Nova Reader - Subject https://www.novareader.co Default RSS Feed en-us Newgen KnowledgeWorks <![CDATA[Crystal structure and functional implication of a bacterial cyclic AMP–AMP–GMP synthetase]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab165 Mammalian cyclic GMP-AMP synthase (cGAS) and its homologue dinucleotide cyclase in Vibrio cholerae (VcDncV) produce cyclic dinucleotides (CDNs) that participate in the defense against viral infection. Recently, scores of new cGAS/DncV-like nucleotidyltransferases (CD-NTases) were discovered, which produce various CDNs and cyclic trinucleotides (CTNs) as second messengers. Here, we present the crystal structures of EcCdnD, a CD-NTase from Enterobacter cloacae that produces cyclic AMP-AMP-GMP, in its apo-form and in complex with ATP, ADP and AMPcPP, an ATP analogue. Despite the similar overall architecture, the protein shows significant structural variations from other CD-NTases. Adjacent to the donor substrate, another nucleotide is bound to the acceptor binding site by a non-productive mode. Isothermal titration calorimetry results also suggest the presence of two ATP binding sites. GTP alone does not bind to EcCdnD, which however binds to pppApG, a possible intermediate. The enzyme is active on ATP or a mixture of ATP and GTP, and the best metal cofactor is Mg2+. The conserved residues Asp69 and Asp71 are essential for catalysis, as indicated by the loss of activity in the mutants. Based on structural analysis and comparison with VcDncV and RNA polymerase, a tentative catalytic pathway for the CTN-producing EcCdnD is proposed.

]]> <![CDATA[Molecular mechanism of RNase R substrate sensitivity for RNA ribose methylation]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab202 RNA 2′-O-methylation is widely distributed and plays important roles in various cellular processes. Mycoplasma genitalium RNase R (MgR), a prokaryotic member of the RNase II/RNB family, is a 3′-5′ exoribonuclease and is particularly sensitive to RNA 2′-O-methylation. However, how RNase R interacts with various RNA species and exhibits remarkable sensitivity to substrate 2′-O-methyl modifications remains elusive. Here we report high-resolution crystal structures of MgR in apo form and in complex with various RNA substrates. The structural data together with extensive biochemical analysis quantitively illustrate MgR’s ribonuclease activity and significant sensitivity to RNA 2′-O-methylation. Comparison to its related homologs reveals an exquisite mechanism for the recognition and degradation of RNA substrates. Through structural and mutagenesis studies, we identified proline 277 to be responsible for the significant sensitivity of MgR to RNA 2′-O-methylation within the RNase II/RNB family. We also generated several MgR variants with modulated activities. Our work provides a mechanistic understanding of MgR activity that can be harnessed as a powerful RNA analytical tool that will open up a new venue for RNA 2′-O-methylations research in biological and clinical samples.

]]> <![CDATA[Water structure around a left-handed Z-DNA fragment analyzed by cryo neutron crystallography]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab264 Even in high-quality X-ray crystal structures of oligonucleotides determined at a resolution of 1 Å or higher, the orientations of first-shell water molecules remain unclear. We used cryo neutron crystallography to gain insight into the H-bonding patterns of water molecules around the left-handed Z-DNA duplex [d(CGCGCG)]2. The neutron density visualized at 1.5 Å resolution for the first time allows us to pinpoint the orientations of most of the water molecules directly contacting the DNA and of many second-shell waters. In particular, H-bond acceptor and donor patterns for water participating in prominent hydration motifs inside the minor groove, on the convex surface or bridging nucleobase and phosphate oxygen atoms are finally revealed. Several water molecules display entirely unexpected orientations. For example, a water molecule located at H-bonding distance from O6 keto oxygen atoms of two adjacent guanines directs both its deuterium atoms away from the keto groups. Exocyclic amino groups of guanine (N2) and cytosine (N4) unexpectedly stabilize waters H-bonded to O2 keto oxygens from adjacent cytosines and O6 keto oxygens from adjacent guanines, respectively. Our structure offers the most detailed view to date of DNA solvation in the solid-state undistorted by metal ions or polyamines.

]]> <![CDATA[Nucleosome composition regulates the histone H3 tail conformational ensemble and accessibility]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab246 Hexasomes and tetrasomes are intermediates in nucleosome assembly and disassembly. Their formation is promoted by histone chaperones, ATP-dependent remodelers, and RNA polymerase II. In addition, hexasomes are maintained in transcribed genes and could be an important regulatory factor. While nucleosome composition has been shown to affect the structure and accessibility of DNA, its influence on histone tails is largely unknown. Here, we investigate the conformational dynamics of the H3 tail in the hexasome and tetrasome. Using a combination of NMR spectroscopy, MD simulations, and trypsin proteolysis, we find that the conformational ensemble of the H3 tail is regulated by nucleosome composition. As has been found for the nucleosome, the H3 tails bind robustly to DNA within the hexasome and tetrasome, but upon loss of the H2A/H2B dimer, we determined that the adjacent H3 tail has an altered conformational ensemble, increase in dynamics, and increase in accessibility. Similar to observations of DNA dynamics, this is seen to be asymmetric in the hexasome. Our results indicate that nucleosome composition has the potential to regulate chromatin signaling and ultimately help shape the chromatin landscape.

