The SH3 domain of HS1 protein recognizes lysine-rich polyproline motifs

HS1 is a protein involved in erythroid proliferation and apoptotic cell death, containing several structurally significant motifs including a C-terminal SH3 domain. HPK1 is a member of the Ste20-related kinase family, which contains four proline-rich sequences and is constitutively associated with HS1 in hematopoietic cells. Recombinant fusion protein GST-SH3_HS1 was expressed to assess the binding properties of 16 peptides derived from the HPK1 proline-rich regions. The binding affinities were determined by non-immobilized ligand interaction assay by circular dichroism. Our results revealed that the classical PxxPxK class II binding motif is not sufficient to induce the interaction with the GST-SH3_HS1 domain, an event dependent on the presence of additional basic residue(s) located at the C-terminus of the PxxPxK motif: Lys₋₅ in P2 peptide and Lys₋₈ in P4c peptide. Lys replacement by Arg residues decreases the ligand binding affinity. The finding that both SH3_HS1 domain and full-length HS1 protein bind to P2 peptide with similar affinity demonstrates that the whole protein sequence does not affect the interaction properties of the domain. In silico models of SH3_HS1 as a complex with P2 or P4c highlight the domain residues that interact with the recognition determinants of the peptide ligand and that cooperate in the complex stabilization.     

Reconstitution of a native-like SH2 domain from disordered peptide fragments examined by multidimensional heteronuclear NMR

The N-terminal SH2 domain from the p85alpha subunit of phosphatidylinositol 3' kinase is cleaved specifically into 9- and 5-kD fragments by limited proteolytic digestion with trypsin. The noncovalent SH2 domain complex and its constituent tryptic peptides have been investigated using high-resolution heteronuclear magnetic resonance (NMR). These studies have established the viability of the SH2 domain as a fragment complementation system. The individual peptide fragments are predominantly unstructured in solution. In contrast, the noncovalent 9-kD + 5-kD complex shows a native-like (1)H-(15)N HSQC spectrum, demonstrating that the two fragments fold into a native-like structure on binding. Chemical shift analysis of the noncovalent complex compared to the native SH2 domain reveals that the highest degree of perturbation in the structure occurs at the cleavage site within a flexible loop and along the hydrophobic interface between the two peptide fragments. Mapping of these chemical shift changes on the structure of the domain reveals changes consistent with the reduction in affinity for the target peptide ligand observed in the noncovalent complex relative to the intact protein. The 5-kD fragment of the homologous Src protein is incapable of structurally complementing the p85 9-kD fragment, either in complex formation or in the context of the full-length protein. These high-resolution structural studies of the SH2 domain fragment complementation features establish the suitability of the system for further protein-folding and design studies.     

Synthesis and evaluation of phosphopeptides containing iminodiacetate groups as binding ligands of the Src SH2 domain

Phosphopeptide pTyr-Glu-Glu-Ile (pYEEI) has been introduced as an optimal Src SH2 domain ligand. Peptides, Ac-K(IDA)pYEEIEK(IDA) (1), Ac-KpYEEIEK (2), Ac-K(IDA)pYEEIEK (3), and Ac-KpYEEIEK(IDA) (4), containing 0–2 iminodiacetate (IDA) groups at the N- and C-terminal lysine residues were synthesized and evaluated as the Src SH2 domain binding ligands. Fluorescence polarization assays showed that peptide 1 had a higher binding affinity (K_d = 0.6 μM) to the Src SH2 domain when compared with Ac-pYEEI (K_d = 1.7 μM), an optimal Src SH2 domain ligand, and peptides 2–4 (K_d = 2.9–52.7 μM). The binding affinity of peptide 1 to the SH2 domain was reduced by more than 2-fold (K_d = 1.6 μM) upon addition of Ni²⁺ (300 μM), possibly due to modest structural effect of Ni²⁺ on the protein as shown by circular dichroism experimental results. The binding affinity of 1 was restored in the presence of EDTA (300 μM) (K_d = 0.79 μM). These studies suggest that peptides containing IDA groups may be used for designing novel SH2 domain binding ligands.     

