Crystal structure of human protein tyrosine phosphatase SHP-1 in the open conformation

SHP‐1 belongs to the family of non‐receptor protein tyrosine phosphatases (PTPs) and generally acts as a negative regulator in a variety of cellular signaling pathways. Previously, the crystal structures of the tail‐truncated SHP‐1 and SHP‐2 revealed an autoinhibitory conformation. To understand the regulatory mechanism of SHP‐1, we have determined the crystal structure of the full‐length SHP‐1 at 3.1 Å. Although the tail was disordered in current structure, the huge conformational rearrangement of the N‐SH2 domain and the incorporation of sulfate ions into the ligand‐binding site of each domain indicate that the SHP‐1 is in the open conformation. The N‐SH2 domain in current structure is shifted away from the active site of the PTP domain to the other side of the C‐SH2 domain, resulting in exposure of the active site. Meanwhile, the C‐SH2 domain is twisted anticlockwise by about 110°. In addition, a set of new interactions between two SH2 domains and between the N‐SH2 and the catalytic domains is identified, which could be responsible for the stabilization of SHP‐1 in the open conformation. Based on the structural comparison, a model for the activation of SHP‐1 is proposed. J. Cell. Biochem. 112: 2062–2071, 2011. © 2011 Wiley‐Liss, Inc.     

Syntheses and activities of backbone‐side chain cyclic octapeptide ligands with N‐functionalized phosphotyrosine for the N‐terminal SH2‐domain of the protein tyrosine phosphatase SHP‐1

The protein tyrosine phosphatase SHP‐1 plays an important role in many physiological and pathophysiological processes. This phosphatase is activated through binding of ligands to its SH2‐domains, mainly to the N‐terminal one. Based on a theoretical docking model, backbone‐to‐side chain cyclized octapeptides were designed as ligands. Assembly of such modelled structures required the synthesis of N‐functionalized tyrosine derivatives and their incorporation into the sequence. Because of difficulties encountered in the condensation of N‐protected amino acids to the N‐alkylated tyrosine‐peptide we synthesized and used preformed dipeptide building units. As all attempts to obtain phosphorylated dipeptide units failed, the syntheses had to be performed with a free phenolic function. Use of different N‐alkyl or cycloalkyl residues in the N‐functionalized side chains allowed to investigate the effect of ring size, flexibility and hydrophobicity of formed lactam bridges on stimulatory activity. All tested linear and cyclic octapeptides stimulate the phosphatase activity of SHP‐1. Stimulatory activities of cyclic ligands increase with the chain length of the lactam bridges resulting in increased flexibility and better entropic preformation of the binding conformation. The strong activity of some cyclic octapeptides supports the modelled structure. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Molecular cloning and characterization of a tyrosine phosphatase from Monosiga brevicollis

Protein tyrosine phosphorylation is thought to be a unique feature of multicellular animals. Interestingly, the genome of the unicellular protist Monosiga brevicollis reveals a surprisingly high number and diversity of protein tyrosine kinases, protein tyrosine phosphatases (PTPs), and phosphotyrosine-binding domains. Our study focuses on a hypothetical SH2 domain-containing PTP (SHP), which interestingly has a predicted structure that is distinct from SHPs found in animals. In this study, we isolated cDNA of the enzyme and discovered that its actual sequence was different from the predicted sequence as a result of non-consensus RNA splicing. Contrary to the predicted structure with one SH2 domain and a disrupted phosphatase domain, Monosiga brevicollis SHP (MbSHP) contains two SH2 domains and an intact PTP domain, closely resembling SHP enzymes found in animals. We further expressed the full-length and SH2 domain-truncated forms of the enzyme in Escherichiacoli cells and characterized their enzymatic activities. The double-SH2 domain-truncated form of the enzyme effectively dephosphorylated a common PTP substrate with a specific activity among the highest in characterized PTPs, while the full-length and the N-terminal SH2 domain-truncated forms of the enzyme showed much lower activity with altered pH dependency and responses to ionic strength and common PTP inhibitors. This indicates that SH2 domains suppress the catalytic activity. SHP represents a highly conserved ancient PTP, and studying MbSHP should provide a better understanding about the evolution of tyrosine phosphorylation.     

