Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60607, USA.
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, USA.
N Biotechnol. 2018 Oct 25;45:36-44. doi: 10.1016/j.nbt.2018.05.001. Epub 2018 May 12.
Transcription factor c-Myc is an oncoprotein that is regulated at the post-translational level through phosphorylation of two conserved residues, Serine 62 (Ser62) and Threonine 58 (Thr58). A highly specific tool capable of recognizing Myc via pThr58 is needed to monitor activation and localization. Through phage display, we have isolated 10 engineered Forkhead-associated (FHA) domains that selectively bind to a phosphothreonine (pThr)-containing peptide (53-FELLPpTPPLSPS-64) segment of human c-Myc. One domain variant was observed to bind to the Myc-pThr58 peptide with a K value of 800 nM and had >1000-fold discrimination between the phosphorylated and non-phosphorylated peptide. The crystal structure of the engineered FHA Myc-pThr-binding domain (Myc-pTBD) was solved in complex with its cognate ligand. The Myc-pTBD was observed to be structurally similar to the yeast Rad9 FHA1 domain, except that its β4-β5 and β10-β11 loops form a hydrophobic pocket to facilitate the interaction between the domain and the peptide ligand. The Myc-pTBD's specificity for its cognate ligand was demonstrated to be on a par with 3 commercial polyclonal antibodies, suggesting that this recombinant reagent is a viable alternative to antibodies for monitoring Myc regulation.
转录因子 c-Myc 是一种癌蛋白，通过两个保守残基丝氨酸 62（Ser62）和苏氨酸 58（Thr58）的磷酸化在翻译后水平受到调节。需要一种能够通过 pThr58 识别 Myc 的高度特异性工具来监测其激活和定位。通过噬菌体展示，我们分离到了 10 种工程化的 Forkhead 相关（FHA）结构域，它们能够选择性地结合人 c-Myc 的一个含有磷酸苏氨酸（pThr）的肽（53-FELLPpTPPLSPS-64）片段。一种结构域变体被观察到与 Myc-pThr58 肽的结合 K 值为 800 nM，并且在磷酸化和非磷酸化肽之间具有 >1000 倍的区分能力。该工程化的 FHA Myc-pThr 结合结构域（Myc-pTBD）与它的同源配体的晶体结构得到解决。观察到 Myc-pTBD 的结构与酵母 Rad9 FHA1 结构域相似，只是其β4-β5 和β10-β11 环形成一个疏水性口袋，有利于结构域与肽配体之间的相互作用。Myc-pTBD 对其同源配体的特异性与 3 种商业多克隆抗体相当，表明这种重组试剂是监测 Myc 调节的抗体的可行替代品。
