Bernard A. Liu

SH2 Domains Recognize Contextual Peptide Sequence Information to Determine Selectivity

Selective ligand recognition by modular protein interaction domains is a primary determinant of specificity in signaling pathways. Src homology 2 (SH2) domains fulfill this capacity immediately downstream of tyrosine kinases, acting to recruit their host polypeptides to ligand proteins harboring phosphorylated tyrosine residues. The degree to which SH2 domains are selective and the mechanisms underlying selectivity are fundamental to understanding phosphotyrosine signaling networks. An examination of interactions between 50 SH2 domains and a set of 192 phosphotyrosine peptides corresponding to physiological motifs within FGF, insulin, and IGF-1 receptor pathways indicates that individual SH2 domains have distinct recognition properties and exhibit a remarkable degree of selectivity beyond that predicted by previously described binding motifs. The underlying basis for such selectivity is the ability of SH2 domains to recognize both permissive amino acid residues that enhance binding and non-permissive amino acid residues that oppose binding in the vicinity of the essential phosphotyrosine. Neighboring positions affect one another so local sequence context matters to SH2 domains. This complex linguistics allows SH2 domains to distinguish subtle differences in peptide ligands. This newly appreciated contextual dependence substantially increases the accessible information content embedded in the peptide ligands that can be effectively integrated to determine binding. This concept may serve more broadly as a paradigm for subtle recognition of physiological ligands by protein interaction domains.

The SH2 Domain–Containing Proteins in 21 Species Establish the Provenance and Scope of Phosphotyrosine Signaling in Eukaryotes

Analysis of SH2 domain–containing proteins provides insights into the evolution of modular protein domains and organismal complexity. Building Complexity with Phosphotyrosine Regulated protein-protein interactions are crucial to cellular signal transduction. SH2 domains recognize specific sequences containing phosphorylated tyrosine residues. Signaling through tyrosine phosphorylation networks has expanded as organisms have become more complex. Liu et al. combined sequence alignment, analysis of intron and exon boundaries, and domain organization of 21 living species, ranging from amoeba and slime mold to humans, to classify the hundreds of known SH2 domain–containing proteins into 38 families. Their analysis suggests mechanisms by which SH2 and tyrosine kinase signaling networks have evolved and contributed to the development of organismal complexity. The Src homology 2 (SH2) domains are participants in metazoan signal transduction, acting as primary mediators for regulated protein-protein interactions with tyrosine-phosphorylated substrates. Here, we describe the origin and evolution of SH2 domain proteins by means of sequence analysis from 21 eukaryotic organisms from the basal unicellular eukaryotes, where SH2 domains first appeared, through the multicellular animals and increasingly complex metazoans. On the basis of our results, SH2 domains and phosphotyrosine signaling emerged in the early Unikonta, and the numbers of SH2 domains expanded in the choanoflagellate and metazoan lineages with the development of tyrosine kinases, leading to rapid elaboration of phosphotyrosine signaling in early multicellular animals. Our results also indicated that SH2 domains coevolved and the number of the domains expanded alongside protein tyrosine kinases and tyrosine phosphatases, thereby coupling phosphotyrosine signaling to downstream signaling networks. Gene duplication combined with domain gain or loss produced novel SH2-containing proteins that function within phosphotyrosine signaling, which likely have contributed to diversity and complexity in metazoans. We found that intra- and intermolecular interactions within and between SH2 domain proteins increased in prevalence along with organismal complexity and may function to generate more highly connected and robust phosphotyrosine signaling networks.

The language of SH2 domain interactions defines phosphotyrosine-mediated signal transduction

Natural languages arise in an unpremeditated fashion resulting in words and syntax as individual units of information content that combine in a manner that is both complex and contextual, yet intuitive to a native reader. In an analogous manner, protein interaction domains such as the Src Homology 2 (SH2) domain recognize and “read” the information contained within their cognate peptide ligands to determine highly selective protein–protein interactions that underpin much of cellular signal transduction. Herein, we discuss how contextual sequence information, which combines the use of permissive and non‐permissive residues within a parent motif, is a defining feature of selective interactions across SH2 domains. Within a system that reads phosphotyrosine modifications this provides crucial information to distinguish preferred interactions. This review provides a structural and biochemical overview of SH2 domain binding to phosphotyrosine‐containing peptide motifs and discusses how the diverse set of SH2 domains is able to differentiate phosphotyrosine ligands.