Importance of the spatial display of charged residues in heparin–peptide interactions

Many studies have examined consensus sequences required for protein‐glycosaminoglycan interactions. Through the synthesis of helical heparin binding peptides, this study probes the relationship between spatial arrangement of positive charge and heparin binding affinity. Peptides with a linear distribution of positive charge along one face of the α‐helix had the highest affinity for heparin. Moving the basic residues away from a single face resulted in drastic changes in heparin binding affinity of up to three orders of magnitude. These findings demonstrate that amino acid sequences, different from the known heparin binding consensus sequences, will form high affinity protein‐heparin binding interactions when the charged residues are aligned linearly. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 290–298, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Deciphering interactions of the aminoglycoside phosphotransferase(3′)‐IIIa with its ligands

Aminoglycoside phosphotransferase(3′)‐IIIa (APH) is the enzyme with broadest substrate range among the phosphotransferases that cause resistance to aminoglycoside antibiotics. In this study, the thermodynamic characterization of interactions of APH with its ligands are done by determining dissociation constants of enzyme–substrate complexes using electron paramagnetic resonance and fluorescence spectroscopy. Metal binding studies showed that three divalent cations bind to the apo‐enzyme with low affinity. In the presence of AMPPCP, binding of the divalent cations occurs with 7‐to‐37‐fold higher affinity to three additional sites dependent on the presence and absence of different aminoglycosides. Surprisingly, when both ligands, AMPPCP and aminoglycoside, are present, the number of high affinity metal binding sites is reduced to two with a 2‐fold increase in binding affinity. The presence of divalent cations, with or without aminoglycoside present, shows only a small effect (<3‐fold) on binding affinity of the nucleotide to the enzyme. The presence of metal–nucleotide, but not nucleotide alone, increases the binding affinity of aminoglycosides to APH. Replacement of magnesium (II) with manganese (II) lowered the catalytic rates significantly while affecting the substrate selectivity of the enzyme such that the aminoglycosides with 2′‐NH₂ become better substrates (higher V_max) than those with 2′‐OH. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 801–809, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Diversity in peptide recognition by the SH2 domain of SH2B1

SH2B1 is a multidomain protein that serves as a key adaptor to regulate numerous cellular events, such as insulin, leptin, and growth hormone signaling pathways. Many of these protein‐protein interactions are mediated by the SH2 domain of SH2B1, which recognizes ligands containing a phosphorylated tyrosine (pY), including peptides derived from janus kinase 2, insulin receptor, and insulin receptor substrate‐1 and ?2. Specificity for the SH2 domain of SH2B1 is conferred in these ligands either by a hydrophobic or an acidic side chain at the +3 position C‐terminal to the pY. This specificity for chemically disparate species suggests that SH2B1 relies on distinct thermodynamic or structural mechanisms to bind to peptides. Using binding and structural strategies, we have identified unique thermodynamic signatures for each peptide binding mode, and several SH2B1 residues, including K575 and R578, that play distinct roles in peptide binding. The high‐resolution structure of the SH2 domain of SH2B1 further reveals conformationally plastic protein loops that may contribute to the ability of the protein to recognize dissimilar ligands. Together, numerous hydrophobic and electrostatic interactions, in addition to backbone conformational flexibility, permit the recognition of diverse peptides by SH2B1. An understanding of this expanded peptide recognition will allow for the identification of novel physiologically relevant SH2B1/peptide interactions, which can contribute to the design of obesity and diabetes pharmaceuticals to target the ligand‐binding interface of SH2B1 with high specificity.     

Identification of novel peptide ligands for the cancer‐specific receptor mutation EFGRvIII using a mixture‐based synthetic combinatorial library

We report here, the design and synthesis of a positional scanning synthetic combinatorial library for the identification of novel peptide ligands targeted against the cancer‐specific epidermal growth factor tyrosine kinase receptor mutation variant III (EGFRvIII). This receptor is expressed in several kinds of cancer, in particular, ovarian, glioblastomas, and breast cancer, but not in normal tissue. The library consisted of six individual positional sublibraries in the format, H‐O_1–6XXXXX‐NH₂, O being one of the 19 proteinogenic amino acids (cysteine omitted) and X an equimolar mixture of these. The library consisted of 114 mixtures in total. Using a biotin‐streptavidin assay, the binding of each sublibrary to NR6M, NR6W‐A, and NR6 cells was tested. These cells express EGFRvIII, EGFR, and neither of the receptors, respectively. The result from each sublibrary was examined to identify the most active amino acid residue at each position. On the basis of this knowledge, eight peptides were synthesized and tested for binding to EGFRvIII. We identified one peptide, H‐FALGEA‐NH₂, that showed more selective binding to the mutated receptor than the EGFRvIII specific peptide PEPHC1. This study demonstrates the value of using mixture‐based combinatorial positional scanning libraries for the identification of novel peptide ligands targeted against the cancer‐specific EGFRvIII. Our best candidate H‐FALGEA‐NH₂ will be radioactively labeled and evaluated as an imaging agent for positron emission tomography investigation for diagnosis, staging, and monitoring of therapy of various types of cancer. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 201–206, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

