The lysine (K) tRNA synthetase C-terminal (KTSC) domain containing proteins are widely spread in Bacteria, Archaea and Viruses, but the function of this short domain is unclear. The occurrence of the fusion of KTSC domain to a catalytic domain or domains related to DNA or RNA metabolisms suggests its potential role in DNA or RNA binding. Here, we report the characterization of Mvu8s from Methanolobus vulcani, which consists of a single KTSC domain. Mvu8s binds specifically to ssDNA with an affinity approximately 40- and 10-fold higher than those for dsDNA and ssRNA in vitro, respectively. It shows a slight preference to the G-rich DNA sequence but barely binds the A-stretch. Crystal structure of Mvu8s shows that it forms a homo-tetramer, with each monomer composed of a four-strand antiparallel ß-sheet and a helix-turn-helix in the order of ß1-ß2-ß3-α1-α2-ß4. Four basic residues (R3, R7, K54 and K58) were found to serve important roles in ssDNA-binding. And, the spiral arrangement of the DNA interfaces in Mvu8s homo-tetramer presumably results in ssDNA wrapping. Our results not only offer clues of the functions of the KTSC domain containing proteins but also expand our knowledge on the non-oligonucleotide-binding (OB) fold single-stranded DNA-binding proteins in Archaea.
DNA, Single-Stranded , DNA-Binding Proteins , Catalytic Domain , DNA/metabolism , DNA-Binding Proteins/metabolism , Protein Binding/genetics , RNA/metabolism
Studies on the structure-function relationship of protein greatly help to understand not only the principles of protein folding but also the rationales of protein engineering. Crenarchaeal chromatin protein Cren7 provides an excellent research model for this issue. The small protein adopts a 'ß-barrel' fold, formed by the double-stranded antiparallel ß-sheet 1 tightly packing with the triple-stranded antiparallel ß-sheet 2. The simple structure of Cren7 is stabilized by the hydrophobic core between the ß-sheets, consisting of the side chains of V8, V10, L20, V25, F41 and F50. In the present work, mutation analyses by alanine substitution of each of the residues in the hydrophobic core were performed. Circular dichroism spectra and nuclear magnetic resonance analyses showed that mutation of F41 led to a significant misfolding of Cren7 through disruption of the ß-sheets. Meanwhile, the mutant F41A showed a reduced thermostatility (Tm of 53.2 °C), as compared with the wild-type Cren7 (Tm > 80 °C). Biolayer interferometry and nick-closure assays showed the largely unchanged activities in DNA binding and supercoiling of F41A, indicating the DNA interface of Cren7 was generally retained in F41A. However, F41A was unable to mediate DNA bridging, probably due to the impairment in forming oligomers/polymers on DNA. Atomic force microscopic images of the F41A-DNA complexes also revealed that F41A nearly completely lost the ability to compact DNA into highly condensed structures. Our results not only reveal the critical role of F41 in protein folding of Cren7 but also provide new insights into the structure-function relationships of thermostable proteins.
Archaea , Archaeal Proteins , Archaea/metabolism , Archaeal Proteins/chemistry , Chromatin , DNA/chemistry , DNA-Binding Proteins/metabolism , Hydrophobic and Hydrophilic Interactions , Protein Folding
The winged helix superfamily comprises a large number of structurally related nucleic acid-binding proteins. While these proteins are often shown to bind dsDNA, few are known to bind ssDNA. Here, we report the identification and characterization of Sul7s, a novel winged-helix single-stranded DNA binding protein family highly conserved in Sulfolobaceae. Sul7s from Sulfolobus islandicus binds ssDNA with an affinity approximately 15-fold higher than that for dsDNA in vitro. It prefers binding oligo(dT)30 over oligo(dC)30 or a dG-rich 30-nt oligonucleotide, and barely binds oligo(dA)30. Further, binding by Sul7s inhibits DNA strand annealing, but shows little effect on the melting temperature of DNA duplexes. The solution structure of Sul7s determined by NMR shows a winged helix-turn-helix fold, consisting of three α-helices, three ß-strands, and two short wings. It interacts with ssDNA via a large positively charged binding surface, presumably resulting in ssDNA deformation. Our results shed significant light on not only non-OB fold single-stranded DNA binding proteins in Archaea, but also the divergence of the winged-helix proteins in both function and structure during evolution.
DNA-Binding Proteins , Sulfolobus , Archaea/metabolism , DNA/chemistry , DNA, Single-Stranded , DNA-Binding Proteins/metabolism , Sulfolobus/genetics
Cren7 and Sis7d, two chromatin proteins from Sulfolobus islandicus, undergo extensive methylations at multiple lysine residues to various extents. Whether this highly conserved protein serves an epigenetic role in the regulation of the structure and function of the chromosome remains unclear. In the present study, we show that methylation significantly affects Cren7, but not Sis7d, in the ability to bind DNA and to constrain negative DNA supercoils. Strikingly, methylated Cren7 was significantly less efficient in forming oligomers or mediating intermolecular DNA bridging. Single-site substitution mutation with glutamine reveals that methylation of the four lysine residues (K24, K31, K42, and K48) of Cren7 at the protein-DNA interface, which are variably conserved among Cren7 homologues from different branches of the Crenarchaeota, influenced Cren7-DNA interactions in different manners. We suggest that dynamic methylation of Cren7 may represent a potential epigenetic mechanism involved in the chromosomal regulation in crenarchaea.
