Browse
Submit Data
Databases
API
Help

Dataset Information

28 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Structural basis for the nuclease activity of a bacteriophage large terminase.

ABSTRACT: The DNA-packaging motor in tailed bacteriophages requires nuclease activity to ensure that the genome is packaged correctly. This nuclease activity is tightly regulated as the enzyme is inactive for the duration of DNA translocation. Here, we report the X-ray structure of the large terminase nuclease domain from bacteriophage SPP1. Similarity with the RNase H family endonucleases allowed interactions with the DNA to be predicted. A structure-based alignment with the distantly related T4 gp17 terminase shows the conservation of an extended beta-sheet and an auxiliary beta-hairpin that are not found in other RNase H family proteins. The model with DNA suggests that the beta-hairpin partly blocks the active site, and in vivo activity assays show that the nuclease domain is not functional in the absence of the ATPase domain. Here, we propose that the nuclease activity is regulated by movement of the beta-hairpin, altering active site access and the orientation of catalytically essential residues.

SUBMITTER: Smits C

PROVIDER: S-EPMC2685612 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Bacteriophage N4 large terminase: expression, purification and X-ray crystallographic analysis.

Project description:Genome packaging is a critical step in the assembly of dsDNA bacteriophages and is carried out by a powerful molecular motor known as the large terminase. To date, wild-type structures of only two large terminase proteins are available, and more structural information is needed to understand the genome-packaging mechanism. Towards this goal, the large and small terminase proteins from bacteriophage N4, which infects the Escherichia coli K12 strain, have been cloned, expressed and purified. The purified putative large terminase protein hydrolyzes ATP, and this is enhanced in the presence of the small terminase. The large terminase protein was crystallized using the sitting-drop vapour-diffusion method and the crystal diffracted to 2.8?Å resolution using a home X-ray source. Analysis of the X-ray diffraction data showed that the crystal belonged to space group P212121, with unit-cell parameters a = 53.7, b = 93.6, c = 124.9?Å, ? = ? = ? = 90°. The crystal had a solvent content of 50.2% and contained one molecule in the asymmetric unit.

| S-EPMC5894105 | biostudies-literature

Large terminase conformational change induced by connector binding in bacteriophage T7.

Project description:During bacteriophage morphogenesis DNA is translocated into a preformed prohead by the complex formed by the portal protein, or connector, plus the terminase, which are located at an especial prohead vertex. The terminase is a powerful motor that converts ATP hydrolysis into mechanical movement of the DNA. Here, we have determined the structure of the T7 large terminase by electron microscopy. The five terminase subunits assemble in a toroid that encloses a channel wide enough to accommodate dsDNA. The structure of the complete connector-terminase complex is also reported, revealing the coupling between the terminase and the connector forming a continuous channel. The structure of the terminase assembled into the complex showed a different conformation when compared with the isolated terminase pentamer. To understand in molecular terms the terminase morphological change, we generated the terminase atomic model based on the crystallographic structure of its phage T4 counterpart. The docking of the threaded model in both terminase conformations showed that the transition between the two states can be achieved by rigid body subunit rotation in the pentameric assembly. The existence of two terminase conformations and its possible relation to the sequential DNA translocation may shed light into the molecular bases of the packaging mechanism of bacteriophage T7.

| S-EPMC3675631 | biostudies-literature

Structural basis for the substrate selectivity of Helicobacter pylori NucT nuclease activity.

Project description:The Phospholipase D (PLD) superfamily of proteins includes a group of enzymes with nuclease activity on various nucleic acid substrates. Here, with the aim of better understanding the substrate specificity determinants in this subfamily, we have characterised the enzymatic activity and the crystal structure of NucT, a nuclease implicated in Helicobacter pylori purine salvage and natural transformation and compared them to those of its bacterial and mammalian homologues. NucT exhibits an endonuclease activity with a strong preference for single stranded nucleic acids substrates. We identified histidine124 as essential for the catalytic activity of the protein. Comparison of the NucT crystal structure at 1.58 Å resolution reported here with those of other members of the sub-family suggests that the specificity of NucT for single-stranded nucleic acids is provided by the width of a positively charged groove giving access to the catalytic site.

| S-EPMC5714352 | biostudies-literature

Architecture of the Complex Formed by Large and Small Terminase Subunits from Bacteriophage P22.

