Cell Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CH, Utrecht, The Netherlands.
Sci Rep. 2017 Oct 18;7(1):13453. doi: 10.1038/s41598-017-13663-y.
Characterization of protein interaction domains is crucial for understanding protein functions. Here we combine cross-linking mass spectrometry (XL-MS) with deletion analysis to accurately locate minimal protein interaction domains. As a proof of concept, we investigated in detail the binding interfaces of two protein assemblies: the complex formed by MICAL3, ELKS and Rab8A, which is involved in exocytosis, and the complex of SLAIN2, CLASP2 and ch-TOG, which controls microtubule dynamics. We found that XL-MS provides valuable information to efficiently guide the design of protein fragments that are essential for protein interaction. However, we also observed a number of cross-links between polypeptide regions that were dispensable for complex formation, especially among intrinsically disordered sequences. Collectively, our results indicate that XL-MS, which renders distance restrains of linked residue pairs, accelerates the characterization of protein binding regions in combination with other biochemical approaches.
蛋白质相互作用结构域的特征对于理解蛋白质功能至关重要。在这里，我们将交联质谱（XL-MS）与缺失分析相结合，以准确确定最小的蛋白质相互作用结构域。作为概念验证，我们详细研究了两种蛋白质复合物的结合界面：MICAL3、ELKS 和 Rab8A 形成的复合物，该复合物参与胞吐作用，以及 SLAIN2、CLASP2 和 ch-TOG 形成的复合物，该复合物控制微管动力学。我们发现，XL-MS 提供了有价值的信息，可有效地指导设计对于蛋白质相互作用至关重要的蛋白质片段。然而，我们还观察到许多多肽区域之间的交联对于复合物形成是可有可无的，尤其是在固有无序序列中。总的来说，我们的结果表明，XL-MS 可以确定连接残基对的距离限制，与其他生化方法相结合，可以加速蛋白质结合区域的特征描述。
