Recently, "superbinder" SH2 domain variants with three amino acid substitutions (sSH2) were reported to have 100-fold or greater affinity for protein-phosphotyrosine (pY) than natural SH2 domains. Here we report a protocol in which His-tagged Src sSH2 efficiently captures pY-peptides from protease-digested HeLa cell total protein extracts. Affinity purification of pY-peptides by this method shows little bias for pY-proximal amino acid sequences, comparable to that achieved by using antibodies to pY, but with equal or higher yield. Superbinder-SH2 affinity purification mass spectrometry (sSH2-AP-MS) therefore provides an efficient and economical approach for unbiased pY-directed phospho-proteome profiling without the use of antibodies.
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.
Ubiquitin (Ub)-binding domains embedded in intracellular proteins act as readers of the complex Ub code and contribute to regulation of numerous eukaryotic processes. Ubinteracting motifs (UIMs) are short α-helical modular recognition elements whose role in controlling proteostasis and signal transduction has been poorly investigated. Moreover, impaired or aberrant activity of UIM-containing proteins has been implicated in numerous diseases, but targeting modular recognition elements in proteins remains a major challenge. To overcome this limitation, we developed Ub variants (UbVs) that bind to 42 UIMs in the human proteome with high affinity and specificity. Structural analysis of a UbV:UIM complex revealed the molecular determinants of enhanced affinity and specificity. Furthermore, we showed that a UbV targeting a UIM in the cancer-associated Ub-specific protease 28 potently inhibited catalytic activity. Our work demonstrates the versatility of UbVs to target short α-helical Ub receptors with high affinity and specificity. Moreover, the UbVs provide a toolkit to investigate the role of UIMs in regulating and transducing Ub signals and establish a general strategy for the systematic development of probes for Ub-binding domains.
Histone methylation is an important post-translational modification that plays a crucial role in regulating cellular functions, and its dysregulation is implicated in cancer and developmental defects. Therefore, systematic characterization of histone methylation is necessary to elucidate complex biological processes, identify biomarkers, and ultimately, enable drug discovery. Studying histone methylation relies on the use of antibodies, but these suffer from lot-to-lot variation, are costly, and cannot be used in live cells. Chromatin-modification reader domains are potential affinity reagents for methylated histones, but their application is limited by their modest affinities. We used phage display to identify key residues that greatly enhance the affinities of Cbx chromodomains for methylated histone marks and develop a general strategy for enhancing the affinity of chromodomains of the human Cbx protein family. Our strategy allows us to develop powerful probes for genome-wide binding analysis and live-cell imaging. Furthermore, we use optimized chromodomains to develop extremely potent CRISPR-based repressors for tailored gene silencing. Our results highlight the power of engineered chromodomains for analyzing protein interaction networks involving chromatin and represent a modular platform for efficient gene silencing.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.