]]> <![CDATA[Crystal structures of N-terminally truncated telomerase reverse transcriptase from fungi<a href="#FN2"><sup>‡</sup></a>]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab261 Telomerase plays critical roles in cellular aging, in the emergence and/or development of cancer, and in the capacity for stem-cell renewal, consists of a catalytic telomerase reverse transcriptase (TERT) and a template-encoding RNA (TER). TERs from diverse organisms contain two conserved structural elements: the template-pseudoknot (T-PK) and a helical three-way junction (TWJ). Species-specific features of the structure and function of telomerase make obtaining a more in-depth understanding of the molecular mechanism of telomerase particularly important. Here, we report the first structural studies of N-terminally truncated TERTs from Candida albicans and Candida tropicalis in apo form and complexed with their respective TWJs in several conformations. We found that Candida TERT proteins perform only one round of telomere addition in the presence or absence of PK/TWJ and display standard reverse transcriptase activity. The C-terminal domain adopts at least two extreme conformations and undergoes conformational interconversion, which regulates the catalytic activity. Most importantly, we identified a conserved tertiary structural motif, called the U-motif, which interacts with the reverse transcriptase domain and is crucial for catalytic activity. Together these results shed new light on the structure and mechanics of fungal TERTs, which show common TERT characteristics, but also display species-specific features.

]]> <![CDATA[Structures of <i>Neisseria gonorrhoeae</i> MtrR-operator complexes reveal molecular mechanisms of DNA recognition and antibiotic resistance-conferring clinical mutations]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab213 Mutations within the mtrR gene are commonly found amongst multidrug resistant clinical isolates of Neisseria gonorrhoeae, which has been labelled a superbug by the Centers for Disease Control and Prevention. These mutations appear to contribute to antibiotic resistance by interfering with the ability of MtrR to bind to and repress expression of its target genes, which include the mtrCDE multidrug efflux transporter genes and the rpoH oxidative stress response sigma factor gene. However, the DNA-recognition mechanism of MtrR and the consensus sequence within these operators to which MtrR binds has remained unknown. In this work, we report the crystal structures of MtrR bound to the mtrCDE and rpoH operators, which reveal a conserved, but degenerate, DNA consensus binding site 5′-MCRTRCRN4YGYAYGK-3′. We complement our structural data with a comprehensive mutational analysis of key MtrR-DNA contacts to reveal their importance for MtrR-DNA binding both in vitro and in vivo. Furthermore, we model and generate common clinical mutations of MtrR to provide plausible biochemical explanations for the contribution of these mutations to multidrug resistance in N. gonorrhoeae. Collectively, our findings unveil key biological mechanisms underlying the global stress responses of N. gonorrhoeae.

]]> <![CDATA[Structural basis for substrate recognition and cleavage by the dimerization-dependent CRISPR–Cas12f nuclease]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab179 Cas12f, also known as Cas14, is an exceptionally small type V-F CRISPR–Cas nuclease that is roughly half the size of comparable nucleases of this type. To reveal the mechanisms underlying substrate recognition and cleavage, we determined the cryo-EM structures of the Cas12f-sgRNA-target DNA and Cas12f-sgRNA complexes at 3.1 and 3.9 Å, respectively. An asymmetric Cas12f dimer is bound to one sgRNA for recognition and cleavage of dsDNA substrate with a T-rich PAM sequence. Despite its dimerization, Cas12f adopts a conserved activation mechanism among the type V nucleases which requires coordinated conformational changes induced by the formation of the crRNA-target DNA heteroduplex, including the close-to-open transition in the lid motif of the RuvC domain. Only one RuvC domain in the Cas12f dimer is activated by substrate recognition, and the substrate bound to the activated RuvC domain is captured in the structure. Structure-assisted truncated sgRNA, which is less than half the length of the original sgRNA, is still active for target DNA cleavage. Our results expand our understanding of the diverse type V CRISPR–Cas nucleases and facilitate potential genome editing applications using the miniature Cas12f.

]]> <![CDATA[Structural and functional studies of SF1B Pif1 from <i>Thermus oshimai</i> reveal dimerization-induced helicase inhibition]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab188 Pif1 is an SF1B helicase that is evolutionarily conserved from bacteria to humans and plays multiple roles in maintaining genome stability in both nucleus and mitochondria. Though highly conserved, Pif1 family harbors a large mechanistic diversity. Here, we report crystal structures of Thermus oshimai Pif1 (ToPif1) alone and complexed with partial duplex or single-stranded DNA. In the apo state and in complex with a partial duplex DNA, ToPif1 is monomeric with its domain 2B/loop3 adopting a closed and an open conformation, respectively. When complexed with a single-stranded DNA, ToPif1 forms a stable dimer with domain 2B/loop3 shifting to a more open conformation. Single-molecule and biochemical assays show that domain 2B/loop3 switches repetitively between the closed and open conformations when a ToPif1 monomer unwinds DNA and, in contrast with other typical dimeric SF1A helicases, dimerization has an inhibitory effect on its helicase activity. This mechanism is not general for all Pif1 helicases but illustrates the diversity of regulation mechanisms among different helicases. It also raises the possibility that although dimerization results in activation for SF1A helicases, it may lead to inhibition for some of the other uncharacterized SF1B helicases, an interesting subject warranting further studies.

]]> <![CDATA[Crystal structures of influenza nucleoprotein complexed with nucleic acid provide insights into the mechanism of RNA interaction]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab203 The nucleoprotein (NP) of influenza virus is the core component of the ribonucleoprotein (RNP) and performs multiple structural and functional roles. Structures of the influenza A, B and D NP molecules have been solved previously, but structural information on how NP interacts with RNA remains elusive. Here we present the crystal structure of an obligate monomer of H5N1 NP in complex with RNA nucleotides to 2.3 Å, and a C-terminal truncation of this mutant, also in complex with RNA nucleotides, to 3 Å. In both structures, three nucleotides were identified near two positive grooves of NP suggested to be important for RNA binding. Structural evidence supports that conformational changes of flexible loops and the C-terminal tail both play important roles in the binding of RNA. Based on the structure, we propose a mechanism by which NP captures RNA by flexible loops and transfers it onto the positive binding grooves. Binding of RNA by NP is a crucial step for template re-encapsidation during transcription and replication and cRNP formation. Our structures thus provide insights into the molecular virology of the influenza virus.

