ABSTRACT
[This corrects the article DOI: 10.1371/journal.pone.0035263.].
ABSTRACT
[This corrects the article DOI: 10.1371/journal.pone.0035263.].
ABSTRACT
In this work, we study dynamical properties of an extremophilic protein, Initiation Factor 6 (IF6), produced by the archeabacterium Methanocaldococcus jannascii, which thrives close to deep-sea hydrothermal vents where temperatures reach 80 °C and the pressure is up to 750 bar. Molecular dynamics simulations (MD) and quasi-elastic neutron scattering (QENS) measurements give new insights into the dynamical properties of this protein with respect to its eukaryotic and mesophilic homologue. Results obtained by MD are supported by QENS data and are interpreted within the framework of a fractional Brownian dynamics model for the characterization of protein relaxation dynamics. IF6 from M. jannaschii at high temperature and pressure shares similar flexibility with its eukaryotic homologue from S. cerevisieae under ambient conditions. This work shows for the first time, to our knowledge, that the very common pattern of corresponding states for thermophilic protein adaptation can be extended to thermo-barophilic proteins. A detailed analysis of dynamic properties and of local structural fluctuations reveals a complex pattern for "corresponding" structural flexibilities. In particular, in the case of IF6, the latter seems to be strongly related to the entropic contribution given by an additional, C-terminal, 20 amino-acid tail which is evolutionary conserved in all mesophilic IF6s.
Subject(s)
Archaeal Proteins/chemistry , Prokaryotic Initiation Factors/chemistry , Hydrodynamics , Methanocaldococcus , Molecular Dynamics Simulation , Neutron Diffraction , Pliability , Pressure , Saccharomyces cerevisiae Proteins/chemistry , TemperatureABSTRACT
Nucleoplasmin is a histone chaperone that consists of a pentameric N-terminal domain and an unstructured C-terminal tail. The pentameric core domain, a doughnut-like structure with a central pore, is only found in the nucleoplasmin family. Here, we report the first structure of a nucleoplasmin-like domain (NPL) from the unrelated Drosophila protein, FKBP39, and we present evidence that this protein associates with chromatin. Furthermore, we show that two other chromatin proteins, Arabidopsis thaliana histone deacetylase type 2 (HD2) and Saccharomyces cerevisiae Fpr4, share the NPL fold and form pentamers, or a dimer of pentamers in the case of HD2. Thus, we propose a new family of proteins that share the pentameric nucleoplasmin-like NPL domain and are found in protists, fungi, plants and animals.
Subject(s)
Chromatin/metabolism , Drosophila Proteins/chemistry , Histone Chaperones/chemistry , Histone Deacetylase 2/chemistry , Histones/metabolism , Nucleoplasmins/chemistry , Recombinant Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Tacrolimus Binding Proteins/chemistry , Amino Acid Sequence , Animals , Arabidopsis/metabolism , Cross-Linking Reagents , Crystallography, X-Ray , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Histone Chaperones/metabolism , Histone Deacetylase 2/metabolism , Immunoprecipitation , Models, Molecular , Molecular Sequence Data , Nucleoplasmins/metabolism , Phylogeny , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Sequence Homology, Amino Acid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Structure-Activity Relationship , Tacrolimus Binding Proteins/metabolismABSTRACT
Type I restriction-modification (RM) systems are comprised of two multi-subunit enzymes, the methyltransferase (â¼160 kDa), responsible for methylation of DNA, and the restriction endonuclease (â¼400 kDa), responsible for DNA cleavage. Both enzymes share a number of subunits. An engineered RM system, EcoR124I(NT), based on the N-terminal domain of the specificity subunit of EcoR124I was constructed that recognises the symmetrical sequence GAAN(7)TTC and is active as a methyltransferase. Here, we investigate the restriction endonuclease activity of R. EcoR124I(NT)in vitro and the subunit assembly of the multi-subunit enzyme. Finally, using small-angle neutron scattering and selective deuteration, we present a low-resolution structural model of the endonuclease and locate the motor subunits within the multi-subunit enzyme. We show that the covalent linkage between the two target recognition domains of the specificity subunit is not required for subunit assembly or enzyme activity, and discuss the implications for the evolution of Type I enzymes.
