-SmtB winged-helix-turn-helix fold and forms a vicinal disulfide bond to orchestrate a response to H 2 O 2 . The importance of the disulfide-forming Cys residues was corroborated using site-directed mutagenesis, mass spectrometry, and H 2 O 2 -consumption assays. Furthermore, an entrance channel for H 2 O 2 was identified and key residues implicated in H 2 O 2 activation were pinpointed. Finally, bioinformatics analysis of the ArsR-SmtB family indicates that the vicinal disulfide “redox switch” is a unique feature of cyanobacteria in the Nostocales order, presenting an interesting case where an ArsR-SmtB protein scaffold has been evolved to showcase peroxidatic activity and facilitate redox-based regulation.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

Nucleic acid mediated activation of a short prokaryotic Argonaute immune system

A short prokaryotic Argonaute (pAgo) TIR-APAZ (SPARTA) defense system, activated by invading DNA to unleash its TIR domain for NAD(P) + hydrolysis, was recently identified in bacteria. We report the crystal structure of SPARTA heterodimer in the absence of guide-RNA/target-ssDNA (2.66 Å) and a cryo-EM structure of the SPARTA oligomer (tetramer of heterodimers) bound to guide-RNA/target-ssDNA at nominal 3.15–3.35 Å resolution. The crystal structure provides a high-resolution view of SPARTA, revealing the APAZ domain as equivalent to the N, L1, and L2 regions of long pAgos and the MID domain containing a unique insertion (insert57). Cryo-EM structure reveals regions of the PIWI (loop10-9) and APAZ (helix αN) domains that reconfigure for nucleic-acid binding and decrypts regions/residues that reorganize to expose a positively charged pocket for higher-order assembly. The TIR domains amass in a parallel-strands arrangement for catalysis. We visualize SPARTA before and after RNA/ssDNA binding and uncover the basis of its active assembly leading to abortive infection.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

An autoinhibited state of 53BP1 revealed by small molecule antagonists and protein engineering

The recruitment of 53BP1 to chromatin, mediated by its recognition of histone H4 dimethylated at lysine 20 (H4K20me2), is important for DNA double-strand break repair. Using a series of small molecule antagonists, we demonstrate a conformational equilibrium between an open and a pre-existing lowly populated closed state of 53BP1 in which the H4K20me2 binding surface is buried at the interface between two interacting 53BP1 molecules. In cells, these antagonists inhibit the chromatin recruitment of wild type 53BP1, but do not affect 53BP1 variants unable to access the closed conformation despite preservation of the H4K20me2 binding site. Thus, this inhibition operates by shifting the conformational equilibrium toward the closed state. Our work therefore identifies an auto-associated form of 53BP1—autoinhibited for chromatin binding—that can be stabilized by small molecule ligands encapsulated between two 53BP1 protomers. Such ligands are valuable research tools to study the function of 53BP1 and have the potential to facilitate the development of new drugs for cancer therapy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structure of an RNA G-quadruplex from the West Nile virus genome

Potential G-quadruplex sites have been identified in the genomes of DNA and RNA viruses and proposed as regulatory elements. The genus Orthoflavivirus contains arthropod-transmitted, positive-sense, single-stranded RNA viruses that cause significant human disease globally. Computational studies have identified multiple potential G-quadruplex sites that are conserved across members of this genus. Subsequent biophysical studies established that some G-quadruplexes predicted in Zika and tickborne encephalitis virus genomes can form and known quadruplex binders reduced viral yields from cells infected with these viruses. The susceptibility of RNA to degradation and the variability of loop regions have made structure determination challenging. Despite these difficulties, we report a high-resolution structure of the NS5-B quadruplex from the West Nile virus genome. Analysis reveals two stacked tetrads that are further stabilized by a stacked triad and transient noncanonical base pairing. This structure expands the landscape of solved RNA quadruplex structures and demonstrates the diversity and complexity of biological quadruplexes. We anticipate that the availability of this structure will assist in solving further viral RNA quadruplexes and provides a model for a conserved antiviral target in Orthoflavivirus genomes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Structural analyses of the PKA RIIβ holoenzyme containing the oncogenic DnaJB1-PKAc fusion protein reveal protomer asymmetry and fusion-induced allosteric perturbations in fibrolamellar hepatocellular carcinoma

When the J-domain of the heat shock protein DnaJB1 is fused to the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), replacing exon 1, this fusion protein, J-C subunit (J-C), becomes the driver of fibrolamellar hepatocellular carcinoma (FL-HCC). Here, we use cryo-electron microscopy (cryo-EM) to characterize J-C bound to RIIβ, the major PKA regulatory (R) subunit in liver, thus reporting the first cryo-EM structure of any PKA holoenzyme. We report several differences in both structure and dynamics that could not be captured by the conventional crystallography approaches used to obtain prior structures. Most striking is the asymmetry caused by the absence of the second cyclic nucleotide binding (CNB) domain and the J-domain in one of the RIIβ:J-C protomers. Using molecular dynamics (MD) simulations, we discovered that this asymmetry is already present in the wild-type (WT) RIIβ 2 C 2 but had been masked in the previous crystal structure. This asymmetry may link to the intrinsic allosteric regulation of all PKA holoenzymes and could also explain why most disease mutations in PKA regulatory subunits are dominant negative. The cryo-EM structure, combined with small-angle X-ray scattering (SAXS), also allowed us to predict the general position of the Dimerization/Docking (D/D) domain, which is essential for localization and interacting with membrane-anchored A-Kinase-Anchoring Proteins (AKAPs). This position provides a multivalent mechanism for interaction of the RIIβ holoenzyme with membranes and would be perturbed in the oncogenic fusion protein. The J-domain also alters several biochemical properties of the RIIβ holoenzyme: It is easier to activate with cAMP, and the cooperativity is reduced. These results provide new insights into how the finely tuned allosteric PKA signaling network is disrupted by the oncogenic J-C subunit, ultimately leading to the development of FL-HCC.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The Single Residue K12 Governs the Exceptional Voltage Sensitivity of Mitochondrial Voltage-Dependent Anion Channel Gating

The voltage-dependent anion channel (VDAC) is a β-barrel channel of the mitochondrial outer membrane (MOM) that passively transports ions, metabolites, polypeptides, and single-stranded DNA. VDAC responds to a transmembrane potential by “gating,” i.e. transitioning to one of a variety of low-conducting states of unknown structure. The gated state results in nearly complete suppression of multivalent mitochondrial metabolite (such as ATP and ADP) transport, while enhancing calcium transport. Voltage gating is a universal property of β-barrel channels, but VDAC gating is anomalously sensitive to transmembrane potential. Here, we show that a single residue in the pore interior, K12, is responsible for most of VDAC’s voltage sensitivity. Using the analysis of over 40 μs of atomistic molecular dynamics (MD) simulations, we explore correlations between motions of charged residues inside the VDAC pore and geometric deformations of the β-barrel. Residue K12 is bistable; its motions between two widely separated positions along the pore axis enhance the fluctuations of the β-barrel and augment the likelihood of gating. Single channel electrophysiology of various K12 mutants reveals a dramatic reduction of the voltage-induced gating transitions. The crystal structure of the K12E mutant at a resolution of 2.6 Å indicates a similar architecture of the K12E mutant to the wild type; however, 60 μs of atomistic MD simulations using the K12E mutant show restricted motion of residue 12, due to enhanced connectivity with neighboring residues, and diminished amplitude of barrel motions. We conclude that β-barrel fluctuations, governed particularly by residue K12, drive VDAC gating transitions.

59 BASIC BIOLOGICAL SCIENCES↗