Identification and Optimization of Protein Tyrosine Phosphatase Inhibitors Via Fragment Ligation.

Phosphotyrosine biomimetics are starting points for potent inhibitors of protein tyrosine phosphatases (PTPs) and, thus, crucial for drug development. Their identification, however, has been heavily driven by rational design, limiting the discovery of diverse, novel, and improved mimetics. In this chapter, we describe two screening approaches utilizing fragment ligation methods: one to identify new mimetics and the other to optimize existing mimetics into more potent and selective inhibitors.

Custom Workflow for the Confident Identification of Sulfotyrosine-Containing Peptides and Their Discrimination from Phosphopeptides.

Protein tyrosine sulfation (sY) is a post-translational modification (PTM) catalyzed by Golgi-resident tyrosyl protein sulfo transferases (TPSTs). Information on sY in humans is currently limited to ∼50 proteins, with only a handful having verified sites of sulfation. As such, the contribution of sulfation to the regulation of biological processes remains poorly defined. Mass spectrometry (MS)-based proteomics is the method of choice for PTM analysis but has yet to be applied for systematic investigation of the "sulfome", primarily due to issues associated with discrimination of sY-containing from phosphotyrosine (pY)-containing peptides. In this study, we developed an MS-based workflow for sY-peptide characterization, incorporating optimized Zr4+ immobilized metal-ion affinity chromatography (IMAC) and TiO2 enrichment strategies. Extensive characterization of a panel of sY- and pY-peptides using an array of fragmentation regimes (CID, HCD, EThcD, ETciD, UVPD) highlighted differences in the generation of site-determining product ions and allowed us to develop a strategy for differentiating sulfated peptides from nominally isobaric phosphopeptides based on low collision energy-induced neutral loss. Application of our "sulfomics" workflow to a HEK-293 cell extracellular secretome facilitated identification of 21 new sulfotyrosine-containing proteins, several of which we validate enzymatically, and reveals new interplay between enzymes relevant to both protein and glycan sulfation.

Structure-based design of a phosphotyrosine-masked covalent ligand targeting the E3 ligase SOCS2.

The Src homology 2 (SH2) domain recognizes phosphotyrosine (pY) post translational modifications in partner proteins to trigger downstream signaling. Drug discovery efforts targeting the SH2 domains have long been stymied by the poor drug-like properties of phosphate and its mimetics. Here, we use structure-based design to target the SH2 domain of the E3 ligase suppressor of cytokine signaling 2 (SOCS2). Starting from the highly ligand-efficient pY amino acid, a fragment growing approach reveals covalent modification of Cys111 in a co-crystal structure, which we leverage to rationally design a cysteine-directed electrophilic covalent inhibitor MN551. We report the prodrug MN714 containing a pivaloyloxymethyl (POM) protecting group and evidence its cell permeability and capping group unmasking using cellular target engagement and in-cell 19F NMR spectroscopy. Covalent engagement at Cys111 competitively blocks recruitment of cellular SOCS2 protein to its native substrate. The qualified inhibitors of SOCS2 could find attractive applications as chemical probes to understand the biology of SOCS2 and its CRL5 complex, and as E3 ligase handles in proteolysis targeting chimera (PROTACs) to induce targeted protein degradation.

Unveiling Metastable Ensembles of GRB2 and the Relevance of Interdomain Communication during Folding.

