Mechanistic understanding of DNA recombination in the Cre-loxP system has largely been guided by crystallographic structures of tetrameric synaptic complexes. Those studies have suggested a role for protein conformational dynamics that has not been well characterized at the atomic level. We used solution nuclear magnetic resonance (NMR) spectroscopy to discover the link between intrinsic flexibility and function in Cre recombinase. Transverse relaxation-optimized spectroscopy (TROSY) NMR spectra show the N-terminal and C-terminal catalytic domains (Cre^NTD and Cre^Cat) to be structurally independent. Amide ¹⁵N relaxation measurements of the Cre^Cat domain reveal fast-timescale dynamics in most regions that exhibit conformational differences in active and inactive Cre protomers in crystallographic tetramers. However, the C-terminal helix αN, implicated in assembly of synaptic complexes and regulation of DNA cleavage activity via trans protein–protein interactions, is unexpectedly rigid in free Cre. Chemical shift perturbations and intra- and intermolecular paramagnetic relaxation enhancement (PRE) NMR data reveal an alternative autoinhibitory conformation for the αN region of free Cre, wherein it packs in cis over the protein DNA binding surface and active site. Moreover, binding to loxP DNA induces a conformational change that dislodges the C terminus, resulting in a cis-to-trans switch that is likely to enable protein–protein interactions required for assembly of recombinogenic Cre intasomes. These findings necessitate a reexamination of the mechanisms by which this widely utilized gene-editing tool selects target sites, avoids spurious DNA cleavage activity, and controls DNA recombination efficiency.

对Cre- loxP系统中DNA重组的机理的理解主要是由四聚体突触复合物的晶体学结构指导的。这些研究表明，蛋白质构象动力学的作用尚未在原子水平上得到很好的表征。我们使用溶液核磁共振（NMR）光谱来发现Cre重组酶的固有柔性与功能之间的联系。横向弛豫优化光谱（TROSY）NMR光谱显示N端和C端催化域（Cre ^NTD和Cre ^Cat）在结构上是独立的。Cre ^Cat的酰胺¹⁵ N弛豫测量结构域揭示了大多数区域的快速时标动力学，这些区域在晶体四聚体中的有活性和无活性Cre原发子中显示出构象差异。但是，C末端螺旋αN与突触复合物的组装和通过反式蛋白质-蛋白质相互作用调节DNA切割活性有关，在游离Cre中出乎意料地刚性。化学位移扰动以及分子内和分子间顺磁弛豫增强（PRE）NMR数据揭示了游离Cre的αN区的另一种自抑制构象，其中它顺式堆积在蛋白质DNA结合表面和活性位点上。此外，结合于loxP序列的DNA诱导了变位C末端，导致构象变化顺式-到-反式开关可能使重组重组Cre酶体组装所需的蛋白质间相互作用。这些发现需要重新研究这种广泛使用的基因编辑工具选择靶位点的机制，避免伪造的DNA裂解活性，并控制DNA重组效率。