Protein and DNA Conformational Changes Contribute to 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 loxP 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 loxP 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.