Structural analysis of cysteine-free Nt.BspD6 nicking endonuclease and its functional features

• Published in: Biochimica et biophysica acta. Proteins and proteomics 2022-03, Vol.1870 (3), p.140756-140756, Article 140756
• Main Authors: Artyukh, Rimma I., Fatkhullin, Bulat F., Kachalova, Galina S., Antipova, Valeriya N., Perevyazova, Tatyana A., Yunusova, Alfiya K.
• Format: Article
• Language: English
• Subjects: Bacillus - enzymology; Binding Sites; Crystal structure; Crystallography, X-Ray - methods; Cysteine - chemistry; Deoxyribonuclease I - chemistry; Deoxyribonuclease I - genetics; Deoxyribonuclease I - metabolism; Dimerization; DNA - chemistry; DNA - metabolism; Endonucleases - chemistry; Endonucleases - genetics; Endonucleases - metabolism; Heterodimeric restriction endonuclease; Hydrogen Bonding; Hydrolysis; Molecular Structure; Mutagenesis, Site-Directed; Nickase; Protein Subunits - chemistry; Serine - chemistry; Site-directed mutagenesis; Small subunit
• ISSN: 1570-9639; 1878-1454
• DOI: 10.1016/j.bbapap.2022.140756

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. •Crystal structures of the mutated forms of the nicking endonuclease Nt.BspD6I•Changes of the electrostatic field of the molecule cysteine-free Nt.BspD6I•Mutation of cysteines in nickase changes the interaction of subunits of R.BspD6I.•Mutation of cysteines for serine in nickase decreases the activity of R.BspD6I.