Amino acid selective cross-saturation method for identification of proximal residue pairs in a protein-protein complex
Amino acid selective cross-saturation method for identification of proximal residue pairs in a protein-protein complex
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DOI：
10.1021/ja804062t
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发表时间：
2008-09-10
影响因子：
15
通讯作者：
Shimada, Ichio
中科院分区：
文献类型：
--
作者：
Igarashi, Shunsuke;Osawa, Masanori;Shimada, Ichio
我们描述了一种基于NMR的方法，氨基酸选择性交叉饱和（ASCS）方法，以确定蛋白质-蛋白质复合物的界面残基对。ASCS使用“交叉饱和（CS）-供体”蛋白，其中在H-2-背景中仅一个氨基酸被选择性地H-1-标记，和具有均匀H-2、N-15标记的“CS-受体”蛋白。仅存在于供体中的H-1标记的氨基酸的照射通过CS现象降低了邻近H-1标记的CS源残基的受体残基的H-1-N-15 HSQC信号的强度。考虑到复合物中每种蛋白质的三维结构，但不是复合物结构，基于CS供体上的CS源残基和受体上的交叉饱和酰胺质子之间的空间互补性，多个ASCS结果的组合分析指定了CS源残基。NMR研究了E.大肠杆菌宿主的ASCS的关键蛋白的表达，揭示了Ala、Arg、His、Ile、Leu、Lys、Met、Phe、Pro、Trp和Tyr被选择性地标记，具有高的H-1/H-2比率。然后使用酵母泛素（Ub）和酵母泛素水解酶1（YUH 1）的已知结构确认ASCS的观察。相反，通过对ASCS结果的分析，参考YUH 1和Ub的个体结构，提出了CS源残基的合理候选物。通过ASCS获得的CS-源残基和交叉饱和酰胺基团之间的成对距离信息将用于建模蛋白质-蛋白质复合物。
We describe an NMR-based approach, the amino acid selective cross-saturation (ASCS) method, to identify the pairs of the interface residues of protein-protein complexes. ASCS uses a "cross-saturation (CS)-donor" protein, in which only one amino acid is selectively H-1-labeled in a H-2-background, and a "CS-acceptor" protein with uniform H-2, N-15 labeling. Irradiation of the H-1-labeled amino acid, which exists only in the donor, decreases the intensity of the H-1-N-15 HSQC signals of the acceptor residues proximal to the H-1-labeled CS-source residue(s) through the CS phenomenon. Given the three-dimensional structure of each protein in the complex, but not the complex structure, the combinatorial analysis of multiple ASCS results specify the CS-source residue(s), based on the spatial complementarity between the CS-source residues on the CS donor and the cross-saturated amide protons on the acceptor. NMR investigations of the labeling selectivity and efficiency in an E. coli host, which are critical for ASCS, revealed that Ala, Arg, His, Ile, Leu, Lys, Met, Phe, Pro, Trp, and Tyr are selectively labeled with a high H-1/H-2 ratio. The observation of the ASCS was then confirmed using the known structure of the yeast ubiquitin (Ub) and yeast ubiquitin hydrolase 1 (YUH1). Conversely, reasonable candidates for the CS-source residues were suggested by the analysis of the ASCS results, with reference to the individual structures of YUH1 and Ub. The pairwise distance information between the CS-source residues and the cross-saturated amide groups obtained by ASCS will be useful for modeling protein-protein complexes.