Modulation of protein–ligand interactions by photocleavage of a cyclic peptide using phosphatidylinositol 3‐kinase SH3 domain as model system

To photomodulate the interaction of the phosphatidylinositol 3‐kinase SH3 domain with a peptide ligand, a cyclic peptide (cyclic‐1) with a photolabile side chain‐to‐side chain linker was synthesized. The conformation of cyclic‐1 differs from that of the parent linear peptide, but becomes identical by UV‐irradiation. Accordingly, the binding affinity of cyclic‐1 to the SH3 domain increased upon conversion of the cyclic to a linear flexible structure by irradiation (K_d: 3.4 ± 1.7 and 0.9 ± 0.3 mM , respectively). These results confirm the usefulness of a photocleavable peptide for photocontrol of peptide–protein interactions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Modeling and prediction of binding affinities between the human amphiphysin SH3 domain and its peptide ligands using genetic algorithm-Gaussian processes

In this article, we discuss the application of the Gaussian process (GP) and other statistical methods (PLS, ANN, and SVM) for the modeling and prediction of binding affinities between the human amphiphysin SH3 domain and its peptide ligands. Divided physicochemical property scores of amino acids, involving significant hydrogen bond, electronic, hydrophobic, and steric properties, was used to characterize the peptide structures, and quantitative structure-affinity relationship models were then constructed by PLS, ANN, SVM, and GP coupled with genetic algorithm-variable selection. The results show that: (i) since the significant flexibility and high complexity possessed in polypeptide structures, linear PLS method was incapable of fulfilling a satisfying behavior on SH3 domain binding peptide dataset; (ii) the overfitting involved in training process has decreased the predictive power of ANN model to some extent; (iii) both SVM and GP have a good performance for SH3 domain binding peptide dataset. Moreover, by combining linear and nonlinear terms in the covariance function, the GP is capable of handling linear and nonlinear-hybrid relationship, and which thus obtained a more stable and predictable model than SVM. Analyses of GP models showed that diversified properties contribute remarkable effect to the interactions between the SH3 domain and the peptides. Particularly, steric property and hydrophobicity of P(2), electronic property of P(0), and electronic property and hydrogen bond property of P(-3) in decapeptide (P(4)P(3)P(2)P(1)P(0)P(-1)P(-2)P(-3)P(-4)P(-5)) significantly contribute to the binding affinities of SH3 domain-peptide interactions.     

Synthesis and evaluation of conformationally constrained peptide analogues as the Src SH3 domain binding ligands

Src kinase activity is regulated by the interaction of SH3 domain with protein sequences that are rich in proline residues. Identification of more potent SH3 domain binding ligands that can regulate Src kinase activity is a subject of major interest. Conformationally constrained peptides have been previously used for improving the binding potency of the Src SH2 domain binding peptide ligands and peptide substrates of the substrate-binding site of Src. A series of peptide analogues of Ac-VSLARRPLPPLP (1, Ac-VSL12, K_d = 0.34 μM) were synthesized by introducing conformational constraints to improve the binding affinity towards the Src SH3 domain. Peptides synthesized through cyclization between N-terminal to C-terminal [VSLARRPLPPLP] or N-terminal to side chain flanking residues (i.e., [^βAVS]LARRPLPPLP and [VSLE]RRPLPPLP) exhibited at least 6.4-fold less binding affinity (K_d = 2.19–4.85 μM) when compared to 1. The data suggest upon N-terminal cyclization with C-terminal or flanking residues, the interactions of the amino acids in the core RPLPPLP reduce significantly with the residues within the Src SH3 domain. Conformationally constrained peptide V[SLARRPLPPLP] (5) was synthesized through cyclization of C-terminal to the serine side chain and displayed a comparable binding affinity (K_d = 0.35 μM) towards the Src SH3 domain versus that of 1. Thus, this template may be used to optimize and generate more potent analogues with higher stability.     

