Inaba-Inoue Satomi, Sabei Afra, Molza Anne-Elisabeth, Prentiss Mara, Mikawa Tsutomu, Sekiguchi Hiroshi, Prévost Chantal, Takahashi Masayuki
Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan.
Université Paris-Cité, CNRS UPR9080, Laboratoire de Biochimie Théorique, Institut de Biologie Physico-Chimique, F-75005 Paris, France.
Molecules. 2025 Apr 16;30(8):1793. doi: 10.3390/molecules30081793.
Structural analyses of protein filaments formed by self-assembly, such as actin, tubulin, or recombinase filaments, have suffered for decades from technical issues due to difficulties in crystallization, their large size, or the dynamic behavior inherent to their cellular function. The advent of cryo-electron microscopy has finally enabled us to obtain structures at different stages of the existence of these filaments. However, these structures correspond to frozen states, and the possibility of observations in solution is still lacking, especially for filaments characterized by a high plasticity, such as the RecA protein for homologous recombination. Here, we use a combination of SAXS measurements and integrative modeling to generate the solution structure of two known forms of the RecA nucleoprotein filament, previously characterized by electron microscopy and resolved by X-ray crystallography. The two forms differ in the cofactor bound to RecA-RecA interfaces, either ATP or ADP. Cooperative transition from one form to the other has been observed during single-molecule experiments by pulling on the filament but also in solution by modifying solvent conditions. We first compare the SAXS data against known structural information. While the crystal structure of the ATP form matches well with the SAXS data, we deduce from the SAXS profiles of the ADP-form values of the pitch (72.0 Å) and the number of monomers per turn (6.4) that differ with respect to the crystal structure (respectively, 82.7 Å and 6.0). We then monitor the transition between the two states driven by the addition of magnesium, and we show this transition occurs with 0.3 mM Mg ions with a high cooperativity.
由自组装形成的蛋白质细丝，如肌动蛋白、微管蛋白或重组酶细丝，由于结晶困难、尺寸较大或细胞功能固有的动态行为等技术问题，其结构分析已经困扰了几十年。低温电子显微镜的出现最终使我们能够获得这些细丝在不同存在阶段的结构。然而，这些结构对应于冻结状态，仍然缺乏在溶液中进行观察的可能性，特别是对于具有高可塑性的细丝，如用于同源重组的RecA蛋白。在这里，我们结合小角X射线散射（SAXS）测量和整合建模来生成RecA核蛋白细丝两种已知形式的溶液结构，这两种形式先前已通过电子显微镜表征并通过X射线晶体学解析。这两种形式在与RecA-RecA界面结合的辅因子上有所不同，要么是ATP，要么是ADP。在单分子实验中，通过拉伸细丝观察到了从一种形式到另一种形式的协同转变，在溶液中通过改变溶剂条件也观察到了这种转变。我们首先将SAXS数据与已知的结构信息进行比较。虽然ATP形式的晶体结构与SAXS数据匹配良好，但我们从ADP形式的SAXS曲线推导出螺距（72.0 Å）和每圈单体数（6.4）的值，这与晶体结构（分别为82.7 Å和6.0）不同。然后，我们监测由添加镁驱动的两种状态之间的转变，并表明这种转变在0.3 mM镁离子存在下以高协同性发生。