Subject(s)
DNA/chemistry , Deoxyribonucleases, Type I Site-Specific/chemistry , Escherichia coli/enzymology , Methyltransferases/chemistry , Base Sequence , Binding Sites , DNA/genetics , Deoxyribonucleases, Type I Site-Specific/genetics , Deoxyribonucleases, Type I Site-Specific/metabolism , Electrophoretic Mobility Shift Assay , Escherichia coli/genetics , Methyltransferases/genetics , Methyltransferases/metabolism , Models, Molecular , Molecular Sequence Data , Neutron Diffraction , Protein Conformation , Protein Engineering , Protein Multimerization , Protein Structure, Tertiary , Protein Subunits , Scattering, Small AngleABSTRACT
Type I DNA restriction/modification (RM) enzymes are molecular machines found in the majority of bacterial species. Their early discovery paved the way for the development of genetic engineering. They control (restrict) the influx of foreign DNA via horizontal gene transfer into the bacterium while maintaining sequence-specific methylation (modification) of host DNA. The endonuclease reaction of these enzymes on unmethylated DNA is preceded by bidirectional translocation of thousands of base pairs of DNA toward the enzyme. We present the structures of two type I RM enzymes, EcoKI and EcoR124I, derived using electron microscopy (EM), small-angle scattering (neutron and X-ray), and detailed molecular modeling. DNA binding triggers a large contraction of the open form of the enzyme to a compact form. The path followed by DNA through the complexes is revealed by using a DNA mimic anti-restriction protein. The structures reveal an evolutionary link between type I RM enzymes and type II RM enzymes.
Subject(s)
DNA Restriction Enzymes/chemistry , DNA Restriction Enzymes/ultrastructure , Models, Molecular , Deoxyribonucleases, Type I Site-Specific/chemistry , Deoxyribonucleases, Type I Site-Specific/ultrastructure , Microscopy, Electron , Negative Staining , Protein Structure, TertiaryABSTRACT
Human cytosolic seryl-tRNA synthetase (hsSerRS) is responsible for the covalent attachment of serine to its cognate tRNA(Ser). Significant differences between the amino-acid sequences of eukaryotic, prokaryotic and archaebacterial SerRSs indicate that the domain composition of hsSerRS differs from that of its eubacterial and archaebacterial analogues. As a consequence of an N-terminal insertion and a C-terminal extra-sequence, the binding mode of tRNA(Ser) to hsSerRS is expected to differ from that in prokaryotes. Recombinant hsSerRS protein was purified to homogeneity and crystallized. Diffraction data were collected to 3.13â Å resolution. The structure of hsSerRS has been solved by the molecular-replacement method.
Subject(s)
Cytosol/enzymology , Serine-tRNA Ligase/chemistry , Crystallization , Crystallography, X-Ray , HumansABSTRACT
Epstein-Barr virus (EBV) causes infectious mononucleosis and is linked to several human malignancies. EBV has a biphasic infection cycle consisting of a latent and a lytic, replicative phase. The switch from latent to lytic infection is triggered by the EBV immediate-early transcription factor ZEBRA (BZLF1, Zta, Z, EB1). We present the crystal structure of ZEBRA's DNA binding domain bound to an EBV lytic gene promoter element. ZEBRA exhibits a variant of the basic-region leucine zipper (bZIP) fold in which a C-terminal moiety stabilizes the coiled coil involved in dimer formation. The structure provides insights into ZEBRA's broad target site specificity, preferential activation of specific EBV promoters in their methylated state, ability to dimerize despite lacking a leucine zipper motif, and failure to heterodimerize with cellular bZIP proteins. The structure will allow for the design of new therapeutic agents that block activation of the EBV lytic cycle.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Herpesvirus 4, Human/physiology , Protein Structure, Quaternary , Trans-Activators/chemistry , Trans-Activators/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism , Amino Acid Sequence , Crystallography, X-Ray , DNA/chemistry , DNA/metabolism , DNA-Binding Proteins/genetics , Dimerization , Gene Expression Regulation, Viral , Humans , Macromolecular Substances , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Promoter Regions, Genetic , Sequence Alignment , Trans-Activators/genetics , Viral Proteins/geneticsABSTRACT
Rat gammaE-crystallin was overexpressed, purified under different labelling conditions and crystallized and X-ray data were collected at resolutions between 1.71 and 1.36 A. The structures were determined by molecular replacement. In these structures, the cd loop of the Greek-key motif 3, which is the major structural key motif of the two phase-transition groups of gamma-crystallins, presents a double conformation. The influence of the perdeuteration on the protein structure was determined by comparison of the atomic positions and temperature factors of the different models. The perdeuterated proteins have a similar structure to their hydrogenated counterparts, but partial or full deuteration may have some effect on the atomic B-factor values.
