A novel chemical footprinting approach identifies critical lysine residues involved in the binding of receptor-associated protein to cluster II of LDL receptor-related protein
A novel chemical footprinting approach identifies critical lysine residues involved in the binding of receptor-associated protein to cluster II of LDL receptor-related protein
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DOI：
10.1042/bj20140977
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发表时间：
2015-05-15
影响因子：
4.1
通讯作者：
Meijer, Alexander B.
中科院分区：
文献类型：
--
作者：
Bloem, Esther;Ebberink, Eduard H. T. M.;Meijer, Alexander B.
串联质量标签（TMT）被用于一种新的化学足迹法，以确定介导的受体相关蛋白（RAP）与LDL（低密度脂蛋白）受体（LDLR）相关蛋白（LRP）的簇II的相互作用的赖氨酸残基。分离的RAP D3结构域用TMT 126修饰，D3结构域-簇II复合物用TMT-127修饰。Nano-LC-MS分析显示TMT-127对包括Lys（256）、Lys（270）和Lys（305）-Lys（306）的肽的修饰减少，表明这些残基有助于簇II结合。这与先前的发现一致，即Lys（256）和Lys（270）对于结合簇II亚结构域是关键的[Fisher，Beglova和Blacklow（2006）Mol. Cell 22，277-283]。利用D3结构域变体K（256）A、K（305）A和K（306）A进行的簇II结合研究现在表明，Lys（306）也有助于簇II结合。对于全长RAP，我们观察到包括Lys（60）、Lys（191）、Lys（256）、Lys（270）和Lys（305）-Lys（306）的肽在RAP-簇II复合物中显示出减少的TMT修饰。值得注意的是，赖氨酸（60）先前已涉及介导D1结构域与簇II的相互作用。我们的结果表明，D2结构域的赖氨酸（191）也有助于簇II结合。然而，使用RAP变体K（191）A、K（256）A、K（305）A和K（306）A的结合研究揭示了仅K（256）A变体的簇II结合的适度降低。这表明其他赖氨酸残基可以补偿单个赖氨酸残基的缺失，以实现有效的复合物组装。总的来说，新的见解已获得RAP的赖氨酸残基簇II结合的贡献。此外，我们建议，TMT可用于确定蛋白质复合物形成的关键赖氨酸残基。
Tandem mass tags (TMTs) were utilized in a novel chemical footprinting approach to identify lysine residues that mediate the interaction of receptor-associated protein (RAP) with cluster II of LDL (low-density lipoprotein) receptor (LDLR)-related protein (LRP). The isolated RAP D3 domain was modified with TMT126 and the D3 domain-cluster II complex with TMT-127. Nano-LC-MS analysis revealed reduced modification with TMT-127 of peptides including Lys(256), Lys(270) and Lys(305)-Lys(306) suggesting that these residues contribute to cluster II binding. This agrees with previous findings that Lys(256) and Lys(270) are critical for binding cluster II sub-domains [Fisher, Beglova and Blacklow (2006) Mol. Cell 22, 277-283]. Cluster II-binding studies utilizing D3 domain variants K(256)A, K(305)A and K(306)A now showed that Lys(306) contributes to cluster II binding as well. For full-length RAP, we observed that peptides including Lys(60), Lys(191), Lys(256), Lys(270) and Lys(305)-Lys(306) exhibited reduced modification with TMT in the RAP-cluster II complex. Notably, Lys(60) has previously been implicated to mediate D1 domain interaction with cluster II. Our results suggest that also Lys(191) of the D2 domain contributes to cluster II binding. Binding studies employing the RAP variants K(191)A, K(256)A, K(305)A and K(306)A, however, revealed a modest reduction in cluster II binding for the K(256)A variant only. This suggests that the other lysine residues can compensate for the absence of a single lysine residue for effective complex assembly. Collectively, novel insight has been obtained into the contribution of lysine residues of RAP to cluster II binding. In addition, we propose that TMTs can be utilized to identify lysine residues critical for protein complex formation.