]]> <![CDATA[Mechanism of forkhead transcription factors binding to a novel palindromic DNA site]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab086 Forkhead transcription factors bind a canonical consensus DNA motif, RYAAAYA (R = A/G, Y = C/T), as a monomer. However, the molecular mechanisms by which forkhead transcription factors bind DNA as a dimer are not well understood. In this study, we show that FOXO1 recognizes a palindromic DNA element DIV2, and mediates transcriptional regulation. The crystal structure of FOXO1/DIV2 reveals that the FOXO1 DNA binding domain (DBD) binds the DIV2 site as a homodimer. The wing1 region of FOXO1 mediates the dimerization, which enhances FOXO1 DNA binding affinity and complex stability. Further biochemical assays show that FOXO3, FOXM1 and FOXI1 also bind the DIV2 site as homodimer, while FOXC2 can only bind this site as a monomer. Our structural, biochemical and bioinformatics analyses not only provide a novel mechanism by which FOXO1 binds DNA as a homodimer, but also shed light on the target selection of forkhead transcription factors.

]]> <![CDATA[Evolving models for assembling and shaping clathrin-coated pits]]> https://www.novareader.co/book/isbn/10.1083/jcb.202005126 Integrating recent findings, Chen and Schmid present a more dynamic, flexible, and nonlinear model for clathrin-coated vesicle formation.

Clathrin-mediated endocytosis occurs via the assembly of clathrin-coated pits (CCPs) that invaginate and pinch off to form clathrin-coated vesicles (CCVs). It is well known that adaptor protein 2 (AP2) complexes trigger clathrin assembly on the plasma membrane, and biochemical and structural studies have revealed the nature of these interactions. Numerous endocytic accessory proteins collaborate with clathrin and AP2 to drive CCV formation. However, many questions remain as to the molecular events involved in CCP initiation, stabilization, and curvature generation. Indeed, a plethora of recent evidence derived from cell perturbation, correlative light and EM tomography, live-cell imaging, modeling, and high-resolution structural analyses has revealed more complexity and promiscuity in the protein interactions driving CCP maturation than anticipated. After briefly reviewing the evidence supporting prevailing models, we integrate these new lines of evidence to develop a more dynamic and flexible model for how redundant, dynamic, and competing protein interactions can drive endocytic CCV formation and suggest new approaches to test emerging models.

]]> <![CDATA[ <i>In vitro</i> selected GUAA tetraloop-binding receptors with structural plasticity and evolvability towards natural RNA structural modules]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab021 GNRA tetraloop-binding receptor interactions are key components in the macromolecular assembly of a variety of functional RNAs. In nature, there is an apparent bias for GAAA/11nt receptor and GYRA/helix interactions, with the former interaction being thermodynamically more stable than the latter. While past in vitro selections allowed isolation of novel GGAA and GUGA receptors, we report herein an in vitro selection that revealed several novel classes of specific GUAA receptors with binding affinities comparable to those from natural GAAA/11nt interactions. These GUAA receptors have structural homology with double-locked bulge RNA modules naturally occurring in ribosomal RNAs. They display mutational robustness that enables exploration of the sequence/phenotypic space associated to GNRA/receptor interactions through epistasis. Their thermodynamic self-assembly fitness landscape is characterized by a rugged neutral network with possible evolutionary trajectories toward natural GNRA/receptor interactions. High throughput sequencing analysis revealed synergetic mutations located away from the tertiary interactions that positively contribute to assembly fitness. Our study suggests that the repertoire of GNRA/receptor interactions is much larger than initially thought from the analysis of natural stable RNA molecules and also provides clues for their evolution towards natural GNRA/receptors.

]]> <![CDATA[Insight into formation propensity of pseudocircular DNA G-hairpins]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab029 We recently showed that Saccharomyces cerevisiae telomeric DNA can fold into an unprecedented pseudocircular G-hairpin (PGH) structure. However, the formation of PGHs in the context of extended sequences, which is a prerequisite for their function in vivo and their applications in biotechnology, has not been elucidated. Here, we show that despite its ‘circular’ nature, PGHs tolerate single-stranded (ss) protrusions. High-resolution NMR structure of a novel member of PGH family reveals the atomistic details on a junction between ssDNA and PGH unit. Identification of new sequences capable of folding into one of the two forms of PGH helped in defining minimal sequence requirements for their formation. Our time-resolved NMR data indicate a possibility that PGHs fold via a complex kinetic partitioning mechanism and suggests the existence of K+ ion-dependent PGH folding intermediates. The data not only provide an explanation of cation-type-dependent formation of PGHs, but also explain the unusually large hysteresis between PGH melting and annealing noted in our previous study. Our findings have important implications for DNA biology and nanotechnology. Overrepresentation of sequences able to form PGHs in the evolutionary-conserved regions of the human genome implies their functionally important biological role(s).

