Solid-phase synthesis of Stat3 inhibitors incorporating O-carbamoylserine and O-carbamoylthreonine as glutamine mimics

O-Carbamoylserine and O-carbamoylthreonine are glutamine analogues that were incorporated into a Stat3 inhibitory peptide to probe the requirements of Gln at the pY+3 position. Fmoc-Ser-NHBn and Fmoc-Thr-NHBn were converted to nitrophenyl carbonates and were attached to Rink resin via a side-chain carbamate linkage. After assembly of the peptide, acid treatment resulted in O-carbamoylserine and O-carbamoylthreonine-containing peptides. The order of affinity for Stat3 was Gln > Ser(CONH2) > Thr(CONH2) suggesting a relatively tight binding pocket for the side chain of glutamine.     

Polyproline, beta-turn helices. Novel secondary structures proposed for the tandem repeats within rhodopsin, synaptophysin, synexin, gliadin, RNA polymerase II, hordein, and gluten

Seven proteins each contain 8 to 52 tandem repeats of a unique class of oligopeptide. The consensus peptide for each is rhodopsin Tyr Pro Pro Gln Gly synaptophysin Tyr Gly Pro Gln Gly synexin Tyr Pro Pro Pro Pro Gly gliadin Tyr Pro Pro Pro Gln Pro RNA polymerase II Tyr Ser Pro Thr Ser Pro Ser hordein Phe Pro Gln Gln Pro Gln Gln Pro gluten Tyr Pro Thr Ser Pro Gln Gln Gly Tyr Although there is obvious variation of sequence and of length, the penta- to nonapeptides share an initial Tyr (or Phe) and have high Pro contents and abundant Gly, Gln, and Ser. We have evaluated helical models that both recognize the uniqueness of these sequence repeats and accommodate variations on the basic theme. We have developed a group of related helical models for these proteins with about three oligopeptide repeats per turn of 10-20 A. These models share several common features: Most of the phi dihedral angles are -54 degrees, to accommodate Pro at all positions except the first (Tyr). Except for the beta-turns, most psi dihedral angles are near +140 degrees as found in polyproline. Each oligopeptide has at least one beta-turn; several have two. Some contain a cis-Tyr, Pro peptide bond; a few have a cis-bond plus one beta-turn. Tyr side chains vary from totally exposed to buried within the helices and could move to accommodate either external hydrophobic interactions or phosphorylation. The several related structures seem to be readily interconverted without major change in the overall helical parameters, and therein may lie the key to their functions.     

Reexamination of the recognition preference of the specificity pocket of the Abl SH3 domain

Src homology-3 (SH3) domains mediate important protein-protein interactions in a variety of normal and pathological cellular processes, thus providing an attractive target for the selective interference of SH3-dependent signaling events that govern these processes. Most SH3 domains recognize proline-rich peptides with low affinity and poor selectivity, and the goal to design potent and specific ligands for various SH3 domains remains elusive. Better understanding of the molecular basis for SH3 domain recognition is needed in order to design such ligands with potency and specificity. In this report, we seek to define a clear recognition preference of the specificity pocket of the Abl SH3 domain using targeted synthetic peptide libraries. High-resolution affinity panning coupled with mass spectrometric readout allows for quick identification of Trp as the preferred fourth residue in the decapeptide ligand APTWSPPPPP, which binds to Abl SH3 four times stronger than does the decapeptide containing Tyr or Phe in the fourth position. This finding is in contrast to several reports that Tyr is the only residue selected from phage displayed peptide libraries that interacts with the specificity pocket of Abl SH3. This simple, unbiased approach can fine-tune the affinity and selectivity of both natural and unnatural SH3 ligands whose consensus binding sequence has been pre-defined by combinatorial library methods.     

Reciprocal regulation of Hck activity by phosphorylation of Tyr(527) and Tyr(416). Effect of introducing a high affinity intramolecular SH2 ligand

The Src family tyrosine kinase Hck possesses two phosphorylation sites, Tyr(527) and Tyr(416), that affect the catalytic activity in opposite ways. When phosphorylated, Tyr(527) and residues C-terminal to it are involved in an inhibitory intramolecular interaction with the SH2 domain. However, this sequence does not conform to the sequence of the high affinity SH2 ligand, pYEEI. We mutated this sequence to YEEI and show that this mutant form of Hck cannot be activated by exogenous SH2 ligands. The SH3 domain of Hck is also involved in an inhibitory interaction with the catalytic domain. The SH3 ligand Nef binds to and activates YEEI-Hck mutant in a similar manner to wild-type Hck, indicating that disrupting the SH3 interaction overrides the strengthened SH2 interaction. The other phosphorylation site, Tyr(416), is the autophosphorylation site in the activation loop. Phosphorylation of Tyr(416) is required for Hck activation. We mutated this residue to alanine and characterized its catalytic activity. The Y416A mutant shows a higher K(m) value for peptide and a lower V(max) than autophosphorylated wild-type Hck. We also present evidence for cross-talk between the activation loop and the intramolecular binding of the SH2 and SH3 domains.     