Large-scale interaction profiling of PDZ domains through proteomic peptide-phage display using human and viral phage peptidomes

Significance Although knowledge about the human interactome is increasing in coverage because of the development of high-throughput technologies, fundamental gaps remain. In particular, interactions mediated by short linear motifs are of great importance for signaling, but systematic experimental approaches for their detection are missing. We fill this important gap by developing a dedicated approach that combines bioinformatics, custom oligonucleotide arrays and peptide-phage display. We computationally design a library of all possible motifs in a given proteome, print representatives of these on custom oligonucleotide arrays, and identify natural peptide binders for a given protein using phage display. Our approach is scalable and has broad application. Here, we present a proof-of-concept study using both designed human and viral peptide libraries. The human proteome contains a plethora of short linear motifs (SLiMs) that serve as binding interfaces for modular protein domains. Such interactions are crucial for signaling and other cellular processes, but are difficult to detect because of their low to moderate affinities. Here we developed a dedicated approach, proteomic peptide-phage display (ProP-PD), to identify domain–SLiM interactions. Specifically, we generated phage libraries containing all human and viral C-terminal peptides using custom oligonucleotide microarrays. With these libraries we screened the nine PSD-95/Dlg/ZO-1 (PDZ) domains of human Densin-180, Erbin, Scribble, and Disks large homolog 1 for peptide ligands. We identified several known and putative interactions potentially relevant to cellular signaling pathways and confirmed interactions between full-length Scribble and the target proteins β-PIX, plakophilin-4, and guanylate cyclase soluble subunit α-2 using colocalization and coimmunoprecipitation experiments. The affinities of recombinant Scribble PDZ domains and the synthetic peptides representing the C termini of these proteins were in the 1- to 40-μM range. Furthermore, we identified several well-established host–virus protein–protein interactions, and confirmed that PDZ domains of Scribble interact with the C terminus of Tax-1 of human T-cell leukemia virus with micromolar affinity. Previously unknown putative viral protein ligands for the PDZ domains of Scribble and Erbin were also identified. Thus, we demonstrate that our ProP-PD libraries are useful tools for probing PDZ domain interactions. The method can be extended to interrogate all potential eukaryotic, bacterial, and viral SLiMs and we suggest it will be a highly valuable approach for studying cellular and pathogen–host protein–protein interactions.

Evolution of SH2 domains and phosphotyrosine signalling networks.

Src homology 2 (SH2) domains mediate selective protein–protein interactions with tyrosine phosphorylated proteins, and in doing so define specificity of phosphotyrosine (pTyr) signalling networks. SH2 domains and protein-tyrosine phosphatases expand alongside protein-tyrosine kinases (PTKs) to coordinate cellular and organismal complexity in the evolution of the unikont branch of the eukaryotes. Examination of conserved families of PTKs and SH2 domain proteins provides fiduciary marks that trace the evolutionary landscape for the development of complex cellular systems in the proto-metazoan and metazoan lineages. The evolutionary provenance of conserved SH2 and PTK families reveals the mechanisms by which diversity is achieved through adaptations in tissue-specific gene transcription, altered ligand binding, insertions of linear motifs and the gain or loss of domains following gene duplication. We discuss mechanisms by which pTyr-mediated signalling networks evolve through the development of novel and expanded families of SH2 domain proteins and the elaboration of connections between pTyr-signalling proteins. These changes underlie the variety of general and specific signalling networks that give rise to tissue-specific functions and increasingly complex developmental programmes. Examination of SH2 domains from an evolutionary perspective provides insight into the process by which evolutionary expansion and modification of molecular protein interaction domain proteins permits the development of novel protein-interaction networks and accommodates adaptation of signalling networks.

High-throughput analysis of peptide-binding modules.

Modular protein interaction domains (PIDs) that recognize linear peptide motifs are found in hundreds of proteins within the human genome. Some PIDs such as SH2, 14–3‐3, Chromo, and Bromo domains serve to recognize posttranslational modification (PTM) of amino acids (such as phosphorylation, acetylation, methylation, etc.) and translate these into discrete cellular responses. Other modules such as SH3 and PSD‐95/Discs‐large/ZO‐1 (PDZ) domains recognize linear peptide epitopes and serve to organize protein complexes based on localization and regions of elevated concentration. In both cases, the ability to nucleate‐specific signaling complexes is in large part dependent on the selectivity of a given protein module for its cognate peptide ligand. High‐throughput (HTP) analysis of peptide‐binding domains by peptide or protein arrays, phage display, mass spectrometry, or other HTP techniques provides new insight into the potential protein–protein interactions prescribed by individual or even whole families of modules. Systems level analyses have also promoted a deeper understanding of the underlying principles that govern selective protein–protein interactions and how selectivity evolves. Lastly, there is a growing appreciation for the limitations and potential pitfalls associated with HTP analysis of protein–peptide interactomes. This review will examine some of the common approaches utilized for large‐scale studies of PIDs and suggest a set of standards for the analysis and validation of datasets from large‐scale studies of peptide‐binding modules. We will also highlight how data from large‐scale studies of modular interaction domain families can provide insight into systems level properties such as the linguistics of selective interactions.