]]> <![CDATA[Structure-based analyses of <i>Salmonella</i> RcsB variants unravel new features of the Rcs regulon]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab060 RcsB is a transcriptional regulator that controls expression of numerous genes in enteric bacteria. RcsB accomplishes this role alone or in combination with auxiliary transcriptional factors independently or dependently of phosphorylation. To understand the mechanisms by which RcsB regulates such large number of genes, we performed structural studies as well as in vitro and in vivo functional studies with different RcsB variants. Our structural data reveal that RcsB binds promoters of target genes such as rprA and flhDC in a dimeric active conformation. In this state, the RcsB homodimer docks the DNA-binding domains into the major groove of the DNA, facilitating an initial weak read-out of the target sequence. Interestingly, comparative structural analyses also show that DNA binding may stabilize an active conformation in unphosphorylated RcsB. Furthermore, RNAseq performed in strains expressing wild-type or several RcsB variants provided new insights into the contribution of phosphorylation to gene regulation and assign a potential role of RcsB in controlling iron metabolism. Finally, we delimited the RcsB box for homodimeric active binding to DNA as the sequence TN(G/A)GAN4TC(T/C)NA. This RcsB box was found in promoter, intergenic and intragenic regions, facilitating both increased or decreased gene transcription.

]]> <![CDATA[Dynamics of the HD regulatory subdomain of PARP-1; substrate access and allostery in PARP activation and inhibition]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab020 PARP-1 is a key early responder to DNA damage in eukaryotic cells. An allosteric mechanism links initial sensing of DNA single-strand breaks by PARP-1’s F1 and F2 domains via a process of further domain assembly to activation of the catalytic domain (CAT); synthesis and attachment of poly(ADP-ribose) (PAR) chains to protein sidechains then signals for assembly of DNA repair components. A key component in transmission of the allosteric signal is the HD subdomain of CAT, which alone bridges between the assembled DNA-binding domains and the active site in the ART subdomain of CAT. Here we present a study of isolated CAT domain from human PARP-1, using NMR-based dynamics experiments to analyse WT apo-protein as well as a set of inhibitor complexes (with veliparib, olaparib, talazoparib and EB-47) and point mutants (L713F, L765A and L765F), together with new crystal structures of the free CAT domain and inhibitor complexes. Variations in both dynamics and structures amongst these species point to a model for full-length PARP-1 activation where first DNA binding and then substrate interaction successively destabilise the folded structure of the HD subdomain to the point where its steric blockade of the active site is released and PAR synthesis can proceed.

]]> <![CDATA[Structural determinants of the IRF4/DNA homodimeric complex]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1287 Interferon regulatory factor 4 (IRF4) is a key transcription factor (TF) in the regulation of immune cells, including B and T cells. It acts by binding DNA as both a homodimer and, in conjunction with other TFs, as a heterodimer. The choice of homo and heterodimeric/ DNA interactions is a critical aspect in the control of the transcriptional program and cell fate outcome. To characterize the nature of this interaction in the homodimeric complex, we have determined the crystal structure of the IRF4/ISRE homodimeric complex. We show that the complex formation is aided by a substantial DNA deformation with co-operative binding achieved exclusively through protein–DNA contact. This markedly contrasts with the heterodimeric form where DNA bound IRF4 is shown to physically interact with PU.1 TF to engage EICE1. We also show that the hotspot residues (Arg98, Cys99 and Asn102) contact both consensus and non-consensus sequences with the L1 loop exhibiting marked flexibility. Additionally, we identified that IRF4L116R, a mutant associated with chronic lymphocytic leukemia, binds more robustly to DNA thereby providing a rationale for the observed gain of function. Together, we demonstrate key structural differences between IRF4 homo and heterodimeric complexes, thereby providing molecular insights into IRF4-mediated transcriptional regulation.

]]> <![CDATA[NMR structural study on the self-trimerization of d(GTTAGG) into a dynamic trimolecular G-quadruplex assembly preferentially in Na<sup>+</sup> solution with a moderate K<sup>+</sup> tolerance]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab028 Vast G-quadruplexes (GQs) are primarily folded by one, two, or four G-rich oligomers, rarely with an exception. Here, we present the first NMR solution structure of a trimolecular GQ (tri-GQ) that is solely assembled by the self-trimerization of d(GTTAGG), preferentially in Na+ solution tolerant to an equal amount of K+ cation. Eight guanines from three asymmetrically folded strands of d(GTTAGG) are organized into a two-tetrad core, which features a broken G-column and two width-irregular grooves. Fast strand exchanges on a timescale of second at 17°C spontaneously occur between folded tri-GQ and unfolded single-strand of d(GTTAGG) that both species coexist in dynamic equilibrium. Thus, this tri-GQ is not just simply a static assembly but rather a dynamic assembly. Moreover, another minor tetra-GQ that has putatively tetrameric (2+2) antiparallel topology becomes noticeable only at an extremely high strand concentration above 18 mM. The major tri-GQ and minor tetra-GQ are considered to be mutually related, and their reversible interconversion pathways are proposed accordingly. The sequence d(GTTAGG) could be regarded as either a reading frame shifted single repeat of human telomeric DNA or a 1.5 repeat of Bombyx mori telomeric DNA. Overall, our findings provide new insight into GQs and expect more functional applications.

]]> <![CDATA[Structural basis of diversity and homodimerization specificity of zinc-finger-associated domains in Drosophila]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab061 In arthropods, zinc finger-associated domains (ZADs) are found at the N-termini of many DNA-binding proteins with tandem arrays of Cys2-His2 zinc fingers (ZAD-C2H2 proteins). ZAD-C2H2 proteins undergo fast evolutionary lineage-specific expansion and functional diversification. Here, we show that all ZADs from Drosophila melanogaster form homodimers, but only certain ZADs with high homology can also heterodimerize. CG2712, for example, is unable to heterodimerize with its paralog, the previously characterized insulator protein Zw5, with which it shares 46% homology. We obtained a crystal structure of CG2712 protein's ZAD domain that, in spite of a low sequence homology, has similar spatial organization with the only known ZAD structure (from Grauzone protein). Steric clashes prevented the formation of heterodimers between Grauzone and CG2712 ZADs. Using detailed structural analysis, site-directed mutagenesis, and molecular dynamics simulations, we demonstrated that rapid evolutionary acquisition of interaction specificity was mediated by the more energy-favorable formation of homodimers in comparison to heterodimers, and that this specificity was achieved by multiple amino acid substitutions resulting in the formation or breaking of stabilizing interactions. We speculate that specific homodimerization of ZAD-C2H2 proteins is important for their architectural role in genome organization.

