An Engineered SS Bridge Blocks the Conformational Change Required for the Nuclease Activity of BfiI.
The type IIS restriction endonuclease BfiI is a homodimer, and each monomer is composed of the N-terminal catalytic and C-terminal DNA recognition domains connected by a 28-residue linker segment. In the crystal in the absence of cognate DNA, BfiI exists in a "closed" conformation, in which an interdomain linker occludes a putative DNA binding surface at the catalytic domain and sterically hinders access to the active site. Cognate DNA binding presumably triggers a conformational change from the inactive "closed" state to the catalytically competent "open" state. Here we show that the disulfide SS bridge engineered at the domain interface locks the enzyme in the "closed" state. In the "closed" SS-linked state, BfiI binds cognate DNA with the same affinity as the wild-type enzyme but does not cut it, indicating that cross-linking introduces a restraint on the conformational transition, which couples DNA recognition and cleavage. Disruption of the interdomain SS bridge by the reducing agent restores the DNA cleavage ability of BfiI.
Institute of Biotechnology, Vilnius University , Graiciuno 8, Vilnius LT-02241, Lithuania.
Mindaugas
M
Zaremba
Institute of Biotechnology, Vilnius University , Graiciuno 8, Vilnius LT-02241, Lithuania.
Virginijus
V
Siksnys
D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D003432:Cross-Linking Reagents; D004269:DNA, Bacterial / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D006358:Hot Temperature; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D056811:Unfolded Protein Response
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D004220:Disulfides; D011994:Recombinant Proteins; C113635:endodeoxyribonuclease BfiI; D015252:Deoxyribonucleases, Type II Site-Specific