A strained DNA binding helix is conserved for site recognition,folding nucleation,and conformational modulation

Nucleic acid recognition is often mediated by α‐helices or disordered regions that fold into α‐helix on binding. A peptide bearing the DNA recognition helix of HPV16 E2 displays type II polyproline (PII) structure as judged by pH, temperature, and solvent effects on the CD spectra. NMR experiments indicate that the canonical α‐helix is stabilized at the N‐terminus, while the PII forms at the C‐terminus half of the peptide. Re‐examination of the dihedral angles of the DNA binding helix in the crystal structure and analysis of the NMR chemical shift indexes confirm that the N‐terminus half is a canonical α‐helix, while the C‐terminal half adopts a 3₁₀ helix structure. These regions precisely match two locally driven folding nucleii, which partake in the native hydrophobic core and modulate a conformational switch in the DNA binding helix. The peptide shows only weak and unspecific residual DNA binding, 10⁴‐fold lower affinity, and 500‐fold lower discrimination capacity compared with the domain. Thus, the precise side chain conformation required for modulated and tight physiological binding by HPV E2 is largely determined by the noncanonical strained α‐helix conformation, “presented” by this unique architecture. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 432–443, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Quantitative relation between intermolecular and intramolecular binding of pro-rich peptides to SH3 domains

Flexible linkers are often found to tether binding sequence motifs or connect protein domains. Here we analyze three usages of flexible linkers: 1), intramolecular binding of proline-rich peptides (PRPs) to SH3 domains for kinase regulation; 2), intramolecular binding of PRP for increasing the folding stability of SH3 domains; and 3), covalent linking of PRPs and other ligands for high-affinity bivalent binding. The basis of these analyses is a quantitative relation between intermolecular and intramolecular binding constants. This relation has the form K(i) = K(e0)p for intramolecular binding and K(e) = K(e01)K(e02)p for bivalent binding. The effective concentration p depends on the length of the linker and the distance between the linker attachment points in the bound state. Several applications illustrate the usefulness of the quantitative relation. These include intramolecular binding to the Itk SH3 domain by an internal PRP and to a circular permutant of the alpha-spectrin SH3 domain by a designed PRP, and bivalent binding to the two SH3 domains of Grb2 by two linked PRPs. These and other examples suggest that flexible linkers and sequence motifs tethered to them, like folded protein domains, are also subject to tight control during evolution.     

Structural Determinants of SHP-2 Function and Specificity in Xenopus Mesoderm Induction

SHP-2 is a positive component of many receptor tyrosine kinase signaling pathways. The related protein-tyrosine phosphatase (PTP) SHP-1 usually acts as a negative regulator. The precise domains utilized by SHP-2 to transmit positive signals in vivo and the basis for specificity between SHP-1 and SHP-2 are not clear. In Xenopus, SHP-2 is required for mesoderm induction and completion of gastrulation. We investigated the effects of SHP-2 mutants and SHP-2/SHP-1 chimeras on basic fibroblast growth factor-induced mesoderm induction. Both SH2 domains and the PTP domain are required for normal SHP-2 function in this pathway. The N-terminal SH2 domain is absolutely required, whereas the C-terminal SH2 contributes to wild-type function. The C-terminal tyrosyl phosphorylation sites and proline-rich region are dispensable, arguing against adapter models of SHP-2 function. Although the SH2 domains contribute to SHP-2 specificity, studies of SHP chimeras reveal that substantial specificity resides in the PTP domain. Thus, PTP domains exhibit biologically relevant specificity in vivo, and noncatalytic and catalytic domains of PTPs contribute to specificity in a combinatorial fashion.     

