Luo X, Fang G, Coldiron M, Lin Y, Yu H, Kirschner M W, Wagner G
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
Nat Struct Biol. 2000 Mar;7(3):224-9. doi: 10.1038/73338.
The checkpoint protein Mad2 inhibits the activity of the anaphase promoting complex by sequestering Cdc20 until all chromosomes are aligned at the metaphase plate. We report the solution structure of human Mad2 and its interaction with Cdc20. Mad2 possesses a novel three-layered alpha/beta fold with three alpha-helices packed between two beta-sheets. Using deletion mutants we identified the minimal Mad2-binding region of human Cdc20 as a 40-residue segment immediately N-terminal to the WD40 repeats. Mutagenesis and NMR titration experiments show that a C-terminal flexible region of Mad2 is required for binding to Cdc20. Mad2 and Cdc20 form a tight 1:1 heterodimeric complex in which the C-terminal segment of Mad2 becomes folded. These results provide the first structural insight into mechanisms of the spindle assembly checkpoint.
检查点蛋白Mad2通过隔离Cdc20来抑制后期促进复合物的活性，直到所有染色体在中期板上排列整齐。我们报道了人Mad2的溶液结构及其与Cdc20的相互作用。Mad2具有一种新颖的三层α/β折叠结构，在两个β折叠之间堆积着三个α螺旋。使用缺失突变体，我们确定人Cdc20的最小Mad2结合区域是WD40重复序列紧邻N端的一个40个残基的片段。诱变和核磁共振滴定实验表明，Mad2的C端柔性区域是与Cdc20结合所必需的。Mad2和Cdc20形成紧密的1:1异二聚体复合物，其中Mad2的C端片段发生折叠。这些结果为纺锤体组装检查点机制提供了首个结构上的见解。
