Role of Cre Dynamics in Autoinhibition and Priming

Cre recombinase, a powerful tool for genome engineering, associates into an intasome, a tetrameric complex of alternate active and inactive monomers that bring together two loxP sequences, stabilized by key protein-protein and protein-DNA interactions. High-resolution structural information for free Cre is still missing, in contrast to the many structures found for Cre-DNA complexes in the Protein Data Bank, hindering understanding of the initial steps in intasome formation. To approach Cre structure and dynamics, we carried out 100 μs of molecular dynamics simulations of free Cre, starting from five Cre structures from different stages of intasome assembly. In the generated ensemble, the linker connecting the CBD and CAT domains is an intrinsically disordered region (IDR) that promotes different orientations of the two domains. The domains remain folded and interact with each other through short-lived interactions, retaining ∼70% of their surface available for interaction with loxP. The C-terminal Helix N in the CAT domain is also an IDR that interacts with the entire protein, including the active site, transiently forming an autoinhibited complex. The active site can be assembled in the absence of DNA, albeit inefficiently. The CAT domain has a clam-like motion, opening and closing the cavity where helix N docks, establishing protein-protein interactions in the intasome. Helix A in the CBD domain slides over the domain like a windshield wiper, sampling intasome-like conformations, among others. The wide range of intramolecular motion sampled by free Cre suggests that it uses conformational selection, using primed DNA-binding surfaces in both domains while assembling into the intasome.