pTyr-containing Peptides Research Articles

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.

Tandem Mass Tag Approach Utilizing Pervanadate BOOST Channels Delivers Deeper Quantitative Characterization of the Tyrosine Phosphoproteome

Dynamic tyrosine phosphorylation is fundamental to a myriad of cellular processes. However, the inherently low abundance of tyrosine phosphorylation in the proteome and the inefficient enrichment of phosphotyrosine(pTyr)-containing peptides has led to poor pTyr peptide identification and quantitation, critically hindering researchers' ability to elucidate signaling pathways regulated by tyrosine phosphorylation in systems where cellular material is limited. The most popular approaches to wide-scale characterization of the tyrosine phosphoproteome use pTyr enrichment with pan-specific, anti-pTyr antibodies from a large amount of starting material. Methods that decrease the amount of starting material and increase the characterization depth of the tyrosine phosphoproteome while maintaining quantitative accuracy and precision would enable the discovery of tyrosine phosphorylation networks in rarer cell populations. To achieve these goals, the BOOST (Broad-spectrum Optimization Of Selective Triggering) method leveraging the multiplexing capability of tandem mass tags (TMT) and the use of pervanadate (PV) boost channels (cells treated with the broad-spectrum tyrosine phosphatase inhibitor PV) selectively increased the relative abundance of pTyr-containing peptides. After PV boost channels facilitated selective fragmentation of pTyr-containing peptides, TMT reporter ions delivered accurate quantitation of each peptide for the experimental samples while the quantitation from PV boost channels was ignored. This method yielded up to 6.3-fold boost in pTyr quantification depth of statistically significant data derived from contrived ratios, compared with TMT without PV boost channels or intensity-based label-free (LF) quantitation while maintaining quantitative accuracy and precision, allowing quantitation of over 2300 unique pTyr peptides from only 1 mg of T cell receptor-stimulated Jurkat T cells. The BOOST strategy can potentially be applied in analyses of other post-translational modifications where treatments that broadly elevate the levels of those modifications across the proteome are available.

Antibody-based fluorescent and fluorescent ratiometric indicators for detection of phosphotyrosine

Fluorescent indicators for protein phosphorylation are very important in not only fundamental biology but also biomedical applications. In this study, we developed novel fluorescent and fluorescent ratiometric indicators for detection of phosphotyrosine (pTyr) derivatives. A single-chain antibody variable fragment (scFv) against phosphotyrosine was fluorescent-labeled by incorporation of tetramethylrhodamine (TAMRA)-linked nonnatural amino acid at the N- or C-terminus. The TAMRA-labeled scFv showed fluorescence enhancement upon addition of pTyr-containing peptides based on antigen-dependent fluorescence quenching effect on TAMRA. The TAMRA-labeled scFv was further fused with enhanced green fluorescent protein (EGFP) to generate a double-labeled scFv for pTyr. In the absence of antigen, fluorescence resonance energy transfer (FRET) occurred from EGFP to TAMRA but TAMRA was quenched. The antigen-binding removed the quenching of TAMRA while FRET occurred without altering its efficiency. As a result of the FRET and antigen-dependent fluorescence quenching effect, the double-labeled scFv exhibited fluorescence ratio enhancement upon the antigen-binding. The fluorescent and fluorescent ratiometric indicators obtained in this study will become a novel tool for analysis of protein phosphorylation. Moreover, this strategy utilizes antibody derivatives, and therefore, can be easily applied to other antigen-antibody pairs to generate fluorescent ratiometric indicators for various target molecules.

Allosteric Communication across STAT3 Domains Associated with STAT3 Function and Disease-Causing Mutation

STAT3 is a member of STAT (signal transducer and activator of transcription) transcription activators. Aberration in STAT3 activity due to constitutive activation or mutations leads to diseases such as cancer and hyper-immunoglobulin E syndrome (HIES). STAT3 contains several structured domains including the Src homology 2 domain (SH2), linker domain (LD), DNA-binding domain (DBD) and the coiled-coil domain. Here we report the discovery of inter-domain allosteric communications in STAT3 from studies using nuclear magnetic resonance (NMR) and other methods. We found that pTyr-peptide interactions with SH2 cause structural and dynamics changes in LD and DBD. The inter-domain allosteric effect is likely mediated by the flexibility in the hydrophobic core. In addition, a mutation in LD found in HIES (I568F) induces NMR chemical shift perturbation in SH2, DBD and the coiled-coil domain, suggesting conformational changes in these domains. Consistent with conformational changes in SH2, the I568F mutant reduces SH2's binding affinity to a pTyr-containing peptide. This study provides an example of dynamics-dependent allosteric effects, and due to the structural conservation of the STAT family of proteins, the inter-domain allosteric communication observed in STAT3 likely occurs in other STATs.

