Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210.
Laboratorio de Bioquímica y Resonancia Magnética Nuclear, Centro de Investigaciones Químicas, Instituto de Investigación en Ciencias Básicas y Aplicadas, Universidad Autónoma del Estado de Morelos, 62209 Cuernavaca, Morelos, Mexico.
Proc Natl Acad Sci U S A. 2020 Oct 6;117(40):24849-24858. doi: 10.1073/pnas.2011448117. Epub 2020 Sep 23.
Mechanistic understanding of DNA recombination in the Cre 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 and Cre) to be structurally independent. Amide N relaxation measurements of the Cre 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 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 over the protein DNA binding surface and active site. Moreover, binding to DNA induces a conformational change that dislodges the C terminus, resulting in a -to- 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.
Cre 系统中 DNA 重组的机制理解在很大程度上受到四聚体突触复合物晶体结构的指导。这些研究表明，蛋白质构象动力学在原子水平上尚未得到很好的描述。我们使用溶液核磁共振（NMR）光谱技术来发现 Cre 重组酶固有灵活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 端催化结构域（Cre 和 Cre）在结构上是独立的。Cre 结构域酰胺 N 弛豫测量显示，在大多数区域中存在快速时间尺度的动力学，这些区域在晶体四聚体中活性和非活性 Cre 前体中表现出构象差异。然而，C 端螺旋 αN 参与突触复合物的组装，并通过蛋白质-蛋白质相互作用调节 DNA 切割活性，但其在游离 Cre 中出人意料地僵硬。化学位移扰动和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 区域的替代自动抑制构象，其中它包裹在蛋白质 DNA 结合表面和活性位点上。此外，与 DNA 的结合诱导构象变化，从而使 C 端脱离，导致 -到-的开关，这可能使组装重组 Cre 内切酶所需的蛋白质-蛋白质相互作用成为可能。这些发现需要重新审视这个广泛使用的基因编辑工具选择靶位点、避免虚假 DNA 切割活性和控制 DNA 重组效率的机制。