Evolution of High-Affinity Peptide Probes to Detect the SH3 Domain of Cancer Biomarker BCR–ABL

The BCR–ABL fusion protein is closely associated with the pathological progression of chronic myelogenous leukaemia and some other myeloproliferative diseases, which has long been recognized as one of the most important cancer biomarkers in the tumor diagnosis community. The SH3 domain of BCR–ABL is a small, conserved protein module that specifically recognizes and binds proline-rich peptide fragments. In the current study, we used a synthetic strategy to discover new peptide probes with high affinity binding to the BCR–ABL SH3 domain. In the procedure, a sequence-based machine learning predictor was developed based on a set of affinity-known SH3 binders, and the predictor was then used to guide the evolutional optimization of numerous virtual peptides to enrich high binding potency for the SH3 domain. Subsequently, a evolved peptide population was generated, from which ten peptides with the highest affinity scores were selected and their interaction free energies with SH3 domain were characterized systematically using a combination of molecular dynamics simulation and binding free energy analysis. Consequently, four peptides were suggested as promising candidates and their affinities toward SH3 domain were assayed; two peptides, APTYTPPPPP and APTYAPPPPP, were identified to have potent binding capability with dissociation constants K _d of 3 and 8 μM, respectively. Further, the structural basis and energetic property of SH3 domain in complex with APTYTPPPPP were examined in detail, revealing a non-specific interaction in SH3–peptide recognition that should render a broad ligand spectrum for the domain.     

Crystal Structure of P13K SH3 Domain at 2.0 Å Resolution

The P13K SH3 domain, residues 1 to 85 of the P1 – 3 kinase p85 subunit, has been characterized by X-ray diffraction. Crystals belonging to space groupP4₃2₁2 diffract to 2.0 Å resolution and the structure was phased by single isomorphous replacement and anomalous scattering (SIRAS). As expected, the domain is a compact β barrel with an over-all conformation very similar to the independently determined NMR structures. The X-ray structure illuminates a discrepancy between the two NMR structures on the conformation of the loop region unique to P13K SH3. Furthermore, the ligand binding pockets of P13K SH3 domain are occupied by amino acid residues from symmetry-related P13K SH3 molecules: the C-terminal residues I(82) SPP of one and R18 of another. The interaction modes clearly resemble those observed for the P13K SH3 domain complexed with the synthetic peptide RLP1, a class 1 ligand, although there are significant differences. The solid-state interactions suggest a model of protein – protein aggregation that could be mediated by SH3 domains.     

Density functional theory study of model complexes for the revised nitrate reductase active site in Desulfovibrio desulfuricans NapA

[Mo(SSCH₃)(S₂C₂(CH₃)₂)₂] ^x complexes with charges x between −3 and +3 were investigated by density functional theory computations as minimal nitrate reductase active-site models. The strongly reduced species (x = −2, −3) exist preferentially as pentacoordinate sulfo complexes separated from a thiolate anion. The oxidized extremes (x > 0) clearly prefer hexacoordinate complexes with an η²-MeSS ligand. Among the neutral and especially for the singly negatively charged species structures with η²-MeSS and η¹-MeSS ligands are energetically close to the sulfo methyl sulfide complex without SS bonding. For x = −1 the three isomers lie in a 1.5 kcal mol⁻¹ energy range. Putative mechanistic pathways for nitrate reduction from the literature were investigated computationally: (1) reduction at a pentacoordinate sulfo complex, (2) reduction at the ligand, and (3) reduction at the molybdenum center with an R–S–S ligand. All three pathways could be traced at least for some overall charges but no definite conclusion can be drawn about the mechanism. Complexes with larger dithiolato ligands were also computed in order to model the tricyclic metallopterin framework more accurately: the first heterocyclus (5,6-dihydro-2H-pyran) stabilizes the nitrate complex and the molybdenum oxo product complex by approximately 10 kcal mol⁻¹ and also reduces the activation barrier (by approximately 5 kcal mol⁻¹). The effect of the second (1,2,3,4-tetrahydropyrazin) and third heterocyclus (2-amino-3H-pyrimidin-4-one) on the relative energies is relatively small. For bigger models derived from an experimental protein structure, nitrate reduction at a persulfo molybdenum(IV) complex fragment (mechanism 3) is clearly favored over the oxidation of a molybdenum-bound sulfur atom (mechanism 2). Mechanism 1 could not be investigated for the big models but seems the least favorable on the basis of the results from smaller models.     

