Mapping the Homodimer Interface of an Optimized, Artificial, Transmembrane Protein Activator of the Human Erythropoietin Receptor
Mapping the Homodimer Interface of an Optimized, Artificial, Transmembrane Protein Activator of the Human Erythropoietin Receptor
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DOI：
10.1371/journal.pone.0095593
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发表时间：
2014-04-30
期刊：
影响因子：
3.7
通讯作者：
DiMaio, Daniel
中科院分区：
文献类型：
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作者：
Cohen, Emily B.;Jun, Susan J.;DiMaio, Daniel
跨膜蛋白是细胞蛋白质的重要组成部分，跨膜蛋白片段的特异性相互作用在蛋白质齐聚、折叠和功能中起着重要的作用。我们之前分离到了一种人工的，二聚体的，44个氨基酸的跨膜蛋白，它可以激活反式人促红细胞生成素受体(HEPOR)。这种人造蛋白质在体外支持原代人类造血祖细胞有限的红系分化，尽管它不像促红细胞生成素，促红细胞生成素是这种受体的天然配体。在这里，我们使用定向进化的方法来探索跨膜蛋白激活hEPOR的结构基础。对表达数千个跨膜激活剂突变体的文库进行了筛选，以寻找在表达hEPOR的小鼠细胞中诱导生长因子独立方面比原始分离物更活跃的突变体。最活跃的突变体EBC5-16支持人类细胞的红系分化，其活性接近EPO，通过细胞表面糖蛋白A的表达来评估，血糖素A是红系分化的晚期标志。EBC5-16在第25位含有单一的异亮氨酸到丝氨酸的取代，这增加了它形成二聚体的能力。遗传学研究证实了二聚化对活性的重要性，并确定了构成EBC5-16同源二聚体界面的残基。界面需要一个GxxxG二聚体包装基序和25位的小氨基酸才能获得最大活性，这意味着EBC5-16二聚体的紧密包装是活性的关键决定因素。这些实验确定了一种人工蛋白，它能在自然宿主细胞中引起其靶标的强劲激活，证明了该蛋白的二聚化对hEPOR参与的重要性，并为未来对这一新的受体激活机制的结构和功能研究提供了框架。
Transmembrane proteins constitute a large fraction of cellular proteins, and specific interactions involving membrane-spanning protein segments play an important role in protein oligomerization, folding, and function. We previously isolated an artificial, dimeric, 44-amino acid transmembrane protein that activates the human erythropoietin receptor (hEPOR) in trans. This artificial protein supports limited erythroid differentiation of primary human hematopoietic progenitor cells in vitro, even though it does not resemble erythropoietin, the natural ligand of this receptor. Here, we used a directed-evolution approach to explore the structural basis for the ability of transmembrane proteins to activate the hEPOR. A library that expresses thousands of mutants of the transmembrane activator was screened for variants that were more active than the original isolate at inducing growth factor independence in mouse cells expressing the hEPOR. The most active mutant, EBC5-16, supports erythroid differentiation in human cells with activity approaching that of EPO, as assessed by cell-surface expression of glycophorin A, a late-stage marker of erythroid differentiation. EBC5-16 contains a single isoleucine to serine substitution at position 25, which increases its ability to form dimers. Genetic studies confirmed the importance of dimerization for activity and identified the residues constituting the homodimer interface of EBC5-16. The interface requires a GxxxG dimer packing motif and a small amino acid at position 25 for maximal activity, implying that tight packing of the EBC5-16 dimer is a crucial determinant of activity. These experiments identified an artificial protein that causes robust activation of its target in a natural host cell, demonstrated the importance of dimerization of this protein for engagement of the hEPOR, and provided the framework for future structure-function studies of this novel mechanism of receptor activation.