The folding process of multidomain proteins is a highly intricate phenomenon involving the assembly of distinct domains into a functional three-dimensional structure. During this process, each domain may fold independently while interacting with others. The folding of multidomain proteins can be influenced by various factors, including their composition, the structure of each domain, or the presence of disordered regions, as well as the surrounding environment. Misfolding of multidomain proteins can lead to the formation of nonfunctional structures associated with a range of diseases, including cancers or neurodegenerative disorders. Understanding this process is an important step for many biophysical analyses such as stability, interaction, malfunctioning, and rational drug design. One such multidomain protein is growth factor receptor-bound protein 2 (GRB2), an adaptor protein that is essential in regulating cell survival. GRB2 consists of one central Src homology 2 (SH2) domain flanked by two Src homology 3 (SH3) domains. The SH2 domain interacts with phosphotyrosine regions in other proteins, while the SH3 domains recognize proline-rich regions on protein partners during cell signaling. Here, we combined computational and experimental techniques to investigate the folding process of GRB2. Through computational simulations, we sampled the conformational space and mapped the mechanisms involved by the free energy profiles, which may indicate possible intermediate states. From the molecular dynamics trajectories, we used the energy landscape visualization method (ELViM), which allowed us to visualize a three-dimensional (3D) representation of the overall energy surface. We identified two possible parallel folding routes that cannot be seen in a one-dimensional analysis, with one occurring more frequently during folding. Supporting these results, we used differential scanning calorimetry (DSC) and fluorescence spectroscopy techniques to confirm these intermediate states in vitro. Finally, we analyzed the deletion of domains to compare our model outputs to previously published results, supporting the presence of interdomain modulation. Overall, our study highlights the significance of interdomain communication within the GRB2 protein and its impact on the formation, stability, and structural plasticity of the protein, which are crucial for its interaction with other proteins in key signaling pathways.

Methods for Structure Determination of SH2 Domain-Phosphopeptide Complexes by NMR.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique to solve the structure of biomolecular complexes at atomic resolution in solution. Small proteins such as Src-homology 2 (SH2) domains have fast tumbling rates and long-lived NMR signals, making them particularly suited to be studied by standard NMR methods. SH2 domains are modular proteins whose function is the recognition of sequences containing phosphotyrosines. In this chapter, we describe the application of NMR to assess the interaction between SH2 domains and phosphopeptides and determine the structure of the resulting complexes.

Crystal Structure Analysis of SH2 Domains in Complex with Phosphotyrosine Peptides.

While the number of tertiary structures solved by cryoelectron microscopy has rapidly increased, X-ray crystallography is still a popular method to determine the tertiary structure of proteins at atomic resolution. However, there are still problems associated with X-ray crystallography, including crystallization and crystal twinning. Indeed, we encountered crystallization and twinning problems in the crystal structure analysis of the SH2 domains complexed with a phosphorylated peptide derived from the oncoprotein CagA. In this chapter, we describe the methods used to overcome these problems. In addition, we provide details of the optimization of the crystallization conditions and cryo-conditions, which are usually not given in published crystal structure analyses.

Revealing Allostery in PTPN11 SH2 Domains from MD Simulations.

Src-homology 2 (SH2) domains are protein interaction domains that bind to specific peptide motifs containing phosphotyrosine. SHP2, a tyrosine phosphatase encoded by PTPN11 gene, which has been emerged as positive or negative modulator in multiple signaling pathways, contains two SH2 domains, respectively, called N-SH2 and C-SH2. These domains play a relevant role in regulating SHP2 activity, either by recognizing its binding partners or by blocking its catalytic site. Considering the multiple functions that these domains carry out in SHP2, N-SH2 and C-SH2 represent an interesting case of study. In this chapter, we present a methodology that permits, by means of the principal component analysis (PCA), to study and to rationalize the structures adopted by the SH2 domains, in terms of the conformations of their binding sites. The structures can be distinguished, grouped, classified, and reported in a diagram. This approach permits to identify the accessible conformations of the SH2 domains in different binding conditions and to eventually reveal allosteric interactions. The method further reveals that the conformation dynamics of N-SH2 and C-SH2 strongly differ, which likely reflects their distinct functional roles.

Structure Determination of SH2-Phosphopeptide Complexes by X-Ray Crystallography: The Example of p120RasGAP.

The p120RasGAP protein contains two Src homology 2 (SH2) domains, each with phosphotyrosine-binding activity. We describe the crystallization of the isolated and purified p120RasGAP SH2 domains with phosphopeptides derived from a binding partner protein, p190RhoGAP. Purified recombinant SH2 domain protein is mixed with synthetic phosphopeptide at a stoichiometric ratio to form the complex in vitro. Crystallization is then achieved by the hanging drop vapor diffusion method over specific reservoir solutions that yield single macromolecular co-crystals containing SH2 domain protein and phosphopeptide. This protocol yields suitable crystals for X-ray diffraction studies, and our recent X-ray crystallography studies of the two SH2 domains of p120RasGAP demonstrate that the N-terminal SH2 domain binds phosphopeptide in a canonical interaction. In contrast, the C-terminal SH2 domain binds phosphopeptide via a unique atypical binding mode. The crystallographic studies for p120RasGAP illustrate that although the three-dimensional structure of SH2 domains and the molecular details of their binding to phosphotyrosine peptides are well defined, careful structural analysis can continue to yield new molecular-level insights.

