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Abstract：
Genetic code expansion enables site-specific photo-crosslinking by introducing photo-reactive non-canonical amino acids into proteins at defined positions during translation. This technology is widely used for analyzing protein-protein interactions and is applicable in mammalian cells. However, the identification of the crosslinked region still remains challenging. Here, we developed a new method to identify the crosslinked region by pre-installing a site-specific cleavage site, an α-hydroxy acid (Nε -allyloxycarbonyl-α-hydroxyl-l-lysine acid, AllocLys-OH), into the target protein. Alkaline treatment cleaves the crosslinked complex at the position of the α-hydroxy acid residue and thus helps to identify which side of the cleavage site, either closer to the N-terminus or C-terminus, the crosslinked site is located within the target protein. A series of AllocLys-OH introductions narrows down the crosslinked region. By applying this method, we identified the crosslinked regions in lysosomal-associated membrane protein type 2A (LAMP2A), a receptor of chaperone-mediated autophagy, in mammalian cells. The results suggested that at least two interfaces are involved in the homophilic interaction, which requires a trimeric or higher oligomeric assembly of adjacent LAMP2A molecules. Thus, the combination of site-specific crosslinking and site-specific cleavage promises to be useful for revealing binding interfaces and protein complex geometries.
摘要：
遗传密码扩展通过在翻译过程中在定义的位置将光反应性非规范氨基酸引入蛋白质中来实现位点特异性光交联。该技术广泛用于分析蛋白质-蛋白质相互作用，并适用于哺乳动物细胞。然而,交联区域的鉴定仍然具有挑战性。这里,我们开发了一种新的方法，通过预先安装一个位点特异性裂解位点来识别交联区，α-羟基酸（Nε-烯丙氧基羰基-α-羟基-1-赖氨酸酸，AllocLys-OH)，进入目标蛋白。碱性处理在α-羟基酸残基的位置切割交联的复合物，因此有助于识别切割位点的哪一侧，靠近N端或C端，交联位点位于靶蛋白内。一系列AllocLys-OH引入使交联区域变窄。通过应用此方法，我们确定了溶酶体相关膜蛋白2A（LAMP2A）中的交联区域，伴侣介导的自噬受体，在哺乳动物细胞中。结果表明，至少有两个界面参与同型相互作用，这需要相邻LAMP2A分子的三聚体或更高的寡聚组装。因此，位点特异性交联和位点特异性切割的组合有望用于揭示结合界面和蛋白质复合物的几何形状。本文受版权保护。保留所有权利。