Directed discovery of bivalent peptide ligands to an SH3 domain

The Caenorhabditis elegans SEM-5 SH3 domains recognize proline-rich peptide segments with modest affinity. We developed a bivalent peptide ligand that contains a naturally occurring proline-rich binding sequence, tethered by a glycine linker to a disulfide-closed loop segment containing six variable residues. The glycine linker allows the loop segment to explore regions of greatest diversity in sequence and structure of the SH3 domain: the RT and n-Src loops. The bivalent ligand was optimized using phage display, leading to a peptide (PP-G(4)-L) with 1000-fold increased affinity for the SEM-5 C-terminal SH3 domain over that of a natural ligand. NMR analysis of the complex confirms that the peptide loop segment is targeted to the RT and n-Src loops and parts of the beta-sheet scaffold of this SH3 domain. This binding region is comparable to that targeted by a natural non-PXXP peptide to the p67(phox) SH3 domain, a region not known to be targeted in the Grb2 SH3 domain family. PP-G(4)-L may aid in the discovery of additional binding partners of Grb2 family SH3 domains.     

Synthesis and characterization of glucosyl-curcuminoids as Fe3+ suppliers in the treatment of iron deficiency

The Fe³⁺ chelating ability of some curcumin glucosyl derivatives (Glc-H; Glc-OH; Glc-OCH₃) is tested by means of UV and NMR study. The pK _a values of the ligands and the overall stability constants of Fe³⁺ and Ga³⁺ complexes are evaluated from UV spectra. The only metal binding site of the ligand is the β-diketo moiety in the keto-enolic form; the glucosyl moiety does not interact with metal ion but it contributes to the stability of metal/ligand 1:2 complexes by means of hydrophilic interactions. These glucosyl derivatives are able to bind Fe³⁺ in a wide pH rage, forming complex species thermodynamically more stable than those of other ligands commonly used in the treatment of iron deficiency. In addition they demonstrate to have a poor affinity for competitive biological metal ions such as Ca²⁺. All ligands and their iron complexes have a good lypophilicity (log P > −0.7) suggesting an efficient gastrointestinal absorption in view of their possible use as iron supplements in oral therapy. The ligand molecules are also tested for their antioxidant properties in “ex vivo” biological system.     

Damaging mechanisms of chilling- and salt stress to <Emphasis Type="Italic">Arachis hypogaea</Emphasis> L. leaves

To investigate damaging mechanisms of chilling and salt stress to peanut (Arachis hypogaea L.) leaves, LuHua 14 was used in the present work upon exposure to chilling temperature (4°C) accompanied by high irradiance (1,200 μmol m⁻² s⁻¹) (CH), salt stress accompanied by high irradiance (1,200 μmol m⁻² s⁻¹) (SH), and high-irradiance stress (1,200 μmol m⁻² s⁻¹) at room temperature (25°C) (NH), respectively. Additionally, plants under low irradiance (100 μmol m⁻² s⁻¹) at room temperature (25°C) were used as control plants (CK). Relative to CK and NH treatments, both the maximal photochemical efficiency of PSII (F_v/F_m) and the absorbance at 820 nm decreased greatly in peanut leaves under CH and SH stress, which indicated that severe photoinhibition occurred in peanut leaves under such conditions. Initial fluorescence (F_o), 1 − q_P and nonphotochemical quenching (NPQ) in peanut leaves significantly increased under CH- and SH stress. Additionally, the activity of superoxide dismutase (SOD), one of the key enzymes of water-water cycle, decreased greatly, the accumulation of malondialdehyde (MDA) and membrane permeability increased. These results suggested that damages to peanut photosystems might be related to the accumulation of reactive oxygen species (ROS) induced by excess energy, and the water-water cycle could not dissipate energy efficiently under the stress of CH and SH, which caused the accumulation of ROS greatly. CH and SH had similar damaging effects on peanut photosystems, except that CH has more severe effects. All the results showed that CH- and SH stress has similar damaging site and mechanisms in peanut leaves.     

