High-density chemical cross-linking for modeling protein interactions
High-density chemical cross-linking for modeling protein interactions
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DOI：
10.1073/pnas.1902931116
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发表时间：
2020-01-07
影响因子：
11.1
通讯作者：
Gygi, Steven P.
中科院分区：
文献类型：
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作者：
Mintseris, Julian;Gygi, Steven P.
从蛋白质复合物的三维结构中可以更深入地了解蛋白质复合物的功能。传统的蛋白质结构解析方法仍然昂贵且劳动密集，并且需要高度纯化的起始材料。化学交联与质谱联用提供了一种替代方法，这种方法的使用越来越多，特别是与其他实验方法如低温电子显微镜相结合。在这里，我们报告了方法开发方面的进展，结合了几种正交交联化学以及搜索算法，统计分析和计算成本的改进，以1%的错误发现率实现每7个氨基酸1个独特的交联位置对的覆盖。这在没有任何肽水平分级分离或富集的情况下完成。我们应用我们的方法来模拟碳酸酐酶（CA）和它的蛋白质抑制剂之间的复合物，表明交联是自洽的，并定义在高分辨率的相互作用界面。由此产生的模型提出了一个支架开发一类基于蛋白质的抑制剂的CA家族的酶。我们接下来交联酵母蛋白酶体，在3次质谱分析中鉴定出3，893个独特的交联肽。该数据集包括蛋白酶体亚基的1，704个独特的交联位置对，其中一半以上是亚基间的。使用多个最近解决的冷冻EM结构，我们表明，观察到的交联反应的构象动力学和一些蛋白酶体亚基的紊乱。我们进一步证明，这种水平的交联密度是足够的19-亚基调节颗粒从头建模的架构。
Detailed mechanistic understanding of protein complex function is greatly enhanced by insights from its 3-dimensional structure. Traditional methods of protein structure elucidation remain expensive and labor-intensive and require highly purified starting material. Chemical cross-linking coupled with mass spectrometry offers an alternative that has seen increased use, especially in combination with other experimental approaches like cryo-electron microscopy. Here we report advances in method development, combining several orthogonal cross-linking chemistries as well as improvements in search algorithms, statistical analysis, and computational cost to achieve coverage of 1 unique cross-linked position pair for every 7 amino acids at a 1% false discovery rate. This is accomplished without any peptide-level fractionation or enrichment. We apply our methods to model the complex between a carbonic anhydrase (CA) and its protein inhibitor, showing that the cross-links are selfconsistent and define the interaction interface at high resolution. The resulting model suggests a scaffold for development of a class of protein-based inhibitors of the CA family of enzymes. We next cross-link the yeast proteasome, identifying 3,893 unique crosslinked peptides in 3 mass spectrometry runs. The dataset includes 1,704 unique cross-linked position pairs for the proteasome subunits, more than half of them intersubunit. Using multiple recently solved cryo-EM structures, we show that observed cross-links reflect the conformational dynamics and disorder of some proteasome subunits. We further demonstrate that this level of cross-linking density is sufficient to model the architecture of the 19-subunit regulatory particle de novo.