Cellular phosphatase activity of C1-Ten/Tensin2 is controlled by Phosphatidylinositol-3,4,5-triphosphate binding through the C1-Ten/Tensin2 SH2 domain

Regulation of tyrosine phosphorylation on insulin receptor substrate-1 (IRS-1) is essential for insulin signaling. The protein tyrosine phosphatase (PTP) C1-Ten/Tensin2 has been implicated in the regulation of IRS-1, but the molecular basis of this dephosphorylation is not fully understood. Here, we demonstrate that the cellular phosphatase activity of C1-Ten/Tensin2 on IRS-1 is mediated by the binding of the C1-Ten/Tensin2 Src-homology 2 (SH2) domain to phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P₃). We show that the role of C1-Ten/Tensin2 is dependent on insulin-induced phosphoinositide 3-kinase activity. The C1-Ten/Tensin2 SH2 domain showed strong preference and high affinity for PtdIns(3,4,5)P₃. Using site-directed mutagenesis, we identified three basic residues in the C1-Ten/Tensin2 SH2 domain that were critical for PtdIns(3,4,5)P₃ binding but were not involved in phosphotyrosine binding and PTP activity. Using a PtdIns(3,4,5)P₃ binding-deficient mutant, we showed that the specific binding of the C1-Ten/Tensin2 SH2 domain to PtdIns(3,4,5)P₃ allowed C1-Ten/Tensin2 to function as a PTP in cells. Collectively, our findings suggest that the interaction between the C1-Ten/Tensin2 SH2 domain and PtdIns(3,4,5)P₃ produces a negative feedback loop of insulin signaling through IRS-1.     

The Src SH2 domain interacts dynamically with the focal adhesion kinase binding site as demonstrated by paramagnetic NMR spectroscopy

The interaction between the tyrosine kinases Src and focal adhesion kinase (FAK) is a key step in signaling processes from focal adhesions. The phosphorylated tyrosine residue 397 in FAK is able to bind the Src SH2 domain. To establish the extent of the FAK binding motif, the binding affinity of the SH2 domain for phosphorylated and unphosphorylated FAK-derived peptides of increasing length was determined and compared with that of the internal Src SH2 binding site. It is shown that the FAK peptides have higher affinity than the internal binding site and that seven negative residues adjacent to the core SH2 binding motif increase the binding constant 30-fold. A rigid spin-label incorporated in the FAK peptides was used to establish on the basis of paramagnetic relaxation enhancement whether the peptide-protein complex is well defined. A large spread of the paramagnetic effects on the surface of the SH2 domain suggests that the peptide-protein complex exhibits dynamics, despite the high affinity of the peptide. The strong electrostatic interaction between the positive side of the SH2 domain and the negative peptide results in a high affinity but may also favor a dynamic interaction.     

High‐resolution structural analysis shows how different crystallographic environments can induce alternative modes of binding of a phosphotyrosine peptide to the SH2 domain of Fer tyrosine kinase

Fes and Fes‐related (Fer) protein tyrosine kinases (PTKs) comprise a subfamily of nonreceptor tyrosine kinases characterized by a unique multidomain structure composed of an N‐terminal Fer/CIP4 homology‐Bin/Amphiphysin/Rvs (F‐BAR) domain, a central Src homology 2 (SH2) domain, and a C‐terminal PTK domain. Fer is ubiquitously expressed, and upregulation of Fer has been implicated in various human cancers. The PTK activity of Fes has been shown to be positively regulated by the binding of phosphotyrosine‐containing ligands to the SH2 domain. Here, the X‐ray crystal structure of human Fer SH2 domain bound to a phosphopeptide that has D‐E‐pY‐E‐N‐V‐D sequence is reported at 1.37 å resolution. The asymmetric unit (ASU) contains six Fer‐phosphopeptide complexes, and the structure reveals three distinct binding modes for the same phosphopeptide. At four out of the six binding sites in the ASU, the phosphopeptide binds to Fer SH2 domain in a type I β‐turn conformation, and this could be the optimal binding mode of this phosphopeptide. At the other two binding sites in the ASU, it appears that spatial proximity of neighboring SH2 domains in the crystal induces alternative modes of binding of this phosphopeptide.     

NMR studies of an immunomodulatory benzodiazepine binding to its molecular target on the mitochondrial F1F0‐ATPase

