SOD1表现出变构阻挫以促进金属结合亲和力
SOD1 Exhibits Allosteric Frustration to Facilitate Metal Binding Affinity
摘要：
超氧化物歧化酶-1（SOD 1）是一种普遍存在的Cu和Zn结合的自由基防御酶，其错误折叠和聚集在肌萎缩侧索硬化症（一种总是致命的神经退行性疾病）中发挥潜在的关键作用。SOD 1中超过150种突变已被鉴定为家族性疾病，但目前尚不清楚这些突变体共有哪些统一特征（如果有的话）使其致病。在这里，我们开发了几种新的计算分析，用于探测ALS相关和合理设计的SOD 1变体的热机械性能。残余物和金属之间的变构相互作用自由能的计算，和一系列的原子力显微镜实验模拟与可变系链位置，量化的机械刚度“指纹”的SOD 1变体。一系列C-末端截短突变体的机械指纹研究，以及在改变天然约束时平衡动态波动的分析，突变时的势能变化，frustratometer分析，以及对90个残基的甘氨酸扫描的局部挫折和金属结合相互作用之间的耦合分析，揭示了apo蛋白是内部挫折的，这些内应力通过突变而部分缓解，但以金属-结合亲和力为代价，并且残基的挫折与其结合金属的作用直接相关。这一证据表明，载脂蛋白SOD 1是一种应变中间体，具有高金属结合亲和力的“自变构”。因此，SOD 1作为抗氧化剂的功能的先决条件与载脂蛋白状态的热机械稳定性竞争，增加了蛋白质在载脂蛋白状态下错误折叠的敏感性。
英文摘要：
Superoxide dismutase-1 (SOD1) is a ubiquitous, Cu and Zn binding, free radical defense enzyme whose misfolding and aggregation play a potential key role in amyotrophic lateral sclerosis, an invariably fatal neurodegenerative disease. Over 150 mutations in SOD1 have been identified with a familial form of the disease, but it is presently not clear what unifying features, if any, these mutants share to make them pathogenic. Here, we develop several new computational assays for probing the thermo-mechanical properties of both ALS-associated and rationally-designed SOD1 variants. Allosteric interaction free energies between residues and metals are calculated, and a series of atomic force microscopy experiments are simulated with variable tether positions, to quantify mechanical rigidity "fingerprints" for SOD1 variants. Mechanical fingerprinting studies of a series of C-terminally truncated mutants, along with an analysis of equilibrium dynamic fluctuations while varying native constraints, potential energy change upon mutation, frustratometer analysis, and analysis of the coupling between local frustration and metal binding interactions for a glycine scan of 90 residues together reveal that the apo protein is internally frustrated, that these internal stresses are partially relieved by mutation but at the expense of metal-binding affinity, and that the frustration of a residue is directly related to its role in binding metals. This evidence points to apo SOD1 as a strained intermediate with "self-allostery" for high metal-binding affinity. Thus, the prerequisites for the function of SOD1 as an antioxidant compete with apo state thermo-mechanical stability, increasing the susceptibility of the protein to misfold in the apo state.
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