Phosphopeptide immuno-affinity enrichment to enhance detection of tyrosine phosphorylation in plants.

Tyrosine (Tyr) phosphorylation plays an essential role in signaling in animal systems, but the relative contribution of Tyr phosphorylation to plant signal transduction has, until recently, remained an open question. One of the major issues hampering the analysis is the low abundance of Tyr phosphorylation and therefore underrepresentation in most mass spec-based proteomic studies. Here, we describe a working approach to selectively enrich Tyr-phosphorylated peptides from complex plant tissue samples. We describe a detailed protocol that is based on immuno-affinity enrichment step using an anti-phospho-tyrosine (pTyr)-specific antibody. This single enrichment strategy effectively enriches pTyr-containing peptides from complex total plant cell extracts, which can be measured by LC-MS/MS without further fractionation or enrichment.

Ultratrace Enrichment of Tyrosine Phosphorylated Peptides on an Imprinted Polymer

Novel molecularly imprinted polymers (MIPs) designed to bind the side chain of phosphotyrosine can be used as artificial receptors for affinity-based enrichment of proteolytic peptides. In comparison with general enrichment methods for phosphorylated peptides such as TiO(2)-based methods, the pTyr-imprinted polymers offered high selectivity for pTyr-containing peptides down to the low fmol level. This suggests MIPs as a new tool for affinity-based proteomics.

Structural and Functional Characterization of the C-Terminal Domain of the Ecdysteroid Phosphate Phosphatase from Bombyx mori Reveals a New Enzymatic Activity

Here, we present the crystal structure of the ecdysone phosphate phosphatase (EPPase) phosphoglycerate mutase (PGM) homology domain, the first structure of a steroid phosphate phosphatase. The structure reveals an alpha/beta-fold common to members of the two histidine (2H)-phosphatase superfamily with strong homology to the Suppressor of T-cell receptor signaling-1 (Sts-1 PGM) protein. The putative EPPase PGM active site contains signature residues shared by 2H-phosphatase enzymes, including a conserved histidine (His80) that acts as a nucleophile during catalysis. The physiological substrate ecdysone 22-phosphate was modeled in a hydrophobic cavity close to the phosphate-binding site. EPPase PGM shows limited substrate specificity with an ability to hydrolyze steroid phosphates, the phospho-tyrosine (pTyr) substrate analogue para-nitrophenylphosphate ( pNPP) and pTyr-containing peptides and proteins. Altogether, our data demonstrate a new protein tyrosine phosphatase (PTP) activity for EPPase. They suggest that EPPase and its closest homologues can be grouped into a distinct subfamily in the large 2H-phosphatase superfamily of proteins.

Quantitative large‐scale phosphotyrosine proteomics of vasopressin‐sensitive inner medullary collecting duct cells using IVICAT N‐terminal labeling and LC‐MS/MS

Large-scale identification of phosphotyrosyl (pTyr)-proteins by LC-MS methods remains challenging. We previously developed a novel isotopic labeling method, called IVICAT (In Vacuo Isotope-Coded Alkylation Technique) that improves the detection sensitivity of pTyr-containing peptides in positive ion ESI-MS by trimethylating the N-terminus of every peptide, thereby creating a fixed positive charge. Here we use pTyr immunoprecipitation, IVICAT labeling, IMAC, and LC-MS/MS to profile the pTyr proteome of inner medullary collecting duct (IMCD) cells isolated from rat kidneys. These experiments identified 114 pTyr sites, 103 of which have not been previously reported. Using H3- or D3-methylating reagents in IVICAT, we quantified pTyr changes in response to the V2 receptor selective analog dDAVP in IMCD cell suspensions. Use of our in house software (QUIL) for construction of pseudochromatograms from time-dependent LC-MS data resulted in the discovery of several sites modified by vasopressin including phospholipase C gamma-1 (PLCγ1) at Y771 (increase of approximately two-fold confirmed by immunoblotting). Differential centrifugation showed that dDAVP displaces PLCγ1 from membrane to cytosol, consistent with a net inhibitory action. The data indicate that vasopressin signaling includes changes in tyrosine phosphorylation that have potential roles in regulation of aquaporins or urea transporters.

