AbstractThe spindle checkpoint complex is a key surveillance mechanism in cell division that prevents premature separation of sister chromatids. Mad2 is an integral component of this spindle checkpoint complex that recognizes cognate substrates such as Mad1 and Cdc20 in its closed (C‐Mad2) conformation by fastening a “seatbelt” around short peptide regions that bind to the substrate recognition site. Mad2 is also a metamorphic protein that adopts not only the fold found in C‐Mad2, but also a structurally distinct open conformation (O‐Mad2) which is incapable of binding substrates. Here, we show using chemical exchange saturation transfer (CEST) and relaxation dispersion (CPMG) NMR experiments that Mad2 transiently populates three other higher free energy states with millisecond lifetimes, two in equilibrium with C‐Mad2 (E1 and E2) and one with O‐Mad2 (E3). E1 is a mimic of substrate‐bound C‐Mad2 in which the N‐terminus of one C‐Mad2 molecule inserts into the seatbelt region of a second molecule of C‐Mad2, providing a potential pathway for autoinhibition of C‐Mad2. E2 is the “unbuckled” conformation of C‐Mad2 that facilitates the triage of molecules along competing fold‐switching and substrate binding pathways. The E3 conformation that coexists with O‐Mad2 shows fluctuations at a hydrophobic lock that is required for stabilizing the O‐Mad2 fold and we hypothesize that E3 represents an early intermediate on‐pathway towards conversion to C‐Mad2. Collectively, the NMR data highlight the rugged free energy landscape of Mad2 with multiple low‐lying intermediates that interlink substrate‐binding and fold‐switching, and also emphasize the role of molecular dynamics in its function.

中文翻译：

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变态蛋白 Mad2 的瞬时激发态及其对功能的影响

摘要纺锤体检查点复合体是细胞分裂中的关键监视机制，可防止姐妹染色单体过早分离。Mad2 是纺锤体检查点复合体的一个组成部分，通过在与底物识别位点结合的短肽区域周围系上“安全带”，识别其闭合 (C-Mad2) 构象的同源底物，例如 Mad1 和 Cdc20。Mad2 也是一种变态蛋白，不仅采用 C-Mad2 中的折叠，而且采用结构上独特的开放构象 (O-Mad2)，无法结合底物。在这里，我们使用化学交换饱和转移 (CEST) 和弛豫色散 (CPMG) NMR 实验表明，Mad2 瞬时填充了其他三种具有毫秒寿命的更高自由能态，其中两种与 C-Mad2（E1 和 E2）处于平衡，一种与 O ‐疯狂2 (E3)。E1 是底物结合的 C-Mad2 的模拟物，其中一个 C-Mad2 分子的 N 末端插入到第二个 C-Mad2 分子的安全带区域，为 C-Mad2 的自身抑制提供了潜在的途径。E2 是 C-Mad2 的“未扣合”构象，有助于沿着竞争性折叠转换和底物结合途径对分子进行分类。与 O-Mad2 共存的 E3 构象显示出稳定 O-Mad2 折叠所需的疏水锁的波动，我们假设 E3 代表了向 C-Mad2 转化的早期中间途径。总的来说，NMR 数据突出了 Mad2 崎岖的自由能景观，具有多个低位中间体，这些中间体将底物结合和折叠转换相互连接，并且还强调了分子动力学在其功能中的作用。