Engineering SH2 Domains with Tailored Specificities and Affinities.

The Src Homology 2 (SH2) domain is an emerging biotechnology with applications in basic science, drug discovery, and even diagnostics. The SH2 domains rapid uptake into different areas of research is a direct result of the wealth of information generated on its biochemical, biological, and biophysical role in mammalian cell biology. Functionally, the SH2 domain binds and recognizes specific phosphotyrosine (pTyr) residues in the cell to mediate protein-protein interactions (PPIs) that govern signal transduction networks. These signal transduction networks are responsible for relaying growth and stress state signals to the cell's nucleus, ultimately effecting a change in cell biology. Protein engineers have been able to increase the affinity of SH2 domains for pTyr while also tailoring the domains' specificity to unique amino acid sequences flanking the pTyr residue. In this way, it has been possible to develop unique SH2 variants for use in affinity-purification coupled to mass spectrometry (AP-MS) experiments, microscopy, or even synthetic biology. This chapter outlines methods to tailor the affinity and specificity of virtually any human SH2 domain using a combination of rational engineering and phage-display approaches.

Phage-Displayed SH2 Domain Libraries: From Ultrasensitive Tyrosine Phosphoproteome Probes to Translational Research.

Tyrosine phosphorylation is a critical regulator of cell signaling. A large fraction of the tyrosine phosphoproteome, however, remains uncharacterized, largely due to a lack of robust and scalable methods. The Src homology 2 (SH2) domain, a structurally conserved protein domain present in many intracellular signal-transducing proteins, naturally binds phosphorylated tyrosine (pTyr) residues, providing an ideal scaffold for the development of sensitive pTyr probes. Its modest affinity, however, has greatly limited its application. Phage display is an in vitro technique used for identifying ligands for proteins and other macromolecules. Using this technique, researchers have been able to engineer SH2 domains to increase their affinity and customize their specificity. Indeed, highly diverse phage display libraries have enabled the engineering of SH2 domains as affinity-purification (AP) tools for proteomic analysis as well as probes for aberrant tyrosine signaling detection and rewiring, and represent a promising class of novel diagnostics and therapeutics. This review describes the unique structure-function characteristics of SH2 domains, highlights the fundamental contribution of phage display in the development of technologies for the dissection of the tyrosine phosphoproteome, and highlights prospective uses of SH2 domains in basic and translational research.

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.

Panel of Engineered Ubiquitin Variants Targeting the Family of Human Ubiquitin Interacting Motifs.

Ubiquitin (Ub)-binding domains embedded in intracellular proteins act as readers of the complex Ub code and contribute to regulation of numerous eukaryotic processes. Ub-interacting 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.

Protein-phosphotyrosine proteome profiling by superbinder-SH2 domain affinity purification mass spectrometry, sSH2-AP-MS.

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.