Bz‐423 is an inhibitor of the mitochondrial F₁F₀‐ATPase, with therapeutic properties in murine models of immune diseases. Here, we study the binding of a water‐soluble Bz‐423 analog (5‐(3‐(aminomethyl)phenyl)‐7‐chloro‐ 1‐methyl‐3‐(naphthalen‐2‐ylmethyl)‐1H‐benzo][e][1,4]diazepin‐2(3H)‐one); (1) to its target subunit on the enzyme, the oligomycin sensitivity conferring protein (OSCP), by NMR spectroscopy using chemical shift perturbation and cross‐relaxation experiments. Titration experiments with constructs representing residues 1–120 or 1–145 of the OSCP reveals that (a) 1 binds to a region of the protein, at the minimum, comprising residues M51, L56, K65, V66, K75, K77, and N92, and (b) binding of 1 induces conformational changes in the OSCP. Control experiments employing a variant of 1 in which a key binding element on the small molecule was deleted; it had no perturbational effect on the spectra of the OSCP, which indicates that the observed changes with 1 represent specific binding interactions. Collectively, these data suggest that 1 might inhibit the enzyme through an allosteric mechanism where binding results in conformational changes that perturb the OSCP‐F₁ interface resulting in disrupted communication between the peripheral stalk and the F₁‐domain of the enzyme. © 2009 Wiley Periodicals, Inc. Biopolymers 29: 85–92, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Determining folding and binding properties of the C‐terminal SH2 domain of SHP2

SH2 domains are a class of protein–protein interaction modules with the function to recognize and bind sequences characterized by the presence of a phosphorylated tyrosine. SHP2 is a protein phosphatase involved in the Ras‐ERK1/2 signaling pathway that possess two SH2 domains, namely, N‐SH2 and C‐SH2, that mediate the interaction of SHP2 with various partners and determine the regulation of its catalytic activity. One of the main interactors of the SH2 domains of SHP2 is Gab2, a scaffolding protein with critical role in determining cell differentiation. Despite their key biological role and the importance of a correct native fold to ensure it, the mechanism of binding of SH2 domains with their ligands and the determinants of their stability have been poorly characterized. In this article, we present a comprehensive kinetic study of the folding of the C‐SH2 domain and the binding mechanism with a peptide mimicking a region of Gab2. Our data, obtained at different pH and ionic strength conditions and supported by site‐directed mutagenesis, highlight the role of electrostatic interactions in the early events of recognition. Interestingly, our results suggest a key role of a highly conserved histidine residue among SH2 family in the interaction with negative charges carried by the phosphotyrosine of Gab2. Moreover, the analysis of the equilibrium and kinetic folding data of C‐SH2 describes a complex mechanism implying a change in rate‐limiting step at high denaturant concentrations. Our data are discussed under the light of previous works on N‐SH2 domain of SHP2 and other SH2 domains.     

Bivalent inhibitors of the tyrosine kinases ABL and SRC: determinants of potency and selectivity

We recently reported a chemical genetic method for generating bivalent inhibitors of protein kinases. This method relies on the use of the DNA repair enzyme O(6)-alkylguanine-DNA alkyltransferase (AGT) to display an ATP-competitive inhibitor and a ligand that targets a secondary binding domain. With this method potent and selective inhibitors of the tyrosine kinases SRC and ABL were identified. Here, we dissect the molecular determinants of the potency and selectivity of these bivalent ligands. Systematic analysis of ATP-competitive inhibitors with varying linker lengths revealed that SRC and ABL have differential sensitivities to ligand presentation. Generation of bivalent constructs that contain ligands with differential affinities for the ATP-binding sites and SH3 domains of SRC and ABL demonstrated the modular nature of inhibitors based on the AGT scaffold. Furthermore, these studies revealed that the interaction between the SH3 domain ligand and the kinase SH3 domain is the major selectivity determinant amongst closely-related tyrosine kinases. Finally, the potency of bivalent inhibitors against distinct phospho-isoforms of SRC was determined. Overall, these results provide insight into how individual ligands can be modified to provide more potent and selective bivalent inhibitors of protein kinases.     

Study on the inhibitory mechanism and binding mode of the hydroxycoumarin compound NSC158393 to HIV‐1 integrase by molecular modeling

Human immunodeficiency virus type 1 integrase (IN) is an essential enzyme in the life cycle of this virus and also an important target for the study of anti‐HIV drugs. In this work, the binding modes of the wild type IN core domain and the two mutants, that is, W132G and C130S, with the 4‐hydroxycoumarin compound NSC158393 were evaluated by using the “relaxed complex” molecular docking approach combined with molecular dynamics (MD) simulations. Based on the monomer MD simulations, both of the two substitutions affect not only the stability of the 128–136 peptides, but also the flexibility of the functional 140s loop. In principle, NSC158393 binds the 128–136 peptides of IN; however, the specific binding modes for the three systems are various. According to the binding mode of NSC158393 with WT, NSC158393 can effectively interfere with the stability of the IN dimer by causing a steric hindrance around the monomer interface. Additionally, through the comparative analysis of the MD trajectories of the wild type IN and the IN‐NSC158393 complex, we found that NSC15893 may also exert its inhibitory function by diminishing the mobility of the function loop of IN. Three key binding residues, that is, W131, K136, and G134, were discovered by energy decomposition calculated with the Molecular Mechanics Generalized Born Surface Area method. Characterized by the largest binding affinity, W131 is likely to be indispensable for the ligand binding. All the above results are consistent with experiment data, providing us some helpful information for understanding the mechanism of the coumarin‐based inhibitors. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 700–709, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