Phosphotyrosine Proteomic Study of Interferon α Signaling Pathway Using a Combination of Immunoprecipitation and Immobilized Metal Affinity Chromatography

Tyrosine phosphorylation is a type of post-translational modification that plays a crucial role in signal transduction. Thus, the study of this modification at the proteomic level has great biological significance. However, because of the low abundance of tyrosine-phosphorylated proteins in total cell lysate, it is difficult to evaluate the dynamics of tyrosine phosphorylation at a global level. In this work, proteins carrying phosphotyrosine (pTyr) were first purified from whole cell lysate by immunoprecipitation using anti-pTyr monoclonal antibodies. After tryptic digestion, phosphopeptides were further enriched by IMAC and analyzed by LC-MS. Quantitative changes of tyrosine phosphorylation at the global level were evaluated using isotopic labeling (introduced at the methyl esterification step prior to IMAC). Using this double enrichment approach, we characterized interferon alpha (IFNalpha)-induced pTyr proteomic changes in Jurkat cells. We observed induced phosphorylation on several well documented as well as novel tyrosine phosphorylation sites on proteins involved in IFNalpha signal transduction, such as Tyk2, JAK1, and IFNAR subunits. A specific site on alpha-tubulin (Tyr-271) was observed to be phosphorylated upon treatment as well. Furthermore, our results suggest that LOC257106, a CDC42 GAP-like protein, is potentially involved in this pathway.

SRC homology-2 inhibitors: peptidomimetic and nonpeptide.

The structural and functional characterization of Src homology-2 (SH2) domains and their relationship to catalytic proteins (e.g., kinases, phosphatases, and lipases) or non-catalytic proteins (e.g., upstream adapters, and downstream transcription factors) has significantly impacted our understanding of signal transduction pathways and the identification of promising therapeutic targets for drug discovery. Such SH2-containing proteins are known to be intimately involved in the regulation of a number of cellular processes, including growth, mitogenesis, motility, metabolism, and gene transcription. Molecular recognition and biochemical selectivity exists for various SH2 domains based on their binding to phosphotyrosine (pTyr) and contiguous C-terminal amino acids of cognate protein 'partners' in a sequence-dependent manner (i.e., -pTyr-AA(1)-AA(2)-AA(3)-) which result in the formation of signal transduction protein complexes in cells. In recent years, drug discovery efforts have advanced peptidomimetic and nonpeptide inhibitors of such protein-protein interactions based on mimicking pTyr-containing peptide ligands as well as SH2 structure-based de novo design of nonpeptide templates that can capture key binding sites on the target protein. Noteworthy are peptidomimetic and nonpeptide inhibitors of Src, Lck, Grb2, PI-3K, and Zap70 from pioneering efforts that led to the first examples of cellularly and in vivo active SH2 inhibitors. This mini-review highlights key achievements in SH2 inhibitor drug discovery with an emphasis on peptidomimetic and nonpeptide lead compounds in terms of structure-based design, key chemical and biological properties, and proof-of-concept studies relative to further defining the role(s) of SH2 domains in signal transduction processes, cellular functions, and in vivo disease models.

Molecular Basis for the Dephosphorylation of the Activation Segment of the Insulin Receptor by Protein Tyrosine Phosphatase 1B

The protein tyrosine phosphatase PTP1B is responsible for negatively regulating insulin signaling by dephosphorylating the phosphotyrosine residues of the insulin receptor kinase (IRK) activation segment. Here, by integrating crystallographic, kinetic, and PTP1B peptide binding studies, we define the molecular specificity of this reaction. Extensive interactions are formed between PTP1B and the IRK sequence encompassing the tandem pTyr residues at 1162 and 1163 such that pTyr-1162 is selected at the catalytic site and pTyr-1163 is located within an adjacent pTyr recognition site. This selectivity is attributed to the 70-fold greater affinity for tandem pTyr-containing peptides relative to mono-pTyr peptides and predicts a hierarchical dephosphorylation process. Many elements of the PTP1B–IRK interaction are unique to PTP1B, indicating that it may be feasible to generate specific, small molecule inhibitors of this interaction to treat diabetes and obesity.