]]> <![CDATA[Oxidative lesions modulate G-quadruplex stability and structure in the human BCL2 promoter]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab057 Misregulation of BCL2 expression has been observed with many diseases and is associated with cellular exposure to reactive oxygen species. A region upstream of the P1 promoter in the human BCL2 gene plays a major role in regulating transcription. This G/C-rich region is highly polymorphic and capable of forming G-quadruplex structures. Herein we report that an oxidative event simulated with an 8-oxo-7,8-dihydroguanine (oxoG) substitution within a long G-tract results in a reduction of structural polymorphism. Surprisingly, oxoG within a 25-nt construct boosts thermal stability of the resulting G-quadruplex. This is achieved by distinct hydrogen bonding properties of oxoG, which facilitate formation of an antiparallel basket-type G-quadruplex with a three G-quartet core and a G·oxoG·C base triad. While oxoG has previously been considered detrimental for G-quadruplex formation, its stabilizing effect within a promoter described in this study suggests a potential novel regulatory role of oxidative stress in general and specifically in BCL2 gene transcription.

oxoG influences the fold of a G-quadruplex structure within the human BCL2 promoter and enhances the thermal stability of the G4 structure by forming novel structural elements.
Graphical Abstract
oxoG influences the fold of a G-quadruplex structure within the human BCL2 promoter and enhances the thermal stability of the G4 structure by forming novel structural elements.

]]> <![CDATA[DNA G-quadruplexes for native mass spectrometry in potassium: a database of validated structures in electrospray-compatible conditions]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab039 G-quadruplex DNA structures have become attractive drug targets, and native mass spectrometry can provide detailed characterization of drug binding stoichiometry and affinity, potentially at high throughput. However, the G-quadruplex DNA polymorphism poses problems for interpreting ligand screening assays. In order to establish standardized MS-based screening assays, we studied 28 sequences with documented NMR structures in (usually ∼100 mM) potassium, and report here their circular dichroism (CD), melting temperature (Tm), NMR spectra and electrospray mass spectra in 1 mM KCl/100 mM trimethylammonium acetate. Based on these results, we make a short-list of sequences that adopt the same structure in the MS assay as reported by NMR, and provide recommendations on using them for MS-based assays. We also built an R-based open-source application to build and consult a database, wherein further sequences can be incorporated in the future. The application handles automatically most of the data processing, and allows generating custom figures and reports. The database is included in the g4dbr package (https://github.com/EricLarG4/g4dbr) and can be explored online (https://ericlarg4.github.io/G4_database.html).

]]> <![CDATA[Bulges in left-handed G-quadruplexes]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1259 G-quadruplex (G4) DNA structures with a left-handed backbone progression have unique and conserved structural features. Studies on sequence dependency of the structures revealed the prerequisites and some minimal motifs required for left-handed G4 formation. To extend the boundaries, we explore the adaptability of left-handed G4s towards the existence of bulges. Here we present two X-ray crystal structures and an NMR solution structure of left-handed G4s accommodating one, two and three bulges. Bulges in left-handed G4s show distinct characteristics as compared to those in right-handed G4s. The elucidation of intricate structural details will help in understanding the possible roles and limitations of these unique structures.

]]> <![CDATA[First insights into the structural features of Ebola virus methyltransferase activities]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1276 The Ebola virus is a deadly human pathogen responsible for several outbreaks in Africa. Its genome encodes the ‘large’ L protein, an essential enzyme that has polymerase, capping and methyltransferase activities. The methyltransferase activity leads to RNA co-transcriptional modifications at the N7 position of the cap structure and at the 2′-O position of the first transcribed nucleotide. Unlike other Mononegavirales viruses, the Ebola virus methyltransferase also catalyses 2′-O-methylation of adenosines located within the RNA sequences. Herein, we report the crystal structure at 1.8 Å resolution of the Ebola virus methyltransferase domain bound to a fragment of a camelid single-chain antibody. We identified structural determinants and key amino acids specifically involved in the internal adenosine-2′-O-methylation from cap-related methylations. These results provide the first high resolution structure of an ebolavirus L protein domain, and the framework to investigate the effects of epitranscriptomic modifications and to design possible antiviral drugs against the Filoviridae family.

]]> <![CDATA[Crystal structures of the EVE-HNH endonuclease VcaM4I in the presence and absence of DNA]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1218 Many modification-dependent restriction endonucleases (MDREs) are fusions of a PUA superfamily modification sensor domain and a nuclease catalytic domain. EVE domains belong to the PUA superfamily, and are present in MDREs in combination with HNH nuclease domains. Here, we present a biochemical characterization of the EVE-HNH endonuclease VcaM4I and crystal structures of the protein alone, with EVE domain bound to either 5mC modified dsDNA or to 5mC/5hmC containing ssDNA. The EVE domain is moderately specific for 5mC/5hmC containing DNA according to EMSA experiments. It flips the modified nucleotide, to accommodate it in a hydrophobic pocket of the enzyme, primarily formed by P24, W82 and Y130 residues. In the crystallized conformation, the EVE domain and linker helix between the two domains block DNA binding to the catalytic domain. Removal of the EVE domain and inter-domain linker, but not of the EVE domain alone converts VcaM4I into a non-specific toxic nuclease. The role of the key residues in the EVE and HNH domains of VcaM4I is confirmed by digestion and restriction assays with the enzyme variants that differ from the wild-type by changes to the base binding pocket or to the catalytic residues.