Using Linear Motif Database Resources to Identify SH2 Domain Binders.

The SH2-binding phosphotyrosine class of short linear motifs (SLiMs) are key conditional regulatory elements, particularly in signaling protein complexes beneath the cell's plasma membrane. In addition to transmitting cellular signaling information, they can also play roles in cellular hijack by invasive pathogens. Researchers can take advantage of bioinformatics tools and resources to predict the motifs at conserved phosphotyrosine residues in regions of intrinsically disordered protein. A candidate SH2-binding motif can be established and assigned to one or more of the SH2 domain subgroups. It is, however, not so straightforward to predict which SH2 domains are capable of binding the given candidate. This is largely due to the cooperative nature of the binding amino acids which enables poorer binding residues to be tolerated when the other residues are optimal. High-throughput peptide arrays are powerful tools used to derive SH2 domain-binding specificity, but they are unable to capture these cooperative effects and also suffer from other shortcomings. Tissue and cell type expression can help to restrict the list of available interactors: for example, some well-studied SH2 domain proteins are only present in the immune cell lineages. In this article, we provide a table of motif patterns and four bioinformatics strategies that introduce a range of tools that can be used in motif hunting in cellular and pathogen proteins. Experimental followup is essential to determine which SH2 domain/motif-containing proteins are the actual functional partners.

Exploring the Binding Interaction Between Phosphotyrosine Peptides and SH2 Domains by Proximal Crosslinking.

Proximal crosslinking refers to the site-specific conjugation reaction between a synthetic ligand with a bioorthogonal reactive group incorporated at a particular site and a protein of interest (POI). The binding interaction positions a reactive group of a native amino acid of the POI to the proximity of the reactive group in the ligand. The covalent conjugation increases the molecular weight of the POI, shows an upshift in the polyacrylamide gel, and gives a fluorescent band if the ligand is fluorescently labeled. Here, we summarize a method to covalently conjugate phosphotyrosine peptides and SH2 domains that contain cysteine residues. This method yields covalent peptide blockers for a set of SH2 proteins and elucidates the binding interaction between phosphotyrosine peptides and SH2 domains.

Synthesis and Biochemical Evaluation of Monocarboxylic GRB2 SH2 Domain Inhibitors.

This protocol discloses the synthesis of monocarboxylic inhibitors with a macrocyclic peptide scaffold to bind with the GRB2 SH2 domain and disrupt the protein-protein interactions (PPIs) between GRB2 and phosphotyrosine-containing proteins.

Lipid Binding of SH2 Domains.

The Src homology 2 (SH2) domain is a modular protein interaction domain that specifically recognizes the phosphotyrosine (pY) motif of a target molecule. We recently reported that a large majority of human SH2 domains tightly bind membrane lipids, and many show high lipid specificity. Most of them can bind a lipid and the pY motif coincidently because their lipid-binding sites are topologically distinct from pY-binding pockets. Lipid binding of SH2 domain-containing kinases and phosphatases is functionally important because it exerts exquisite spatiotemporal control on protein-protein interaction and cell signaling activities mediated by these proteins. Here, we describe two assays, surface plasmon resonance analysis and fluorescence quenching analysis, which allow quantitative determination of the affinity and specificity of SH2-lipid interaction and high-throughput screening for SH2 domain-lipid-binding inhibitors.

Measuring Protein Tyrosine Phosphatase Activity Dependent on SH2 Domain-Mediated Regulation.

