Protein andDNA Conformational Changes Contributeto Specificity of Cre Recombinase

Cre, a conservative site-specific tyrosine recombinase, is a powerful gene editing tool in the laboratory. Expanded applications in human health are hindered by a lack of understanding of the mechanism by which Cre selectively binds and recombines its cognate <i>loxP</i> sequences. This knowledge is essential for retargeting the enzyme to new sites and for mitigating the effects of off-target recombination. Prior studies have suggested that in addition to a few base-specific contacts to cognate <i>loxP</i> DNA, the enzyme’s specificity is enhanced by (1) autoinhibition involving a conformational change in the protein’s C-terminal helix and (2) indirect readout via binding-coupled conformational changes in the target DNA. We used isothermal titration calorimetry (ITC), circular dichroism (CD), and heteronuclear NMR spectroscopy to investigate DNA site recognition by wild-type Cre and a deletion mutant lacking the C-terminal helix. ITC of Cre and a C-terminal deletion variant against cognate and noncognate DNA recombinase binding elements (RBEs) reveal that the C-terminus reduces DNA binding affinity by 6-fold toward cognate DNA. Additionally, ITC revealed highly unfavorable binding enthalpy, which, when combined with evidence from CD and NMR of structural differences between cognate and noncognate complexes, supports a model in which binding-coupled DNA bending provides a unique structure-thermodynamic signature of cognate complexes. Together, these findings advance our understanding of site recognition by Cre recombinase.