Crystal structures of VcaM4I restriction endonuclease provide the first structural description of the EVE domain-modified DNA interaction. Each protomer of the dimeric enzyme is composed of EVE domain responsible for modification detection and HNH domain responsible for DNA cleavage. The EVE domains flip the modified cytosine bases and bind them in dedicated hydrophobic pockets.
Graphical Abstract
Crystal structures of VcaM4I restriction endonuclease provide the first structural description of the EVE domain-modified DNA interaction. Each protomer of the dimeric enzyme is composed of EVE domain responsible for modification detection and HNH domain responsible for DNA cleavage. The EVE domains flip the modified cytosine bases and bind them in dedicated hydrophobic pockets.

]]> <![CDATA[The solution structures of higher-order human telomere G-quadruplex multimers]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1285 Human telomeres contain the repeat DNA sequence 5′-d(TTAGGG), with duplex regions that are several kilobases long terminating in a 3′ single-stranded overhang. The structure of the single-stranded overhang is not known with certainty, with disparate models proposed in the literature. We report here the results of an integrated structural biology approach that combines small-angle X-ray scattering, circular dichroism (CD), analytical ultracentrifugation, size-exclusion column chromatography and molecular dynamics simulations that provide the most detailed characterization to date of the structure of the telomeric overhang. We find that the single-stranded sequences 5′-d(TTAGGG)n, with n = 8, 12 and 16, fold into multimeric structures containing the maximal number (2, 3 and 4, respectively) of contiguous G4 units with no long gaps between units. The G4 units are a mixture of hybrid-1 and hybrid-2 conformers. In the multimeric structures, G4 units interact, at least transiently, at the interfaces between units to produce distinctive CD signatures. Global fitting of our hydrodynamic and scattering data to a worm-like chain (WLC) model indicates that these multimeric G4 structures are semi-flexible, with a persistence length of ∼34 Å. Investigations of its flexibility using MD simulations reveal stacking, unstacking, and coiling movements, which yield unique sites for drug targeting.

]]> <![CDATA[GTP-dependent formation of straight tubulin oligomers leads to microtubule nucleation]]> https://www.novareader.co/book/isbn/10.1083/jcb.202007033 Ayukawa, Iwata, Imai, et al. visualize the early intermediates in the pathway of spontaneous nucleation of microtubules by using rapid flush negative stain electron microscopy. This study demonstrates that the formation of straight tubulin oligomers of critical size is essential for nucleation.

Nucleation of microtubules (MTs) is essential for cellular activities, but its mechanism is unknown because of the difficulty involved in capturing rare stochastic events in the early stage of polymerization. Here, combining rapid flush negative stain electron microscopy (EM) and kinetic analysis, we demonstrate that the formation of straight oligomers of critical size is essential for nucleation. Both GDP and GTP tubulin form single-stranded oligomers with a broad range of curvatures, but upon nucleation, the curvature distribution of GTP oligomers is shifted to produce a minor population of straight oligomers. With tubulin having the Y222F mutation in the β subunit, the proportion of straight oligomers increases and nucleation accelerates. Our results support a model in which GTP binding generates a minor population of straight oligomers compatible with lateral association and further growth to MTs. This study suggests that cellular factors involved in nucleation promote it via stabilization of straight oligomers.

]]> <![CDATA[Structural dynamics of double-stranded DNA with epigenome modification]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1210 Modification of cytosine plays an important role in epigenetic regulation of gene expression and genome stability. Cytosine is converted to 5-methylcytosine (5mC) by DNA methyltransferase; in turn, 5mC may be oxidized to 5-hydroxymethylcytosine (5hmC) by ten-eleven translocation enzyme. The structural flexibility of DNA is known to affect the binding of proteins to methylated DNA. Here, we have carried out a semi-quantitative analysis of the dynamics of double-stranded DNA (dsDNA) containing various epigenetic modifications by combining data from imino 1H exchange and imino 1H R1ρ relaxation dispersion NMR experiments in a complementary way. Using this approach, we characterized the base-opening (kopen) and base-closing (kclose) rates, facilitating a comparison of the base-opening and -closing process of dsDNA containing cytosine in different states of epigenetic modification. A particularly striking result is the increase in the kopen rate of hemi-methylated dsDNA 5mC/C relative to unmodified or fully methylated dsDNA, indicating that the Watson–Crick base pairs undergo selective destabilization in 5mC/C. Collectively, our findings imply that the epigenetic modulation of cytosine dynamics in dsDNA mediates destabilization of the GC Watson–Crick base pair to allow base-flipping in living cells.

]]> <![CDATA[Water spines and networks in G-quadruplex structures]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1177 Quadruplex DNAs can fold into a variety of distinct topologies, depending in part on loop types and orientations of individual strands, as shown by high-resolution crystal and NMR structures. Crystal structures also show associated water molecules. We report here on an analysis of the hydration arrangements around selected folded quadruplex DNAs, which has revealed several prominent features that re-occur in related structures. Many of the primary-sphere water molecules are found in the grooves and loop regions of these structures. At least one groove in anti-parallel and hybrid quadruplex structures is long and narrow and contains an extensive spine of linked primary-sphere water molecules. This spine is analogous to but fundamentally distinct from the well-characterized spine observed in the minor groove of A/T-rich duplex DNA, in that every water molecule in the continuous quadruplex spines makes a direct hydrogen bond contact with groove atoms, principally phosphate oxygen atoms lining groove walls and guanine base nitrogen atoms on the groove floor. By contrast, parallel quadruplexes do not have extended grooves, but primary-sphere water molecules still cluster in them and are especially associated with the loops, helping to stabilize loop conformations.

