ABSTRACT
Nicking endonuclease Nt.BspD6I (Nt.BspD6I) is the large subunit of the heterodimeric restriction endonuclease R.BspD6I. It recognizes the short specific DNA sequence 5´'- GAGTC and cleaves only the top strand in dsDNA at a distance of four nucleotides downstream the recognition site toward the 3´'-terminus. A mechanism of interaction of this protein with DNA is still unknown. Here we report the crystal structure of Cysteine-free Nt.BspD6I, with four cysteine residues (11, 160, 508, 578) substituted by serine, which was determined with a resolution of 1.93 Å. A comparative structural analysis showed that the substitution of cysteine residues induced marked conformational changes in the N-terminal recognition and the C-terminal cleavage domains. As a result of this changes were formed three new hydrogen bonds and the electrostatic field in these regions changed compared with wild type Nt.BspD6I. The substitution of cysteine residues did not alter the nicking function of Cysteine-free Nt.BspD6I but caused change in the activity of Cysteine-free heterodimeric restriction endonuclease R.BspD6I due to a change in the interaction between its large and small subunits. The results obtained contribute to the identification of factors influencing the interactions of subunits in the heterodimeric restriction enzyme R.BspD6I.
Subject(s)
Cysteine/chemistry , Deoxyribonuclease I/chemistry , Endonucleases/chemistry , Serine/chemistry , Bacillus/enzymology , Binding Sites , Crystallography, X-Ray/methods , DNA/chemistry , DNA/metabolism , Deoxyribonuclease I/genetics , Deoxyribonuclease I/metabolism , Dimerization , Endonucleases/genetics , Endonucleases/metabolism , Hydrogen Bonding , Hydrolysis , Molecular Structure , Mutagenesis, Site-Directed , Protein Subunits/chemistryABSTRACT
The mutated nickase Nt.BspD6I E418A has been obtained by site-directed mutagenesis. The purified protein has been crystallized, and its spatial structure has been determined at 2.45 Å resolution. An analysis of the crystal structures of the wild-type and mutated nickase have shown that the elimination of a carboxyl group due to the E418A mutation initiates marked conformational changes in both the N-terminal recognition domain and the C-terminal catalytic domain of nickase and insignificantly affects its linker domain. This is supported by changes in the functional properties of mutated nickase: an increase in the oligomerization capacity in the presence of a substrate, a reduction in the capacity to bind a substrate, and complete loss of catalytic activity.
Subject(s)
Deoxyribonuclease I/chemistry , Deoxyribonuclease I/metabolism , Catalytic Domain/genetics , Deoxyribonuclease I/genetics , Mutagenesis, Site-Directed , Mutation/geneticsABSTRACT
The heterodimeric restriction endonuclease R.BspD6I from Bacillus species D6 recognizes a pseudosymmetric sequence and cuts both DNA strands outside the recognition sequence. The large subunit, Nt.BspD6I, acts as a type IIS site-specific monomeric nicking endonuclease. The isolated small subunit, ss.BspD6I, does not bind DNA and is not catalytically active. We solved the crystal structures of Nt.BspD6I and ss.BspD6I at high resolution. Nt.BspD6I consists of three domains, two of which exhibit structural similarity to the recognition and cleavage domains of FokI. ss.BspD6I has a fold similar to that of the cleavage domain of Nt.BspD6I, each containing a PD-(D/E)XK motif and a histidine as an additional putative catalytic residue. In contrast to the DNA-bound FokI structure, in which the cleavage domain is rotated away from the DNA, the crystal structure of Nt.BspD6I shows the recognition and cleavage domains in favorable orientations for interactions with DNA. Docking models of complexes of Nt.BspD6I and R.BspD6I with cognate DNA were constructed on the basis of structural similarity to individual domains of FokI, R.BpuJI and HindIII. A three-helix bundle forming an interdomain linker in Nt.BspD6I acts as a rigid spacer adjusting the orientations of the spatially separated domains to match the distance between the recognition and cleavage sites accurately.
Subject(s)
Catalytic Domain , Deoxyribonuclease I/chemistry , Deoxyribonuclease I/metabolism , Deoxyribonucleases, Type II Site-Specific/chemistry , Deoxyribonucleases, Type II Site-Specific/metabolism , Amino Acid Sequence , Catalysis , DNA, Bacterial/metabolism , Dimerization , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Alignment , Structural Homology, Protein , Substrate SpecificityABSTRACT
The heterodimeric restriction endonuclease R.BspD6I is composed of a small subunit with a cleavage site and a large subunit, containing a recognition domain and a cleavage domain, that may function separately as a monomeric nicking endonuclease. Here, the crystallization of the small subunit and diffraction data collection to 1.5 A resolution are reported.
Subject(s)
Deoxyribonuclease I/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , Crystallization , DNA Restriction Enzymes/chemistry , DNA Restriction Enzymes/isolation & purification , Deoxyribonuclease I/isolation & purification , Dimerization , Escherichia coli Proteins/isolation & purification , Protein Subunits/chemistry , X-Ray DiffractionABSTRACT
Crystals of site-specific DNA nickase Nb.BspD6I (of molecular weight 70.8 kDa) have been grown at 291 K using PEG 8000 as precipitant. The diffraction pattern of the crystal extends to 3.3 A resolution at 100 K. The crystal belongs to space group P2(1), with unit-cell parameters a = 57.76, b = 90.67, c = 71.71, beta = 110.1 degrees. There is one molecule in the asymmetric unit and the solvent content is estimated to be 53% by volume.
Subject(s)
Deoxyribonuclease I/metabolism , Bacillus/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Crystallization , Crystallography, X-Ray , Deoxyribonuclease I/chemistry , Deoxyribonuclease I/isolation & purification , Kinetics , Substrate Specificity , X-Ray DiffractionABSTRACT
Crystals of the (cytosine-5)-DNA methyltransferase NlaX from Neisseria lactamica (molecular weight 36.5 kDa) have been grown at 291 K using 2.5 M NaCl as precipitant. The crystals diffract to 3.0 A resolution at 100 K. The crystals belong to space group P321, with unit-cell parameters a = 121.98, b = 121.98, c = 56.71 A. There is one molecule in the asymmetric unit and the solvent content is estimated to be 62.1% by volume.
