Affinity maturation of the RLIP76 Ral binding domain to inform the design of stapled peptides targeting the Ral GTPases.
Affinity maturation of the RLIP76 Ral binding domain to inform the design of stapled peptides targeting the Ral GTPases.
复制标题
DOI：
10.1074/jbc.ra120.015735
复制
发表时间：
2021-01
期刊：
影响因子：
--
通讯作者：
Owen D
中科院分区：
文献类型：
--
作者：
Hurd CA;Brear P;Revell J;Ross S;Mott HR;Owen D
Ral GTP酶已经被认为是许多Ras驱动的癌症中细胞生长和转移的关键驱动因素。我们先前已经报道了基于Ral效应物RLIP 76的钉合肽，其可以破坏Ral信号传导。钉合肽是使用合成支架锁定成其生物活性形式的短肽。在这里，使用RLIP 76 Ral结合结构域的亲和力成熟，我们鉴定了几个序列取代，它们共同提高了与Ral蛋白的结合超过20倍。从选择的命中进行了严格的分析，以确定单个残基的贡献和两个1.5紧密结合的突变体与RalB复合体的共晶结构揭示了关键的相互作用。从这种成熟中获得的见解用于设计基于RLIP 76的第二代钉合肽，与第一代前导肽相比，其表现出对Ral GTP酶的极大改善的选择性。定量第二代肽与Ral蛋白的结合，并通过NMR确定前导肽在RalB上的结合位点。钉合肽成功地与细胞裂解物中的多种Ral效应物相互作用竞争。我们的研究结果表明，如何操纵一个天然的结合伴侣可以帮助合理设计的钉肽抑制剂靶向蛋白质-蛋白质相互作用。
Ral GTPases have been implicated as critical drivers of cell growth and metastasis in numerous Ras-driven cancers. We have previously reported stapled peptides, based on the Ral effector RLIP76, that can disrupt Ral signaling. Stapled peptides are short peptides that are locked into their bioactive form using a synthetic brace. Here, using an affinity maturation of the RLIP76 Ral-binding domain, we identified several sequence substitutions that together improve binding to Ral proteins by more than 20-fold. Hits from the selection were rigorously analyzed to determine the contributions of individual residues and two 1.5 Å cocrystal structures of the tightest-binding mutants in complex with RalB revealed key interactions. Insights gained from this maturation were used to design second-generation stapled peptides based on RLIP76 that exhibited vastly improved selectivity for Ral GTPases when compared with the first-generation lead peptide. The binding of second-generation peptides to Ral proteins was quantified and the binding site of the lead peptide on RalB was determined by NMR. Stapled peptides successfully competed with multiple Ral–effector interactions in cellular lysates. Our findings demonstrate how manipulation of a native binding partner can assist in the rational design of stapled peptide inhibitors targeting a protein–protein interaction.