Molecular design and optimization of hepatic cancer SLP76‐derived PLCγ1 SH3‐binding peptide with the systematic N‐substitution of peptide PXXP motif

The phospholipase Cγ1 (PLCγ1) is essential for T‐cell signaling and activation in hepatic cancer immune response, which has a regulatory Src homology 3 (SH3) domain that can specifically recognize and interact with the PXXP‐containing decapeptide segment (¹⁸⁵QP P VP P QRPM¹⁹⁴, termed as SLP76^185–194 peptide) of adaptor protein SLP76 following T‐cell receptor ligation. The isolated peptide can only bind to the PLCγ1 SH3 domain with a moderate affinity due to lack of protein context support. Instead of the traditional natural residue mutagenesis that is limited by low structural diversity and shifted target specificity, we herein attempt to improve the peptide affinity by replacing the two key proline residues Pro187 and Pro190 of SLP76^185–194 PXXP motif with nonnatural N‐substituted amino acids, as the proline is the only endogenous N‐substituted amino acid. The replacement would increase peptide flexibility but can restore peptide activity by establishing additional interactions with the domain. Structural analysis reveals that the domain pocket can be divided into a large amphipathic region and a small negatively charged region; they accommodate hydrophobic, aromatic, polar, and moderate‐sized N‐substituted amino acid types. A systematic replacement combination profile between the peptide residues Pro187 and Pro190 is created by structural modeling, dynamics simulation, and energetics analysis, from which six improved and two reduced N‐substituted peptides as well as native SLP76^185–194 peptide are identified and tested for their binding affinity to the recombinant protein of the human PLCγ1 SH3 domain using fluorescence‐based assays. Two N‐substituted peptides, SLP76^185–194(N‐Leu187/N‐Gln190) and SLP76^185–194(N‐Thr187/N‐Gln190), are designed to have high potency (K_d = 0.67 ± 0.18 and 1.7 ± 0.3 μM, respectively), with affinity improvement by, respectively, 8.5‐fold and 3.4‐fold relative to native peptide (K_d = 5.7 ± 1.2 μM).     

Density functional theory studies of model complexes for molybdenum-dependent nitrate reductase active sites

Molybdenum and tungsten complexes as models for the active sites of assimilatory or dissimilatory nitrate reductases (NR) were computed at the CPCM-B98/SDDp//B3LYP/Lanl2DZp* plus zero point energy level of density functional theory. The ligands were chosen on the basis of available experimental protein or small chemical model structures. A water molecule is found to bind to assimilatory NR models [(Me₂C₂S₂)MO(YMe)]⁻ (−11.5 kcal mol⁻¹ for M is Mo, Y is S) and may be replaced by nitrate (−4.5 kcal mol⁻¹) (but a hydroxy group may not). Nature’s choice of M is Mo and Y is S for NR has the largest activation energy for protein-free models (13.3 kcal mol⁻¹) and the least exothermic reaction energy for the nitrate reduction (−14.9 kcal mol⁻¹) compared with M is W and Y is O or Se alternatives. Water binding to dissimilatory NR model complexes [(Me₂C₂S₂)₂M(YR)]⁻ is considerably endothermic (10.3 kcal mol⁻¹); nitrate binding is only slightly so (1.5 kcal mol⁻¹ for RY⁻ is MeS⁻). The exchange of an oxo ligand (assimilatory NR) for a dithiolato ligand (dissimilatory NR model) reduces the exothermicity (−8.6 kcal mol⁻¹ relative to the fivefold-coordinate reduced complex) and raises the barrier for oxygen atom transfer (OAT) in the nitrate complex (19.2 kcal mol⁻¹). Not for the mono but only for the bisdithiolato complexes hydrogen bonding involving the coordinated substrate may significantly lower the OAT barrier as shown by explicitly adding water molecules. Substitution of tungsten for molybdenum generally lowers OAT activation energies and makes nitrate reduction reaction energies more negative. Bidentate carboxylato binding identified in Escherichia coli NarGHI is the preferred binding mode also for an acetato model. However, one dithiolato ligand folds when the Mo^VI center is bare of a good π-donor ligand, e.g., an oxo group. Computations on [(mnt)₂Mo^IV(YR)(PPh₃)]⁻ [mnt is (CN)₂C₂S₂ ²⁻] gave a smaller nitrate reduction activation energy for RY⁻ is Cl⁻, compared with RY⁻ is PhS⁻, although experimentally only the phenyl thiolato complex and not the chloro complex was found to be a functional NR model. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Proteome-Wide Inference of Src Homology 3 Domain-Binding Peptides