]]> <![CDATA[Structural basis of sequestration of the anti-Shine-Dalgarno sequence in the Bacteroidetes ribosome]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1195 Genomic studies have indicated that certain bacterial lineages such as the Bacteroidetes lack Shine-Dalgarno (SD) sequences, and yet with few exceptions ribosomes of these organisms carry the canonical anti-SD (ASD) sequence. Here, we show that ribosomes purified from Flavobacterium johnsoniae, a representative of the Bacteroidetes, fail to recognize the SD sequence of mRNA in vitro. A cryo-electron microscopy structure of the complete 70S ribosome from F. johnsoniae at 2.8 Å resolution reveals that the ASD is sequestered by ribosomal proteins bS21, bS18 and bS6, explaining the basis of ASD inhibition. The structure also uncovers a novel ribosomal protein—bL38. Remarkably, in F. johnsoniae and many other Flavobacteriia, the gene encoding bS21 contains a strong SD, unlike virtually all other genes. A subset of Flavobacteriia have an alternative ASD, and in these organisms the fully complementary sequence lies upstream of the bS21 gene, indicative of natural covariation. In other Bacteroidetes classes, strong SDs are frequently found upstream of the genes for bS21 and/or bS18. We propose that these SDs are used as regulatory elements, enabling bS21 and bS18 to translationally control their own production.

In most bacteria, start codon selection is guided by the Shine Dalgarno (SD) sequence. Bacteroidetes fail to recognize the SD sequence of mRNA. However, these bacteria retain the canonical anti-Shine-Dalgarno (ASD) sequence. The cryo-electron microscopy structure of the 70S ribosome from Flavobacterium johnsoniae, a representative of the Bacteroidetes reveals that the ASD is sequestered by ribosomal proteins bS21, bS18 and bS6, explaining the basis of ASD inhibition.
Graphical Abstract
In most bacteria, start codon selection is guided by the Shine Dalgarno (SD) sequence. Bacteroidetes fail to recognize the SD sequence of mRNA. However, these bacteria retain the canonical anti-Shine-Dalgarno (ASD) sequence. The cryo-electron microscopy structure of the 70S ribosome from Flavobacterium johnsoniae, a representative of the Bacteroidetes reveals that the ASD is sequestered by ribosomal proteins bS21, bS18 and bS6, explaining the basis of ASD inhibition.

]]> <![CDATA[Characterization of the first tetrameric transcription factor of the GntR superfamily with allosteric regulation from the bacterial pathogen <i>Agrobacterium fabrum</i>]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1181 A species-specific region, denoted SpG8-1b allowing hydroxycinnamic acids (HCAs) degradation is important for the transition between the two lifestyles (rhizospheric versus pathogenic) of the plant pathogen Agrobacterium fabrum. Indeed, HCAs can be either used as trophic resources and/or as induced-virulence molecules. The SpG8-1b region is regulated by two transcriptional regulators, namely, HcaR (Atu1422) and Atu1419. In contrast to HcaR, Atu1419 remains so far uncharacterized. The high-resolution crystal structures of two fortuitous citrate complexes, two DNA complexes and the apoform revealed that the tetrameric Atu1419 transcriptional regulator belongs to the VanR group of Pfam PF07729 subfamily of the large GntR superfamily. Until now, GntR regulators were described as dimers. Here, we showed that Atu1419 represses three genes of the HCAs catabolic pathway. We characterized both the effector and DNA binding sites and identified key nucleotides in the target palindrome. From promoter activity measurement using defective gene mutants, structural analysis and gel-shift assays, we propose N5,N10-methylenetetrahydrofolate as the effector molecule, which is not a direct product/substrate of the HCA degradation pathway. The Zn2+ ion present in the effector domain has both a structural and regulatory role. Overall, our work shed light on the allosteric mechanism of transcription employed by this GntR repressor.

]]> <![CDATA[A skipping rope translocation mechanism in a widespread family of DNA repair helicases]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1174 Mitomycin repair factor A represents a family of DNA helicases that harbor a domain of unknown function (DUF1998) and support repair of mitomycin C-induced DNA damage by presently unknown molecular mechanisms. We determined crystal structures of Bacillus subtilis Mitomycin repair factor A alone and in complex with an ATP analog and/or DNA and conducted structure-informed functional analyses. Our results reveal a unique set of auxiliary domains appended to a dual-RecA domain core. Upon DNA binding, a Zn2+-binding domain, encompassing the domain of unknown function, acts like a drum that rolls out a canopy of helicase-associated domains, entrapping the substrate and tautening an inter-domain linker across the loading strand. Quantification of DNA binding, stimulated ATPase and helicase activities in the wild type and mutant enzyme variants in conjunction with the mode of coordination of the ATP analog suggest that Mitomycin repair factor A employs similar ATPase-driven conformational changes to translocate on DNA, with the linker ratcheting through the nucleotides like a ‘skipping rope’. The electrostatic surface topology outlines a likely path for the displaced DNA strand. Our results reveal unique molecular mechanisms in a widespread family of DNA repair helicases linked to bacterial antibiotics resistance.

Crystal structures of the DUF1998 DNA helicase, MrfA, were determined in the apo, DNA-bound and DNA/AMPPNP-bound forms, and translocase/helicase mechanisms were interrogated by structure-informed mutational analyses in vitro.
Graphical Abstract
Crystal structures of the DUF1998 DNA helicase, MrfA, were determined in the apo, DNA-bound and DNA/AMPPNP-bound forms, and translocase/helicase mechanisms were interrogated by structure-informed mutational analyses in vitro.

