Peking University, Beijing, China.
PLoS One. 2022 Aug 5;17(8):e0270572. doi: 10.1371/journal.pone.0270572. eCollection 2022.
Cell-cycle dependent proteins are indispensible for the accurate division of cells, a group of proteins called Microtubule-associated proteins (MAPs) are important to cell division as it bind microtubules and participate with other co-factors to form the spindle midbody, which works as the workhorse of cell-division. PRC1 is a distinguishing member of MAPs, as it is a human MAP and works as the key in mediating daughter cell segregation in ana-phase and telo-phase. The physiological significance of PRC1 calls for a high resolution three-dimensional structure. The crystal structure of PRC1 was published but has low resolution (>3 Å) and incomplete sidechains, placing hurdles to understanding the structure-function relationships of PRC1, therefore, we determined the high-resolution solution structure of PRC1's dimerization domain using NMR spectroscopy. Significant differences between the crystal structure and the solution structure can be observed, the main differences center around the N terminus and the end of the alpha-Helix H2. Furthermore, detailed structure analyses revealed that the hydrophobic core packing of the solution and crystal structures are also different. To validate the solution structure, we used Hydrogen-deuterium exchange experiments that address the structural discrepancies between the crystal and solution structure; we also generated mutants that are key to the differences in the crystal and solution structures, measuring its structural or thermal stability by NMR spectroscopy and Fluorescence Thermal Shift Assays. These results suggest that N terminal residues are key to the integrity of the whole protein, and the solution structure of the dimerization domain better reflects the conformation PRC1 adopted in solution conditions.
细胞周期依赖性蛋白对于细胞的精确分裂是必不可少的，一组称为微管相关蛋白（MAPs）的蛋白质对于细胞分裂很重要，因为它们结合微管并与其他共同因子一起形成纺锤体中间体，作为细胞分裂的主力军。PRC1 是 MAPs 的一个独特成员，因为它是一种人类 MAP，作为在中期和末期介导子细胞分离的关键。PRC1 的生理意义需要高分辨率的三维结构。PRC1 的晶体结构已经发表，但分辨率较低（>3 Å）且侧链不完整，这给理解 PRC1 的结构-功能关系带来了障碍，因此，我们使用 NMR 光谱法确定了 PRC1 二聚化结构域的高分辨率溶液结构。可以观察到晶体结构和溶液结构之间存在显著差异，主要差异集中在 N 端和α-螺旋 H2 的末端。此外，详细的结构分析表明，溶液和晶体结构的疏水性核心堆积也不同。为了验证溶液结构，我们使用氢氘交换实验来解决晶体和溶液结构之间的结构差异；我们还生成了关键的突变体，这些突变体是晶体和溶液结构差异的关键，通过 NMR 光谱法和荧光热移位测定法测量其结构或热稳定性。这些结果表明，N 端残基是整个蛋白质完整性的关键，二聚化结构域的溶液结构更好地反映了 PRC1 在溶液条件下采用的构象。
