Crystallographic study of a site-specifically cross-linked protein complex with a genetically incorporated photoreactive amino acid.
Crystallographic study of a site-specifically cross-linked protein complex with a genetically incorporated photoreactive amino acid.
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DOI：
10.1021/bi1016183
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发表时间：
2011-01
期刊：
影响因子：
2.9
通讯作者：
Shin Sato;S. Mimasu;A. Sato;Nobumasa Hino;K. Sakamoto;T. Umehara;S. Yokoyama
中科院分区：
文献类型：
--
作者：
Shin Sato;S. Mimasu;A. Sato;Nobumasa Hino;K. Sakamoto;T. Umehara;S. Yokoyama
二苯甲酮类发光团被广泛应用于大分子的光交联。遗传密码扩展的最新进展使得蛋白质可以在特定的位置与对苯甲酰-L-苯丙氨酸(PBpa)生物合成，从而与相互作用的蛋白质形成共价键。然而，光交联蛋白质复合体的结构还没有被揭示，因此既不知道复合体中“光桥”的实际结构，也不知道这种共价桥对整个复合体结构的影响。在这项研究中，我们测定了肝脏癌蛋白gankyrin和S6蛋白酶体蛋白(S6C)的C-末端结构域的晶体结构，分辨率为2.05？首先，在蛋白质表面的16个位置将光反应性氨基酸分别掺入到甘草蛋白中，发现了两个与S6C形成共价键的变体。通过优化络合物在365 nm光下照射的时间，使其中一个交联物的产率最大化，其中一个交联物以pBpa取代了甘吉林中的Arg85。结构分析表明，在S6C中，pBpa85的二苯甲酮的羰基与Glu356的C-γ原子形成了专属的共价键，表明pBpa具有较高的交联键选择性。此外，与天然复杂结构相比，该交联结构表现出很小的结构扭曲。我们的结果表明，与位点特异性掺入的pBpa交联会保留天然的结合模式，并有助于探测蛋白质-蛋白质的相互作用。
The benzophenone photophore is widely used to photo-cross-link macromolecules. Recent developments in genetic code expansion have allowed the biosynthesis of proteins with p-benzoyl-L-phenylalanine (pBpa) at defined sites, for covalent bonding with interacting proteins. However, the structure of a photo-cross-linked protein complex had not been revealed, and thus neither the actual structure of the "photobridge" in a complex nor the influence of this covalent bridge on the overall complex structure was known. In this study, we determine the crystal structure of the cross-linked complex of the liver oncoprotein gankyrin and the C-terminal domain of S6 proteasomal protein (S6C), at 2.05 Å resolution. First, the photoreactive amino acid was separately incorporated into gankyrin at 16 sites on the protein surface, and two variants that efficiently formed a covalent bond with S6C were found. The yield of one of the cross-linked products, with pBpa in place of Arg85 in gankyrin, was maximized for crystallization via optimization of the duration of complex exposure to 365 nm light. The structure revealed that the carbonyl group of the benzophenone of pBpa85 formed a covalent bond exclusively with the Cγ atom of Glu356 in S6C, showing the high selectivity of formation of cross-links by pBpa. In addition, the cross-linked structure exhibited little structural distortion from the native complex structure. Our results demonstrated that cross-linking with site-specifically incorporated pBpa preserves the native binding mode and is useful for probing protein-protein interactions.