On the benefit of bivalency in peptide ligand/pin1 interactions

The human peptidyl prolyl cis/trans isomerase (PPIase) Pin1 has a key role in developmental processes and cell proliferation. Pin1 consists of an N-terminal WW domain and a C-terminal catalytic PPIase domain both targeted specifically to Ser(PO₃H₂)/Thr(PO₃H₂)-Pro sequences. Here, we report the enhanced affinity originating from bivalent binding of ligands toward Pin1 compared to monovalent binding. We developed composite peptides where an N-terminal segment represents a catalytic site-directed motif and a C-terminal segment exhibits a predominant affinity to the WW domain of Pin1 tethered by polyproline linkers of different chain length. We used NMR shift perturbation experiments to obtain information on the specific interaction of a bivalent ligand to both targeted sites of Pin1. The bivalent ligands allowed a considerable range of thermodynamic investigations using isothermal titration calorimetry and PPIase activity assays. They expressed up to 350-fold improved affinity toward Pin1 in the nanomolar range in comparison to the monovalent peptides. The distance between the two binding motifs was highly relevant for affinity. The optimum in affinity manifested by a linker length of five prolyl residues between active site- and WW domain-directed peptide fragments suggests that the corresponding domains in Pin1 are allowed to adopt preferred spatial arrangement upon ligand binding.     

N-terminal carboxyl and tetrazole-containing amides as adjuvants to Grb2 SH2 domain ligand binding.

High affinity binding of peptides to Src homology 2 (SH2) domains, often requires the presence of phosphotyrosyl (pTyr) or pTyr-mimicking moieties in the N-terminal position of the binding ligand. Several reports have shown that N(alpha)-acylation of the critical pTyr residue can result in increased SH2 domain binding potency. For Grb2 SH2 domains which recognize pTyr-Xxx-Asn-NH(2) motifs, significant potency enhancement can be incurred by N(alpha)-(3-amino)Z derivatization of tripeptides such as pTyr-Ile-Asn-NH(2). Using ligands based on the high affinity pY-Ac(6)c-Asn-(naphthylpropylamide) motif, (where Ac(6)c=1-aminocyclohexanecarboxylic acid), additional reports have shown moderate potentiating effects of N(alpha)-oxalyl derivatization. The current study examined variations of the N(alpha)-oxalyl theme in the context of a Xxx-Ac(6)c-Asn-(naphthylpropylamide) platform, where Xxx=the hydrolytically stable pTyr mimetics phosphonomethyl phenylalanine (Pmp) or carboxymethyl phenylalanine (Cmf). The effects of N(alpha)-(3-amino)Z derivatization were also investigated for this platform, to ascertain whether the large binding enhancement reported for tripeptides such as pTyr-Ile-Asn-NH(2) could be observed. In ELISA-based extracellular Grb2 SH2 domain binding assays, it was found for the Pmp-based series, that extending the oxalyl carboxyl out by one methylene unit or replacing carboxyl functionality with a tetrazole isostere, resulted in binding potency greater than the parent N(alpha)-acetyl-containing compound, with enhancement approximating that observed for the N(alpha)-oxalyl derivative. When Cmf was used as the pTyr mimetic, only modest differences in IC(50) values were observed for the series. Examination of the N(alpha)-(3-amino)Z derivatized Pmp-Ac(6)c-Asn-(naphthylpropylamide), showed that binding affinity was reduced relative to the parent N(alpha)-acetyl analogue, in contrast to the reported significant enhancement of affinity observed with other peptide ligands. Treatment of MDA-453 tumor cells, which are mitogenically driven through erbB-2 tyrosine kinase-dependent pathways, with Pmp-containing inhibitors resulted in growth inhibition, with the N(alpha)-oxalyl and N(alpha)-malonyl-containing compounds exhibiting IC(50) values (4.3 and 4.6 microM, respectively) approximately five-fold lower than the parent N(alpha)-acetyl-containing compound. Tetrazole and N(alpha)-(3-amino)Z-containing inhibitors were from two- to four-fold less potent than these latter analogues in the growth inhibition assays.