Project description:Packaging of viral genomes inside empty procapsids is driven by a powerful ATP-hydrolyzing motor, formed in many double-stranded DNA viruses by a complex of a small terminase (S-terminase) subunit and a large terminase (L-terminase) subunit, transiently docked at the portal vertex during genome packaging. Despite recent progress in elucidating the structure of individual terminase subunits and their domains, little is known about the architecture of an assembled terminase complex. Here, we describe a bacterial co-expression system that yields milligram quantities of the S-terminase:L-terminase complex of the Salmonella phage P22. In vivo assembled terminase complex was affinity-purified and stabilized by addition of non-hydrolyzable ATP, which binds specifically to the ATPase domain of L-terminase. Mapping studies revealed that the N-terminus of L-terminase ATPase domain (residues 1-58) contains a minimal S-terminase binding domain sufficient for stoichiometric association with residues 140-162 of S-terminase, the L-terminase binding domain. Hydrodynamic analysis by analytical ultracentrifugation sedimentation velocity and native mass spectrometry revealed that the purified terminase complex consists predominantly of one copy of the nonameric S-terminase bound to two equivalents of L-terminase (1S-terminase:2L-terminase). Direct visualization of this molecular assembly in negative-stained micrographs yielded a three-dimensional asymmetric reconstruction that resembles a "nutcracker" with two L-terminase protomers projecting from the C-termini of an S-terminase ring. This is the first direct visualization of a purified viral terminase complex analyzed in the absence of DNA and procapsid.

| S-EPMC4587339 | biostudies-literature

Structure and inhibition of herpesvirus DNA packaging terminase nuclease domain.

Project description:During viral replication, herpesviruses package their DNA into the procapsid by means of the terminase protein complex. In human cytomegalovirus (herpesvirus 5), the terminase is composed of subunits UL89 and UL56. UL89 cleaves the long DNA concatemers into unit-length genomes of appropriate length for encapsidation. We used ESPRIT, a high-throughput screening method, to identify a soluble purifiable fragment of UL89 from a library of 18,432 randomly truncated ul89 DNA constructs. The purified protein was crystallized and its three-dimensional structure was solved. This protein corresponds to the key nuclease domain of the terminase and shows an RNase H/integrase-like fold. We demonstrate that UL89-C has the capacity to process the DNA and that this function is dependent on Mn(2+) ions, two of which are located at the active site pocket. We also show that the nuclease function can be inactivated by raltegravir, a recently approved anti-AIDS drug that targets the HIV integrase.

| S-EPMC2941324 | biostudies-literature

The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain.

Project description:Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the adenosine triphosphatase (ATPase) that powers DNA translocation and an endonuclease that cleaves the concatemeric genome at both initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage remains mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nuclease activity. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of DNA cleavage suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the capsid upon completion of packaging unlocks the nuclease domains to cleave DNA.

| S-EPMC5389665 | biostudies-literature

Structural basis of superinfection exclusion by bacteriophage T4 Spackle.

Project description:A bacterial cell infected with T4 phage rapidly establishes resistance against further infections by the same or closely related T-even-type bacteriophages - a phenomenon called superinfection exclusion. Here we show that one of the T4 early gene products and a periplasmic protein, Spackle, forms a stoichiometric complex with the lysozyme domain of T4 tail spike protein gp5 and potently inhibits its activity. Crystal structure of the Spackle-gp5 lysozyme complex shows that Spackle binds to a horseshoe-shaped basic patch surrounding the oligosaccharide-binding cleft and induces an allosteric conformational change of the active site. In contrast, Spackle does not appreciably inhibit the lysozyme activity of cytoplasmic T4 endolysin responsible for cell lysis to release progeny phage particles at the final step of the lytic cycle. Our work reveals a unique mode of inhibition for lysozymes, a widespread class of enzymes in biology, and provides a mechanistic understanding of the T4 bacteriophage superinfection exclusion.

| S-EPMC7677548 | biostudies-literature

Structural basis for the activation of a compact CRISPR-Cas13 nuclease.

