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.