Src-homology-2 (SH2) domains bind selectively to phosphotyrosine (pTyr) residues located in target binding proteins; therefore, they are key elements in pTyr-mediated signaling pathways. The binding of an SH2 domain to a pTyr acts as a docking mechanism that attracts proteins into signaling hubs, and in some cases, it can also regulate the catalytic activity of signaling enzymes such as protein kinases or protein phosphatases. Therefore, compounds that selectively bind SH2 domains can be potentially used to modulate the activity of such SH2 domain-containing enzymes. This chapter describes how to measure the regulation of protein tyrosine phosphatase activity through allosteric binding of peptides to SH2 domains, and uses human recombinant protein tyrosine phosphatase SHP2 (Src homology-2 domain-containing protein tyrosine phosphatase 2) purified from bacteria as a case example. The phosphatase activity against the artificial substrate DiFMUP (6, 8-Difluoro-4-Methylumbelliferyl Phosphate) is measured over time in the presence of a peptide that selectively binds and activates SHP2 at different concentrations to determine the half maximal effective concentration (EC50).

Peptides as Baits for the Coprecipitation of SH2 Domain-Containing Proteins.

Phosphotyrosine (pTyr)-containing amino acid sequences have regulatory effects on proteins that contain pTyr recognition motifs, such as Src Homology 2 (SH2) domains. Using pTyr-containing peptides as a bait for coprecipitation, by immobilization of the synthesized phosphopeptides to beads and incubation with cell lysates, enables to study the binding preference of the SH2 domain for the specific pTyr-sequence obtained from a pTyr-containing protein in a complex biological environment. Using phosphopeptides allows to not only assess the wild-type sequence, but also peptides that can contain modified sequences which carry a nonhydrolyzable pTyr or other modifications varying the binding strength and selectivity, for example, to create strong SH2 domain binders to inhibit their interaction with pTyr-containing proteins. This pulldown experiment can be used as an assay to evaluate the ability of a peptide to bind to the protein of interest in the cell lysate or investigate the selectivity of the peptide. Therefore, immobilizing phosphopeptides and using them as a pulldown tool has a wide range of applications.

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.

Phosphotyrosine proteomics in cells synchronized at monopolar cytokinesis reveals EphA2 as functioning in cytokinesis.

Cytokinesis is the final step of the cell division in which cellular components are separated into two daughter cells. This process is regulated through the phosphorylation of different classes of proteins by serine/threonine (Ser/Thr) kinases such as Aurora B and Polo-like kinase 1 (PLK1). Conversely, the role of phosphorylation at tyrosine residues during cytokinesis has not been studied in detail yet. In this study, we performed a phosphotyrosine proteomic analysis of cells undergoing monopolar cytokinesis synchronized by using the Eg5 inhibitor (+)-S-trityl-l-cysteine (STLC) and the CDK1 inhibitor RO-3306. Phosphotyrosine proteomics gave 362 tyrosine-phosphorylated peptides. Western blot analysis of proteins revealed tyrosine phosphorylation in mitogen-activated protein kinase 14 (MAPK14), vimentin, ephrin type-A receptor 2 (EphA2), and myelin protein zero-like protein 1 (MPZL1) during monopolar cytokinesis. Additionally, we demonstrated that EphA2, a protein with unknown function during cytokinesis, is involved in cytokinesis. EphA2 knockdown accelerated epithelial cell transforming 2 (Ect2) knockdown-induced multinucleation, suggesting that EphA2 plays a role in cytokinesis in a particular situation. The list also included many proteins previously reported to play roles during cytokinesis. These results evidence that the identified phosphopeptides facilitate the identification of novel tyrosine phosphorylation signaling involved in regulating cytokinesis.

Setting sail: Maneuvering SHP2 activity and its effects in cancer.