II. Structure and specificity of the interaction between the FHA2 domain of rad53 and phosphotyrosyl peptides †,1

The forkhead-associated (FHA) domain is a protein module found in many proteins involved in cell signaling in response to DNA damage. It has been suggested to bind to pThr sites of its target protein. Recently we have determined the first structure of an FHA domain, FHA2 from the yeast protein Rad53, and demonstrated that FHA2 binds to a pTyr-containing peptide 826EDI(pY)YLD 832 from Rad9, with a moderate affinity ( K d ca. 100 μM). We now report the solution structure of the complex of FHA2 bound with this pTyr peptide. The structure shows that the phosphate group of pTyr interacts directly with three arginine residues (605, 617, and 620), and that the leucine residue at the +2 position from the pTyr interacts with a hydrophobic surface on FHA2. The sequence specificity of FHA2 was determined by screening a combinatorial pTyr library. The results clearly show that FHA2 recognizes specific sequences C-terminal to pTyr with the following consensus: XX(pY)N 1N 2N 3, where N 1 = Leu, Met, Phe, or Ile, N 2 = Tyr, Phe, Leu, or Met, and N 3 = Phe, Leu, or Met. Two of the selected peptides, GF(pY)LYFIR and DV(pY)FYMIR, bind FHA2 with K d values of 1.1 and 5.0 μM, respectively. The results, along with other recent reports, demonstrate that the FHA domain is a new class of phosphoprotein-binding domain, capable of binding both pTyr and pThr sequences.

Structure and function of a new phosphopeptide-binding domain containing the FHA2 of rad53

The forkhead-associated (FHA) domain is a 55-75 amino acid residue module found in >20 proteins from yeast to human. It has been suggested to participate in signal transduction pathways, perhaps via protein-protein interactions involving recognition of phosphopeptides. Neither the structure nor the ligand of FHA is known. Yeast Rad53, a checkpoint protein involved in DNA damage response, contains two FHA domains, FHA1 (residues 66-116) and FHA2 (residues 601-664), the second of which recognizes phosphorylated Rad9. We herein report the solution structure of an “FHA2-containing domain” of Rad53 (residues 573-730). The structure consists of a β-sandwich containing two antiparallel β-sheets and a short, C-terminal α-helix. Binding experiments suggested that the FHA2-containing domain specifically recognizes pTyr and a pTyr-containing peptide from Rad9, and that the binding site involves residues highly conserved across FHA domains. The results, along with other recent reports, suggest that FHA domains could have pTyr and pSer/Thr dual specificity.

Nonphosphorylated Peptide Ligands for the Grb2 Src Homology 2 Domain

Critical intracellular signals in normal and malignant cells are transmitted by the adaptor protein Grb2 by means of its Src homology 2 (SH2) domain, which binds to phosphotyrosyl (pTyr) residues generated by the activation of tyrosine kinases. To understand this important control point and to design inhibitors, previous investigations have focused on the molecular mechanisms by which the Grb2 SH2 domain selectively binds pTyr containing peptides. In the current study, we demonstrate that the Grb2 SH2 domain can also bind in a pTyr independent manner. Using phage display, an 11-amino acid cyclic peptide, G1, has been identified that binds to the Grb2 SH2 domain but not the src SH2 domain. Synthetic G1 peptide blocks Grb2 SH2 domain association (IC50 10-25 microM) with a 9-amino acid pTyr-containing peptide derived from the SHC protein (pTyr317). These data and amino acid substitution analysis indicate that G1 interacts in the phosphopeptide binding site. G1 peptide requires a YXN sequence similar to that found in natural pTyr-containing ligands, and phosphorylation of the tyrosine increases G1 inhibitory activity. G1 also requires an internal disulfide bond to maintain the active binding conformation. Since the G1 peptide does not contain pTyr, it defines a new type of SH2 domain binding motif that may advance the design of Grb2 antagonists.

High-affinity binding of the Drosophila Numb phosphotyrosine-binding domain to peptides containing a Gly-Pro-(p)Tyr motif.

