Engineered SH2 Domains for Targeted Phosphoproteomics

A comprehensive
analysis of the phosphoproteome is essential for
understanding molecular mechanisms of human diseases. However, current
tools used to enrich phosphotyrosine (pTyr) are limited in their applicability
and scope. Here, we engineered new superbinder Src-Homology 2 (SH2)
domains that enrich diverse sets of pTyr-peptides. We used phage display
to select a Fes-SH2 domain variant (superFes; sFes1) with
high affinity for pTyr and solved its structure bound to a pTyr-peptide.
We performed systematic structure–function analyses of the
superbinding mechanisms of sFes1 and superSrc-SH2 (sSrc1), another SH2 superbinder. We grafted the superbinder motifs
from sFes1 and sSrc1 into 17 additional SH2
domains and confirmed increased binding affinity for specific pTyr-peptides.
Using mass spectrometry (MS), we demonstrated that SH2 superbinders
have distinct specificity profiles and superior capabilities to enrich
pTyr-peptides. Finally, using combinations of SH2 superbinders as
affinity purification (AP) tools we showed that unique subsets of
pTyr-peptides can be enriched with unparalleled depth and coverage.

全面分析磷酸化蛋白质组（phosphoproteome）对于解析人类疾病的分子机制至关重要。然而，当前用于富集磷酸酪氨酸（phosphotyrosine, pTyr）肽段的工具在适用性与覆盖范围上均存在局限。本研究构建了新型超级结合型Src同源2（Src-Homology 2, SH2）结构域，可实现多样化pTyr肽段的富集。我们通过噬菌体展示（phage display）技术筛选出对pTyr具有高亲和力的Fes-SH2结构域变体（superFes；sFes1），并解析了其与pTyr肽段结合的复合物结构。随后，我们对sFes1与另一款SH2超级结合域superSrc-SH2（sSrc1）的超级结合机制开展了系统性的结构-功能分析。我们将sFes1与sSrc1中的超级结合基序移植至另外17种SH2结构域中，并证实这些改造后的结构域对特定pTyr肽段的结合亲和力显著提升。通过质谱（mass spectrometry, MS）实验，我们证明SH2超级结合域具有独特的特异性谱图，且在富集pTyr肽段方面性能更优异。最后，我们将多种SH2超级结合域组合用作亲和纯化（affinity purification, AP）工具，证实可实现前所未有的深度与覆盖度，从而富集得到独特的pTyr肽段子集。