Shu Xin, Asghar Sana, Yang Fan, Li Shang-Tong, Wu Haifan, Yang Bing
Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China.
Cancer Center, Zhejiang University, Hangzhou, China.
Front Chem. 2022 Feb 18;10:815991. doi: 10.3389/fchem.2022.815991. eCollection 2022.
Genetically encoded non-canonical amino acids (ncAAs) with electrophilic moieties are excellent tools to investigate protein-protein interactions (PPIs) both and . These ncAAs, including a series of alkyl bromide-based ncAAs, mainly target cysteine residues to form protein-protein cross-links. Although some reactivities towards lysine and tyrosine residues have been reported, a comprehensive understanding of their reactivity towards a broad range of nucleophilic amino acids is lacking. Here we used a recently developed OpenUaa search engine to perform an in-depth analysis of mass spec data generated for Thioredoxin and its direct binding proteins cross-linked with an alkyl bromide-based ncAA, BprY. The analysis showed that, besides cysteine residues, BprY also targeted a broad range of nucleophilic amino acids. We validated this broad reactivity of BprY with Affibody/Z protein complex. We then successfully applied BprY to map a binding interface between SUMO2 and SUMO-interacting motifs (SIMs). BprY was further applied to probe SUMO2 interaction partners. We identified 264 SUMO2 binders, including several validated SUMO2 binders and many new binders. Our data demonstrated that BprY can be effectively used to probe protein-protein interaction interfaces even without cysteine residues, which will greatly expand the power of BprY in studying PPIs.
带有亲电基团的基因编码非标准氨基酸（ncAAs）是研究蛋白质-蛋白质相互作用（PPI）的优秀工具。这些ncAAs，包括一系列基于烷基溴的ncAAs，主要靶向半胱氨酸残基以形成蛋白质-蛋白质交联。尽管已经报道了它们对赖氨酸和酪氨酸残基的一些反应性，但仍缺乏对其对广泛亲核氨基酸反应性的全面了解。在这里，我们使用最近开发的OpenUaa搜索引擎对用基于烷基溴的ncAA BprY交联的硫氧还蛋白及其直接结合蛋白产生的质谱数据进行了深入分析。分析表明，除了半胱氨酸残基外，BprY还靶向广泛的亲核氨基酸。我们用亲和体/Z蛋白复合物验证了BprY的这种广泛反应性。然后，我们成功地应用BprY绘制了SUMO2与SUMO相互作用基序（SIMs）之间的结合界面。BprY进一步应用于探测SUMO2相互作用伙伴。我们鉴定出264个SUMO2结合蛋白，包括几个经过验证的SUMO2结合蛋白和许多新的结合蛋白。我们的数据表明，即使没有半胱氨酸残基，BprY也可以有效地用于探测蛋白质-蛋白质相互作用界面，这将大大扩展BprY在研究PPI方面的能力。