Src homology 3 (SH3) domain is a versatile protein structure module that participates in mediating various protein?Cprotein binding events by specifically recognizing proline-rich region of diverse plasmatic proteins. Reliable and fast inference of SH3-binding partners over the human proteome are fundamentally important for our understanding of the molecular functions and biological implications underlying SH3-mediated signaling network. Herein, we employ an atom-realistic protocol to perform proteome-wide inference of SH3-binding peptides using the information gained from both the primary sequence of affinity-known peptides and the interaction properties deriving from SH3?Cpeptide complex structures. It is revealed that the binding affinity and specificity of peptides to SH3 domain are co-contributed from electrostatic, steric and hydrophobic effects, and the hydrophobicity and electrostatic property at P₂, P₀ and/or P_?3 play an essential role in determining the binding. In addition, SH3 domain exhibits a broad specificity of recognizing its ligands and thus a large number of protein candidates that might be the potential interacting partners of SH3 domain are extracted from the human proteome, from which several samples are suggested to be the highly promising SH3 binders.     

Exploring the systematic effect of N-substituted PxxP motifs on peptoid affinity to ARHGEF5/TIM SH3 domain and its relationship with ARHGEF5/TIM activation

The TIM protein is a short isoform of full-length Rho guanine nucleotide exchange factor 5 (ARHGEF5), which acts as a functional regulator of Rho-dependent signaling pathways by activating the Rho family of GTPases. The activation is auto-inhibited by a putative helix N-terminal to the DH domain of TIM, which is stabilized by the intramolecular interaction of C-terminal SH3 domain with a proline-rich region ⁴⁷SSPRQP RKAL⁵⁶ (termed as SSP peptide) between the putative helix and the DH domain. Previously, we demonstrate that the auto-inhibitory state of TIM protein can be relieved by targeting its SH3 domain with rationally designed peptide ligands. However, the designed natural peptides have only a moderately increased affinity (~2-fold) as compared to the cognate SH3-SSP interaction and are susceptible to protease degradation. Here, considering that proline is the only endogenous N-substituted amino acid that plays a critical role in SH3-peptide recognition, the two key proline residues Pro49 and Pro52 in the core ⁴⁹PxxP ⁵² motif of SSP peptide are systematically replaced by 19 N-substituted amino acid types to derive a variety of nonnatural peptoid ligands for TIM SH3 domain. Dynamics and energetics analyses reveal that the replacement would impair the active polyproline II (PPII) helical conformation of SSP peptide due to lack of structural constraint introduced by the five-membered ring of native proline side-chains, thus increasing the peptide flexibility that could incur a large entropy penalty upon binding to the domain. However, the impairment is not very significant and the peptide affinity may also be restored and improved if the N-substituted motif of derived peptiod ligands can effectively interact with the PxxP-binding site of TIM SH3 domain. Consequently, a number of potent peptoids are successfully designed by fluorescence spectroscopy confirmation, in which three (ie, SSP[N-Ile49, N-Asn52], SSP[N-Phe49, N-Gln52], and SSP[N-Tyr49, N-Asn52]) exhibit considerably increased affinity (K_d = 0.09, 0.07, and 0.04 μM, respectively) relative to the native SSP peptide (K_d = 0.87 μM). In addition, guanine nucleotide exchange assays also substantiate that the designed SH3-targeted peptiods can effectively enhance TIM-catalyzed RhoA exchange activity (EA), which is observed to present an exponential relationship with the measured SH3-peptoid binding affinity (pK_d).     

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 high-resolution NMR structure of the R21A Spc-SH3:P41 complex: Understanding the determinants of binding affinity by comparison with Abl-SH3

Background  

SH3 domains are small protein modules of 60–85 amino acids that bind to short proline-rich sequences with moderate-to-low affinity and specificity. Interactions with SH3 domains play a crucial role in regulation of many cellular processes (some are related to cancer and AIDS) and have thus been interesting targets in drug design. The decapeptide APSYSPPPPP (p41) binds with relatively high affinity to the SH3 domain of the Abl tyrosine kinase (Abl-SH3), while it has a 100 times lower affinity for the α-spectrin SH3 domain (Spc-SH3).