SRC Homology 2 Domain Binding Sites in Insulin, IGF-1 and FGF receptor mediated signaling networks reveal an extensive potential interactome

Specific peptide ligand recognition by modular interaction domains is essential for the fidelity of information flow through the signal transduction networks that control cell behavior in response to extrinsic and intrinsic stimuli. Src homology 2 (SH2) domains recognize distinct phosphotyrosine peptide motifs, but the specific sites that are phosphorylated and the complement of available SH2 domains varies considerably in individual cell types. Such differences are the basis for a wide range of available protein interaction microstates from which signaling can evolve in highly divergent ways. This underlying complexity suggests the need to broadly map the signaling potential of systems as a prerequisite for understanding signaling in specific cell types as well as various pathologies that involve signal transduction such as cancer, developmental defects and metabolic disorders. This report describes interactions between SH2 domains and potential binding partners that comprise initial signaling downstream of activated fibroblast growth factor (FGF), insulin (Ins), and insulin-like growth factor-1 (IGF-1) receptors. A panel of 50 SH2 domains screened against a set of 192 phosphotyrosine peptides defines an extensive potential interactome while demonstrating the selectivity of individual SH2 domains. The interactions described confirm virtually all previously reported associations while describing a large set of potential novel interactions that imply additional complexity in the signaling networks initiated from activated receptors. This study of pTyr ligand binding by SH2 domains provides valuable insight into the selectivity that underpins complex signaling networks that are assembled using modular protein interaction domains.

Selection of recombinant anti-SH3 domain antibodies by high-throughput phage display

Antibodies are indispensable tools in biochemical research and play an expanding role as therapeutics. While hybridoma technology is the dominant method for antibody production, phage display is an emerging technology. Here, we developed and employed a high‐throughput pipeline that enables selection of antibodies against hundreds of antigens in parallel. Binding selections using a phage‐displayed synthetic antigen‐binding fragment (Fab) library against 110 human SH3 domains yielded hundreds of Fabs targeting 58 antigens. Affinity assays demonstrated that representative Fabs bind tightly and specifically to their targets. Furthermore, we developed an efficient affinity maturation strategy adaptable to high‐throughput, which increased affinity dramatically but did not compromise specificity. Finally, we tested Fabs in common cell biology applications and confirmed recognition of the full‐length antigen in immunoprecipitation, immunoblotting and immunofluorescence assays. In summary, we have established a rapid and robust high‐throughput methodology that can be applied to generate highly functional and renewable antibodies targeting protein domains on a proteome‐wide scale.

Binding Assays Using Recombinant SH2 Domains: Far-Western, Pull-Down, and Fluorescence Polarization

Recognition of phosphotyrosine-containing sequences by SH2 domains confers specificity in tyrosine kinase pathways. By assessing interactions between isolated SH2 domains and their binding proteins, it is possible to gain insight into otherwise inaccessible complex cellular systems. Far-Western, pull-down, and fluorescence polarization (FP) have been frequently used for characterization of phosphotyrosine signaling. Here, we outline standard protocols for these established assays using recombinant SH2 domain, emphasizing the importance of appropriate sample preparation and assay controls.

Expression and Production of SH2 Domain Proteins.

The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating signaling events downstream of receptor tyrosine kinases (RTKs), adaptors, and scaffolds. Over a hundred SH2 domains are present in mammals, each having a unique specificity which determines its interactions with multiple binding partners. One of the essential tools necessary for studying and determining the role of SH2 domains in phosphotyrosine signaling is a set of soluble recombinant SH2 proteins. Here we describe methods, based on a broad experience with purification of all SH2 domains, for the production of SH2 domain proteins needed for proteomic and biochemical-based studies such as peptide arrays, mass-spectrometry, protein microarrays, reverse-phase microarrays, and high-throughput fluorescence polarization (HTP-FP). We describe stepwise protocols for expression and purification of SH2 domains using GST or poly His-tags, two widely adopted affinity tags. In addition, we address alternative approaches, challenges, and validation studies for assessing protein quality and provide general characteristics of purified human SH2 domains.