Project description:The CRISPR-Cas13 ribonucleases have been widely applied for RNA knockdown and transcriptional modulation owing to their high programmability and specificity. However, the large size of Cas13 effectors and their non-specific RNA cleavage upon target activation limit the adeno-associated virus based delivery of Cas13 systems for therapeutic applications. Herein, we report detailed biochemical and structural characterizations of a compact Cas13 (Cas13bt3) suitable for adeno-associated virus delivery. Distinct from many other Cas13 systems, Cas13bt3 cleaves the target and other nonspecific RNA at internal "UC" sites and is activated in a target length-dependent manner. The cryo-electron microscope structure of Cas13bt3 in a fully active state illustrates the structural basis of Cas13bt3 activation. Guided by the structure, we obtain engineered Cas13bt3 variants with minimal off-target cleavage yet maintained target cleavage activities. In conclusion, our biochemical and structural data illustrate a distinct mechanism for Cas13bt3 activation and guide the engineering of Cas13bt3 applications.

| S-EPMC10511502 | biostudies-literature

Structural basis for RNA-genome recognition during bacteriophage Q? replication.

Project description:Upon infection of Escherichia coli by bacteriophage Q?, the virus-encoded ?-subunit recruits host translation elongation factors EF-Tu and EF-Ts and ribosomal protein S1 to form the Q? replicase holoenzyme complex, which is responsible for amplifying the Q? (+)-RNA genome. Here, we use X-ray crystallography, NMR spectroscopy, as well as sequence conservation, surface electrostatic potential and mutational analyses to decipher the roles of the ?-subunit and the first two oligonucleotide-oligosaccharide-binding domains of S1 (OB1-2) in the recognition of Q? (+)-RNA by the Q? replicase complex. We show how three basic residues of the ? subunit form a patch located adjacent to the OB2 domain, and use NMR spectroscopy to demonstrate for the first time that OB2 is able to interact with RNA. Neutralization of the basic residues by mutagenesis results in a loss of both the phage infectivity in vivo and the ability of Q? replicase to amplify the genomic RNA in vitro. In contrast, replication of smaller replicable RNAs is not affected. Taken together, our data suggest that the ?-subunit and protein S1 cooperatively bind the (+)-stranded Q? genome during replication initiation and provide a foundation for understanding template discrimination during replication initiation.

| S-EPMC4678825 | biostudies-literature

Structural basis for the non-self RNA-activated protease activity of the type III-E CRISPR nuclease-protease Craspase.

Project description:The RNA-targeting type III-E CRISPR-gRAMP effector interacts with a caspase-like protease TPR-CHAT to form the CRISPR-guided caspase complex (Craspase), but their functional mechanism is unknown. Here, we report cryo-EM structures of the type III-E gRAMPcrRNA and gRAMPcrRNA-TPR-CHAT complexes, before and after either self or non-self RNA target binding, and elucidate the mechanisms underlying RNA-targeting and non-self RNA-induced protease activation. The associated TPR-CHAT adopted a distinct conformation upon self versus non-self RNA target binding, with nucleotides at positions -1 and -2 of the CRISPR-derived RNA (crRNA) serving as a sensor. Only binding of the non-self RNA target activated the TPR-CHAT protease, leading to cleavage of Csx30 protein. Furthermore, TPR-CHAT structurally resembled eukaryotic separase, but with a distinct mechanism for protease regulation. Our findings should facilitate the development of gRAMP-based RNA manipulation tools, and advance our understanding of the virus-host discrimination process governed by a nuclease-protease Craspase during type III-E CRISPR-Cas immunity.

| S-EPMC9729208 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data