Citron Y Rose, Fagerstrom Carey J, Keszthelyi Bettina, Huang Bo, Rusan Nasser M, Kelly Mark J S, Agard David A
Department of Biochemistry and Biophysics and the Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California, United States of America.
Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
PLoS One. 2018 Jan 9;13(1):e0190530. doi: 10.1371/journal.pone.0190530. eCollection 2018.
The centrosome serves as the main microtubule-organizing center in metazoan cells, yet despite its functional importance, little is known mechanistically about the structure and organizational principles that dictate protein organization in the centrosome. In particular, the protein-protein interactions that allow for the massive structural transition between the tightly organized interphase centrosome and the highly expanded matrix-like arrangement of the mitotic centrosome have been largely uncharacterized. Among the proteins that undergo a major transition is the Drosophila melanogaster protein centrosomin that contains a conserved carboxyl terminus motif, CM2. Recent crystal structures have shown this motif to be dimeric and capable of forming an intramolecular interaction with a central region of centrosomin. Here we use a combination of in-cell microscopy and in vitro oligomer assessment to show that dimerization is not necessary for CM2 recruitment to the centrosome and that CM2 alone undergoes significant cell cycle dependent rearrangement. We use NMR binding assays to confirm this intramolecular interaction and show that residues involved in solution are consistent with the published crystal structure and identify L1137 as critical for binding. Additionally, we show for the first time an in vitro interaction of CM2 with the Drosophila pericentrin-like-protein that exploits the same set of residues as the intramolecular interaction. Furthermore, NMR experiments reveal a calcium sensitive interaction between CM2 and calmodulin. Although unexpected because of sequence divergence, this suggests that centrosomin-mediated assemblies, like the mammalian pericentrin, may be calcium regulated. From these results, we suggest an expanded model where during interphase CM2 interacts with pericentrin-like-protein to form a layer of centrosomin around the centriole wall and that at the onset of mitosis this population acts as a nucleation site of intramolecular centrosomin interactions that support the expansion into the metaphase matrix.
中心体是后生动物细胞中主要的微管组织中心，然而，尽管其功能重要，但从机制上对决定中心体中蛋白质组织的结构和组织原则却知之甚少。特别是，在紧密组织的间期中心体和有丝分裂中心体高度扩展的基质样排列之间发生的大规模结构转变所涉及的蛋白质-蛋白质相互作用，在很大程度上仍未得到充分表征。经历重大转变的蛋白质之一是果蝇蛋白中心体蛋白，它含有一个保守的羧基末端基序CM2。最近的晶体结构表明，该基序是二聚体，能够与中心体蛋白的中央区域形成分子内相互作用。在这里，我们结合细胞内显微镜和体外寡聚体评估，表明二聚化对于CM2募集到中心体不是必需的，并且单独的CM2会发生显著的细胞周期依赖性重排。我们使用核磁共振结合试验来证实这种分子内相互作用，并表明溶液中涉及的残基与已发表的晶体结构一致，并确定L1137对结合至关重要。此外，我们首次展示了CM2与果蝇中心粒外周蛋白样蛋白的体外相互作用，该相互作用利用了与分子内相互作用相同的一组残基。此外，核磁共振实验揭示了CM2与钙调蛋白之间的钙敏感相互作用。尽管由于序列差异而出乎意料，但这表明中心体蛋白介导的组装，如哺乳动物的中心粒外周蛋白，可能受钙调节。根据这些结果，我们提出了一个扩展模型，即在间期CM2与中心粒外周蛋白样蛋白相互作用，在中心粒壁周围形成一层中心体蛋白，并且在有丝分裂开始时，这群蛋白作为分子内中心体蛋白相互作用的成核位点，支持向中期基质的扩展。
