多态性蛋白Mad2与其配体Cdc20^(121-138)的相互作用研究  

Study on The Interaction of Metamorphic Protein Mad2 and Its Ligand Cdc20^(121-138)

作　　者：张会亭[1] 赵园园[1] 葛保胜[1] 黄方[1] 

机构地区：[1]中国石油大学(华东)生物工程与技术中心重质油国家重点实验室,青岛266580

出　　处：《生物化学与生物物理进展》2017年第3期232-241,共10页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(21573289;21373271;21673294)资助项目~~

摘　　要：多态性蛋白Mad2是有丝分裂纺锤体检测点(SAC)的关键蛋白,也是多态性蛋白质家族中研究最广泛的成员之一.Mad2有两种不同的天然构象:O-Mad2和C-Mad2.Mad2构象间的转变及其与配体Cdc20间的相互作用对SAC发挥其生物学功能至关重要.本文利用荧光各向异性技术对O-Mad2和C-Mad2与配体TAMRA-Cdc20^(121-138)间相互作用的热力学及动力学过程进行了系统表征.结果表明:在无盐和低盐溶液(100 mmol/L NaCl)中,Mad2两种构象与Cdc20^(121-138)的平衡解离常数(K_D)均在10^(-6) mol/L,但C-Mad2与Cdc20^(121-138)结合的K_D值约为O-Mad2的1/5;在高盐(300 mmol/L NaCl)溶液中,Mad2两种构象与TAMRA-Cdc20^(121-138)结合的K_D值无明显差别.动力学实验结果显示,在同一种缓冲液中Mad2两种构象与Cdc20^(121-138)相互作用的解离速率常数k_d没有显著差别,而C-Mad2与Cdc20^(121-138)的结合速率常数k_a却比O-Mad2高一个数量级,这表明C-Mad2与Cdc20^(121-138)不仅结合力更强,且结合速率要快很多.Mad2与Cdc20^(121-138)突变体间的相互作用以及离子强度对二者相互作用的影响结果提示,Mad2和Cdc20间的相互作用不是通过静电相互作用,而可能是通过疏水相互作用来实现的.本研究为揭示多态性蛋白Mad2的构象转变机理及其在有丝分裂过程中的作用机制提供了重要的实验基础.Mitotic arrest deficient protein 2 （Mad2） is a typical metamorphic protein, which can adopt two distinct native folds at equilibrium under physiological conditions, an open inactive form （O-Mad2） and a closed active form （C-Mad2）. This unusual two-state behavior of Mad2 and their interactions with cognate ligand Cdc20 plays a critical role in spindle assembly checkpoint signaling during mitosis. In this paper, interactions of O-Mad2 and C-Mad2 with TAMRA-Cdc20 121-138 were systematically investigated using fluorescence anisotropy techniques. As a result, the equilibrium dissociation constant of Mad2 two folds binding with Cdc20 121-138 were both within 10-6 mol/L range in lower ionic strength solutions, and the KD value of C-Mad2 and Cdc20 121-138 was 5 times lower than that of O-Mad2. While in high ionic strength solution, there was no obvious difference on KD value of C-Mad2 and O-Mad2 binding with Cdc20 121-138. The kinetic experiments suggested that the dissociation rate constant （kd） between C-Mad2 and TAMRA-Cdc20 121-138 was similar to that of O-Mad2, but the association rate constant （ka） between C-Mad2 and TAMRA-Cdc20 121-138 was one order of magnitude higher than that of O-Mad2, which suggested that the binding of C-Mad2 with Cdc20 121-138 is thermodynamically more stable and kinetically faster. Studies on interactions between Cdc20 mutants and Mad2 together with influence of ionic strength on their interactions both suggested that the interaction of Mad2 and Cdc20 is possibly not achieved by electrostatic interaction, but through hydrophobic interactions. Our results provide key information for revealing the conformational transition mechanism of metamorphic proteins and their important role in mitosis.

关 键 词：多态性蛋白Mad2 Cdc20 热力学 动力学 相互作用 

分 类 号：Q6[生物学—生物物理学] Q7