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 NMR to discover the link between intrinsic flexibility and function in Cre recombinase. TROSY-NMR spectra show the N-terminal and C-terminal catalytic domains (Cre^NTD, Cre^Cat) to be structurally independent. Amide ¹⁵N relaxation measurements of the Cre^Cat domain reveal fast time scale 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 inter-molecular paramagnetic relaxation enhancement (PRE) NMR data reveal an alternative auto-inhibitory 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 dislodge 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 re-examination of the mechanisms by which this widely-utilized gene editing tool selects target sites, avoids spurious DNA cleavage activity, and controls DNA recombination efficiency.

中文翻译：

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DNA结合诱导顺式在Cre重组酶中进行反式转换，以实现染色体组装。

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NMR来发现Cre重组酶的固有柔性与功能之间的联系。TROSY-NMR谱显示N端和C端催化结构域（Cre ^NTD，Cre ^Cat）在结构上是独立的。Cre ^Cat域的酰胺¹⁵ N弛豫测量结果显示，在大多数区域中，在活性区域和活性区域中显示构象差异的时间尺度动力学很快。晶体四聚体中无活性的Cre原发子。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诱导了移去的C-末端的构象变化，从而导致顺式到反式可能使重组重组Cre内含体组装所需的蛋白质间相互作用的开关。这些发现需要重新检查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。