Since the discovery of tyrosine phosphorylation being a critical modulator of cancer signaling, proteins regulating phosphotyrosine levels in cells have fast become targets of therapeutic intervention. The nonreceptor protein tyrosine phosphatase (PTP) coded by the PTPN11 gene "SHP2" integrates phosphotyrosine signaling from growth factor receptors into the RAS/RAF/ERK pathway and is centrally positioned in processes regulating cell development and oncogenic transformation. Dysregulation of SHP2 expression or activity is linked to tumorigenesis and developmental defects. Even as a compelling anti-cancer target, SHP2 was considered "undruggable" for a long time owing to its conserved catalytic PTP domain that evaded drug development. Recently, SHP2 has risen from the "undruggable curse" with the discovery of small molecules that manipulate its intrinsic allostery for effective inhibition. SHP2's unique domain arrangement and conformation(s) allow for a truly novel paradigm of inhibitor development relying on skillful targeting of noncatalytic sites on proteins. In this review we summarize the biological functions, signaling properties, structural attributes, allostery and inhibitors of SHP2.

Characterization of the folding and binding properties of the PTB domain of FRS2 with phosphorylated and unphosphorylated ligands.

PTB (PhosphoTyrosine Binding) domains are protein domains that exert their function by binding phosphotyrosine residues on other proteins. They are commonly found in a variety of signaling proteins and are important for mediating protein-protein interactions in numerous cellular processes. PTB domains can also exhibit binding to unphosphorylated ligands, suggesting that they have additional binding specificities beyond phosphotyrosine recognition. Structural studies have reported that the PTB domain from FRS2 possesses this peculiar feature, allowing it to interact with both phosphorylated and unphosphorylated ligands, such as TrkB and FGFR1, through different topologies and orientations. In an effort to elucidate the dynamic and functional properties of these protein-protein interactions, we provide a complete characterization of the folding mechanism of the PTB domain of FRS2 and the binding process to peptides mimicking specific regions of TrkB and FGFR1. By analyzing the equilibrium and kinetics of PTB folding, we propose a mechanism implying the presence of an intermediate along the folding pathway. Kinetic binding experiments performed at different ionic strengths highlighted the electrostatic nature of the interaction with both peptides. The specific role of single amino acids in early and late events of binding was pinpointed by site-directed mutagenesis. These results are discussed in light of previous experimental works on these protein systems.

Combinatorial expression of neurexins and LAR-type phosphotyrosine phosphatase receptors instructs assembly of a cerebellar circuit.

Synaptic adhesion molecules (SAMs) shape the structural and functional properties of synapses and thereby control the information processing power of neural circuits. SAMs are broadly expressed in the brain, suggesting that they may instruct synapse formation and specification via a combinatorial logic. Here, we generate sextuple conditional knockout mice targeting all members of the two major families of presynaptic SAMs, Neurexins and leukocyte common antigen-related-type receptor phospho-tyrosine phosphatases (LAR-PTPRs), which together account for the majority of known trans-synaptic complexes. Using synapses formed by cerebellar Purkinje cells onto deep cerebellar nuclei as a model system, we confirm that Neurexins and LAR-PTPRs themselves are not essential for synapse assembly. The combinatorial deletion of both neurexins and LAR-PTPRs, however, decreases Purkinje-cell synapses on deep cerebellar nuclei, the major output pathway of cerebellar circuits. Consistent with this finding, combined but not separate deletions of neurexins and LAR-PTPRs impair motor behaviors. Thus, Neurexins and LAR-PTPRs are together required for the assembly of a functional cerebellar circuit.

Identification of SH2 domain-containing proteins and motifs prediction by a deep learning method.

The Src Homology 2 (SH2) domain plays an important role in the signal transmission mechanism in organisms. It mediates the protein-protein interactions based on the combination between phosphotyrosine and motifs in SH2 domain. In this study, we designed a method to identify SH2 domain-containing proteins and non-SH2 domain-containing proteins through deep learning technology. Firstly, we collected SH2 and non-SH2 domain-containing protein sequences including multiple species. We built six deep learning models through DeepBIO after data preprocessing and compared their performance. Secondly, we selected the model with the strongest comprehensive ability to conduct training and test separately again, and analyze the results visually. It was found that 288-dimensional (288D) feature could effectively identify two types of proteins. Finally, motifs analysis discovered the specific motif YKIR and revealed its function in signal transduction. In summary, we successfully identified SH2 domain and non-SH2 domain proteins through deep learning method, and obtained 288D features that perform best. In addition, we found a new motif YKIR in SH2 domain, and analyzed its function which helps to further understand the signaling mechanisms within the organism.