The phosphotyrosine-binding (PTB) domain is a recently identified protein module that has been characterized as binding to phosphopeptides containing an NPXpY motif (X = any amino acid). We describe here a novel peptide sequence recognized by the PTB domain from Drosophila Numb (dNumb), a protein involved in cell fate determination and asymmetric cell division during the development of the Drosophila nervous system. Using a Tyr-oriented peptide library to screen for ligands, the dNumb PTB domain was found to bind selectively to peptides containing a YIGPYphi motif (phi represents a hydrophobic residue). A synthetic peptide containing this sequence bound specifically to the isolated dNumb PTB domain in solution with a dissociation constant (Kd) of 5.78 +/- 0.74 microM. Interestingly, the affinity of this peptide for the dNumb PTB domain was increased (Kd = 1.41 +/- 0.10 microM) when the second tyrosine in the sequence was phosphorylated. Amino acid substitution studies of the phosphopeptide demonstrated that a core motif of sequence GP(p)Y is required for high-affinity binding to the dNumb PTB domain. Nuclear magnetic resonance experiments performed on isotopically labeled protein complexed with either Tyr- or pTyr-containing peptides suggest that the same set of amino acids in the dNumb PTB domain is involved in binding both phosphorylated and nonphosphorylated forms of the peptide. The in vitro selectivity of the dNumb PTB domain is therefore markedly different from those of the Shc and IRS-1 PTB domains, in that it interacts preferentially with a GP(p)Y motif, rather than NPXpY, and does not absolutely require ligand phosphorylation for binding. Our results suggest that the PTB domain is a versatile protein module, capable of exhibiting varied binding specificities.

Conformational analysis of cyclic hexapeptides designed as constrained ligands for the SH2 domain of the p85 subunit of phosphatidylinositol-3-OH kinase.

The structures of the cyclic hexapeptide cyclo(-Gly-Tyr-Val-Pro-Met-Leu-) (1) and its phosphotyrosyl (pTyr) derivative cyclo[-Gly-Tyr(PO3H2)-Val-Pro-Met-Leu-] (2), designed as constrained models of a sequence that interacts with the src homology 2 (SH2) region of the p85 subunit of phosphatidylinositol-3-OH kinase (PI-3 kinase), were studied in methanol/water solutions by 500 MHz nmr spectroscopy. Compound 1 was found to exist as a 2:1 mixture of isomers about the Val-Pro bond (trans and cis prolyl) between 292-330 K in 75% CD3O(D,H)/(D,H)2O solutions. A third species of undetermined structure (ca. 5%) was also observed. Compound 2, a model of phosphorylated peptide ligand that binds to the PI-3 kinase SH2 domain, exhibited similar conformational isomerism. When either compound was dissolved in pure solvent [i.e., 100% CD3O(H,D) or (H,D)2O] the ratio of cis to trans isomers was ca 1:1. A battery of one- and two-dimensional nmr experiments at different temperatures and solvent compositions allowed a complete assignment of both the cis and trans forms of 1 and indicated the trans compound to be the major isomer. The spectral properties of the phophorylated derivative 2 paralleled those of 1, indicating like conformations for the two compounds. Analysis of rotating frame Overhauser spectroscopy data, coupling constants, amide proton temperature dependence, and amide proton exchange rates generated a set of constraints that were employed in energy minimization and molecular dynamics calculations using the CHARMM force field. The trans isomer exists with the tyrosine and C-terminal Tyr(+3) (Met) residues at opposite corners of the 18-membered ring separated by a distance of 16-18 A, in contrast with the cis isomer where the side chains of these residues are much closer in space (7-14 A). It was previously shown that the pTyr and the third amino acid C-terminal to this residue are the critical recognition elements for pTyr-peptide binding to the PI-3 kinase SH2 domain. Such cyclic structures may offer appropriate scaffolding for positioning important amino acid side chains of pTyr-containing peptides as a means of increasing their binding affinities to SH2 domains, and in turn provide a conceptual approach toward the design of SH2 domain directed peptidomimetics.

Correlation between dynamics and high affinity binding in an SH2 domain interaction.

Protein-protein interfaces can consist of interactions between large numbers of residues of each molecule; some of these interactions are critical in determining binding affinity and conferring specificity, while others appear to play only a marginal role. Src-homology-2 (SH2) domains bind to proteins containing phosphorylated tyrosines, with additional specificity provided by interactions with residues C-terminal to the phosphotyrosine (pTyr) residue. While the C-terminal SH2 domain of phospholipase C-gamma 1 (PLCC SH2) interacts with eight residues of a pTyr-containing peptide from its high affinity binding site on the beta-platelet-derived growth factor receptor, it can still bind tightly to a phosphopeptide containing only three residues. Novel deuterium (2H) based nuclear magnetic resonance (NMR) spin relaxation experiments which probe the nanosecond-picosecond time scale dynamics of methyl containing side chain residues have established that certain regions of the PLCC SH2 domain contacting the residues C-terminal to the pTyr have a high degree of mobility in both the free and peptide complexed states. In contrast, there is significant restriction of motion in the pTyr binding site. These results suggest a correlation between the dynamic behavior of certain groups in the PLCC SH2 complex and their contribution to high affinity binding and binding specificity.

