School of Science, Chongqing University of Posts and Telecommunications, Chongqing, China.
Shenzhen JL Computational Science and Applied Research Institute, Shenzhen, China; Beijing Computational Science Research Center, Beijing, China.
Biophys J. 2021 Aug 3;120(15):3126-3137. doi: 10.1016/j.bpj.2021.06.007. Epub 2021 Jun 29.
Cas1 and Cas2 are highly conserved proteins across clustered-regularly-interspaced-short-palindromic-repeat-Cas systems and play a significant role in protospacer acquisition. Based on crystal structure of twofold symmetric Cas1-Cas2 in complex with dual-forked protospacer DNA (psDNA), we conducted all-atom molecular dynamics simulations to study the psDNA binding, recognition, and response to cleavage on the protospacer-adjacent-motif complementary sequence, or PAMc, of Cas1-Cas2. In the simulation, we noticed that two active sites of Cas1 and Cas1' bind asymmetrically to two identical PAMc on the psDNA captured from the crystal structure. For the modified psDNA containing only one PAMc, as that to be recognized by Cas1-Cas2 in general, our simulations show that the non-PAMc association site of Cas1-Cas2 remains destabilized until after the stably bound PAMc being cleaved at the corresponding association site. Thus, long-range correlation appears to exist upon the PAMc cleavage between the two active sites (∼10 nm apart) on Cas1-Cas2, which can be allosterically mediated by psDNA and Cas2 and Cas2' in bridging. To substantiate such findings, we conducted repeated runs and further simulated Cas1-Cas2 in complex with synthesized psDNA sequences psL and psH, which have been measured with low and high frequency in acquisition, respectively. Notably, such intersite correlation becomes even more pronounced for the Cas1-Cas2 in complex with psH but remains low for the Cas1-Cas2 in complex with psL. Hence, our studies demonstrate that PAMc recognition and cleavage at one active site of Cas1-Cas2 may allosterically regulate non-PAMc association or even cleavage at the other site, and such regulation can be mediated by noncatalytic Cas2 and DNA protospacer to possibly support the ensued psDNA acquisition.
Cas1 和 Cas2 是高度保守的蛋白，存在于成簇规律间隔短回文重复序列- Cas 系统中，在原间隔序列获取中发挥重要作用。基于 Cas1-Cas2 二聚体与双链分叉原间隔序列 DNA（psDNA）复合物的晶体结构，我们进行了全原子分子动力学模拟，以研究 Cas1-Cas2 与原间隔序列邻近基序互补序列（PAMc）结合、识别和对切割的响应。在模拟中，我们注意到 Cas1 和 Cas1' 的两个活性位点不对称地结合到从晶体结构中捕获的 psDNA 上的两个相同的 PAMc。对于仅包含一个 PAMc 的修饰 psDNA，如通常由 Cas1-Cas2 识别的，我们的模拟表明 Cas1-Cas2 的非 PAMc 结合位点在稳定结合的 PAMc 在相应的结合位点被切割之前一直处于不稳定状态。因此，在 Cas1-Cas2 的两个活性位点（相隔约 10nm）之间的 PAMc 切割中似乎存在长程相关性，这种相关性可以通过 psDNA 和 Cas2 和 Cas2' 桥接进行变构调节。为了证实这些发现，我们进行了重复运行，并进一步模拟了 Cas1-Cas2 与合成的 psDNA 序列 psL 和 psH 复合物，这些序列在获取中分别以低和高频率被测量。值得注意的是，这种位点间相关性在 Cas1-Cas2 与 psH 复合物中更为明显，但在 Cas1-Cas2 与 psL 复合物中仍然较低。因此，我们的研究表明，Cas1-Cas2 一个活性位点的 PAMc 识别和切割可能变构调节另一个位点的非 PAMc 结合或甚至切割，这种调节可以通过非催化的 Cas2 和 DNA 原间隔序列来介导，以支持随后的 psDNA 获取。