]]> <![CDATA[Structural basis for inhibition of the type I-F CRISPR–Cas surveillance complex by AcrIF4, AcrIF7 and AcrIF14]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1199 CRISPR–Cas systems are adaptive immune systems in bacteria and archaea to defend against mobile genetic elements (MGEs) and have been repurposed as genome editing tools. Anti-CRISPR (Acr) proteins are produced by MGEs to counteract CRISPR–Cas systems and can be used to regulate genome editing by CRISPR techniques. Here, we report the cryo-EM structures of three type I-F Acr proteins, AcrIF4, AcrIF7 and AcrIF14, bound to the type I-F CRISPR–Cas surveillance complex (the Csy complex) from Pseudomonas aeruginosa. AcrIF4 binds to an unprecedented site on the C-terminal helical bundle of Cas8f subunit, precluding conformational changes required for activation of the Csy complex. AcrIF7 mimics the PAM duplex of target DNA and is bound to the N-terminal DNA vise of Cas8f. Two copies of AcrIF14 bind to the thumb domains of Cas7.4f and Cas7.6f, preventing hybridization between target DNA and the crRNA. Our results reveal structural detail of three AcrIF proteins, each binding to a different site on the Csy complex for inhibiting degradation of MGEs.

]]> <![CDATA[Structural basis for the substrate specificity and catalytic features of pseudouridine kinase from <i>Arabidopsis thaliana</i>]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1144 RNA modifications can regulate the stability of RNAs, mRNA–protein interactions, and translation efficiency. Pseudouridine is a prevalent RNA modification, and its metabolic fate after RNA turnover was recently characterized in eukaryotes, in the plant Arabidopsis thaliana. Here, we present structural and biochemical analyses of PSEUDOURIDINE KINASE from Arabidopsis (AtPUKI), the enzyme catalyzing the first step in pseudouridine degradation. AtPUKI, a member of the PfkB family of carbohydrate kinases, is a homodimeric α/β protein with a protruding small β-strand domain, which serves simultaneously as dimerization interface and dynamic substrate specificity determinant. AtPUKI has a unique nucleoside binding site specifying the binding of pseudourine, in particular at the nucleobase, by multiple hydrophilic interactions, of which one is mediated by a loop from the small β-strand domain of the adjacent monomer. Conformational transition of the dimerized small β-strand domains containing active site residues is required for substrate specificity. These dynamic features explain the higher catalytic efficiency for pseudouridine over uridine. Both substrates bind well (similar Km), but only pseudouridine is turned over efficiently. Our studies provide an example for structural and functional divergence in the PfkB family and highlight how AtPUKI avoids futile uridine phosphorylation which in vivo would disturb pyrimidine homeostasis.

]]> <![CDATA[Structural basis for guide RNA trimming by RNase D ribonuclease in <i>Trypanosoma brucei</i>]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1197 Infection with kinetoplastid parasites, including Trypanosoma brucei (T. brucei), Trypanosoma cruzi (T. cruzi) and Leishmania can cause serious disease in humans. Like other kinetoplastid species, mRNAs of these disease-causing parasites must undergo posttranscriptional editing in order to be functional. mRNA editing is directed by gRNAs, a large group of small RNAs. Similar to mRNAs, gRNAs are also precisely regulated. In T. brucei, overexpression of RNase D ribonuclease (TbRND) leads to substantial reduction in the total gRNA population and subsequent inhibition of mRNA editing. However, the mechanisms regulating gRNA binding and cleavage by TbRND are not well defined. Here, we report a thorough structural study of TbRND. Besides Apo- and NMP-bound structures, we also solved one TbRND structure in complexed with single-stranded RNA. In combination with mutagenesis and in vitro cleavage assays, our structures indicated that TbRND follows the conserved two-cation-assisted mechanism in catalysis. TbRND is a unique RND member, as it contains a ZFD domain at its C-terminus. In addition to T. brucei, our studies also advanced our understanding on the potential gRNA degradation pathway in T. cruzi, Leishmania, as well for as other disease-associated parasites expressing ZFD-containing RNDs.

]]> <![CDATA[Structural insights into G domain dimerization and pathogenic mutation of OPA1]]> https://www.novareader.co/book/isbn/10.1083/jcb.201907098 OPA1 mediates inner mitochondrial fusion and mutations cause optic atrophy. Yu et al. present the crystal structure of a minimal GTPase domain of human OPA1, which sheds light on its dimerization that is physiologically important and provides an explanation for pathogenic mutations.

The fusion of mammalian inner mitochondrial membranes (IMMs) is mediated by dynamin-like GTPase OPA1. Mutations in human OPA1 cause optic atrophy, but the molecular basis for membrane fusion and pathogenesis is not clear. Here, we determined the crystal structure of the minimal GTPase domain (MGD) of human OPA1. A three-helix bundle (HB) domain including two helices extending from the GTPase (G) domain and the last helix of OPA1 tightly associates with the G domain. In the presence of GDP and BeF3−, OPA1-MGD forms a dimer, the interface of which is critical for the maintenance of mitochondrial morphology. The catalytic core of OPA1 possesses unique features that are not present in other dynamin-like proteins. Biochemical experiments revealed that OPA1-MGD forms nucleotide-dependent dimers, which is important for membrane-stimulated GTP hydrolysis, and an N-terminal extension mediates nucleotide-independent dimerization that facilitates efficient membrane association. Our results suggest a multifaceted assembly of OPA1 and explain the effect of most OPA1 mutations on optic atrophy.

]]>