NMR analysis of interactions of a phosphatidylinositol 3'-kinase SH2 domain with phosphotyrosine peptides reveals interdependence of major binding sites.

The interactions of the N-terminal src homology (SH2) domain (N-SH2) of the 85 kDa subunit of phosphatidylinositol 3'-kinase (PI-3K) with phosphotyrosine (ptyr) and a series of ptyr-containing peptides have been examined by NMR spectroscopy. HSQC (heteronuclear single-quantum coherence) NMR spectra of 15N-labeled SH2 were used to evaluate its interactions with ptyr-containing ligands. The ability of ligands to cause chemical shift changes was compared to their potency as competitors in in vitro binding experiments using polyoma virus middle T antigen (MT). The results suggest the interdependence of SH2 binding elements. Chemical shifts of residues involved in the ptyr binding were altered by variations of the sequence of the bound peptide, suggesting that the ptyr fit can be adjusted by the peptide sequence. Perturbations of chemical shifts of residues coordinating the methionine three residues C-terminal to the ptyr (the +3 residue) were affected by substitution in the binding peptide at +1 and vice versa. Such results show synergistic interplay between regions of the SH2 binding residues C-terminal to the ptyr.

L-O-(2-malonyl)tyrosine: a new phosphotyrosyl mimetic for the preparation of Src homology 2 domain inhibitory peptides.

Inhibition of Src homology 2 (SH2) domain-binding interactions affords one potential means of modulating protein-tyrosine kinase-dependent signaling. Small phosphotyrosyl (pTyr)-containing peptides are able to bind to SH2 domains and compete with larger pTyr peptides or native pTyr-containing protein ligands. Such pTyr-containing peptides are limited in their utility as SH2 domain inhibitors in vivo due to their hydrolytic lability to protein-tyrosine phosphatases (PTPs) and the poor cellular penetration of the ionized phosphate moiety. An important aspect of SH2 domain inhibitor design is the creation of pTyr mimetics which are stable to PTPs and have reasonable bioavailability. To date, most PTP-resistant pTyr mimetics which bind to SH2 domains are phosphonates such as (phosphonomethyl)phenylalanine (Pmp, 2), [(monofluorophosphono)methyl]phenylalanine (FPmp, 3) or [(difluorophosphono)methyl]-phenylalanine (F2Pmp, 4). Herein we report the incorporation of a new non-phosphorus-containing pTyr mimetic, L-O-(2-malonyl)tyrosine (L-OMT, 5), into SH2 domain inhibitory peptides using the protected analogue L-N alpha-Fmoc-O'-(O",O"-di-tert-butyl-2-malonyl)tyrosine (6) and solid-phase peptide synthesis techniques. Five OMT-containing peptides were prepared against the following SH2 domains: the PI-3 kinase C-terminal p85 SH2 domain (Ac-D-(L-OMT)-V-P-M-L-amide, 10, IC50 = 14.2 microM), the Src SH2 domain (Ac-Q-(L-OMT)-E-E-I-P-amide, 11, IC50 = 25 microM, and Ac-Q-(L-OMT)-(L-OMT)-E-I-P-amide, 14, IC50 = 23 microM), the Grb2 SH2 domain (Ac-N-(L-OMT)-V-N-I-E-amide, 12, IC50 = 120 microM), and the N-terminal SH-PTP2 SH2 domain (Ac-L-N-(L-OMT)-I-D-L-D-L-V-amide, 13, IC50 = 22.0 microM). These results show that peptides 10, 11, 13, and 14 have reasonable affinity for their respective SH2 domains, with the IC50 value for the SH-PTP2 SH2 domain-directed peptide 13 being equivalent to that previously observed for the corresponding F2Pmp-containing peptide. OMT may afford a new structural starting point for the development of novel and useful SH2 domain inhibitors.