Dissection of the energetic coupling across the Src SH2 domain-tyrosyl phosphopeptide interface

Src Homology (SH2) domains play critical roles in signaling pathways by binding to phosphotyrosine (pTyr)-containing sequences, thereby recruiting SH2 domain-containing proteins to tyrosine-phosphorylated sites on receptor molecules. Investigations of the peptide binding specificity of the SH2 domain of the Src kinase (Src SH2 domain) have defined the EEI motif C-terminal to the phosphotyrosine as the preferential binding sequence. A subsequent study that probed the importance of eight specificity-determining residues of the Src SH2 domain found two residues which when mutated to Ala had significant effects on binding: Tyr beta D5 and Lys beta D3. The mutation of Lys beta D3 to Ala was particularly intriguing, since a Glu to Ala mutation at the first (+1) position of the EEI motif (the residue interacting with Lys beta D3) did not significantly affect binding. Hence, the interaction between Lys beta D3 and +1 Glu is energetically coupled. This study is focused on the dissection of the energetic coupling observed across the SH2 domain-phosphopeptide interface at and around the +1 position of the peptide. It was found that three residues of the SH2 domain, Lys beta D3, Asp beta C8 and AspCD2 (altogether forming the so-called +1 binding region) contribute to the selection of Glu at the +1 position of the ligand. A double (Asp beta C8Ala, AspCD2Ala) mutant does not exhibit energetic coupling between Lys beta D3 and +1 Glu, and binds to the pYEEI sequence 0.3 kcal/mol tighter than the wild-type Src SH2 domain. These results suggest that Lys beta D3 in the double mutant is now free to interact with the +1 Glu and that the role of Lys beta D3 in the wild-type is to neutralize the acidic patch formed by Asp beta C8 and AspCD2 rather than specifically select for a Glu at the +1 position as it had been hypothesized previously. A triple mutant (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) has reduced binding affinity compared to the double (Asp beta C8Ala, AspCD2Ala) mutant, yet binds the pYEEI peptide as well as the wild-type Src SH2 domain. The structural basis for such high affinity interaction was investigated crystallographically by determining the structure of the triple (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) mutant bound to the octapeptide PQpYEEIPI (where pY indicates a phosphotyrosine). This structure reveals for the first time contacts between the SH2 domain and the -1 and -2 positions of the peptide (i.e. the two residues N-terminal to pY). Thus, unexpectedly, mutations in the +1 binding region affect binding of other regions of the peptide. Such additional contacts may account for the high affinity interaction of the triple mutant for the pYEEI-containing peptide.     

Prediction of binding affinities between the human amphiphysin-1 SH3 domain and its peptide ligands using homology modeling, molecular dynamics and molecular field analysis

The SH3 domain of the human protein amphiphysin-1, which plays important roles in clathrin-mediated endocytosis, actin function and signaling transduction, can recognize peptide motif PXRPXR (X is any amino acid) with high affinity and specificity. We have constructed a complex structure of the amphiphysin-1 SH3 domain and a high-affinity peptide ligand PLPRRPPRA using homology modeling and molecular docking, which was optimized by molecular dynamics (MD). Three-dimensional quantitative structure-affinity relationship (3D-QSAR) analyses on the 200 peptides with known binding affinities to the amphiphysin-1 SH3 domain was then performed using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). The best CoMSIA model showed promising predictive power, giving good predictions for about 95% of the peptides in the test set (absolute prediction errors less than 1.0). It was used to validate peptide-SH3 binding structure and provide insight into the structural requirements for binding of peptides to SH3 domains. Finally, MD simulations were performed to analyze the interaction between the SH3 domain and another peptide GFPRRPPPRG that contains with the PXRPXsR (s represents residues with small side chains) motif. MD simulations demonstrated that the binding conformation of GFPRRPPPRG is quite different from that of PLPRRPPRAA especially the four residues at the C terminal, which may explain why the CoMSIA model cannot give good predictions on the peptides of the PXRPXsR motif. Because of its efficiency and predictive power, the 3D-QSAR model can be used as a scoring filter for predicting peptide sequences bound to SH3 domains.     

Mutational investigation of the specificity determining region of the Src SH2 domain

SH2 domains are protein modules which bind tyrosine phosphorylated sequences in many signaling pathways. These domains contain two regions with specialized functions: residues in one region form a deep pocket into which the phosphotyrosine of the target inserts, while the other region contains the so-called "specificity determining residues" which interact with the three residues C-terminal to the phosphotyrosine in the target. Here, titration calorimetry and site-directed mutagenesis have been used to probe the importance of eight specificity determining residues of the SH2 domain of the Src kinase involved in contacts with its tyrosine phosphorylated consensus peptide target (sequence pYEEI where pY indicates a phosphotyrosine). Mutating six of these eight residues to Ala individually, resulted in a threefold or less loss in binding affinity; hence the majority of the residues in the specificity determining region are by themselves of minimal importance for binding. Two residues were found to have significant effects on binding: Tyr betaD5 and Lys betaD3. Tyr betaD5 was the most crucial residue as evidenced by the 30-fold loss in affinity when Tyr betaD5 is mutated to Ile. However, while this mutation eliminated the specificity of the Src SH2 domain for the pYEEI peptide sequence, it was not sufficient to switch the specificity of the Src SH2 domain to that of a related SH2 domain which has an Ile at the betaD5 position. Mutation of Lys betaD3 to an Ala residue resulted in a modest reduction in binding affinity (sevenfold). It is interesting that this mutation resulted in a change of specificity affecting the selection of the +1 position residue C-terminal to the phosphotyrosine. Except for the Lys betaD3-+1 Glu interaction which is significantly coupled, only weak energetic coupling was observed across the binding interface, as assessed using double mutant cycles. The results of this study suggest that interactions involving the specificity determining region of SH2 domains may be insufficient by themselves to target single SH2 domains to particular phosphorylated sites.     

Unique structural determinants for Stat3 recruitment and activation by the granulocyte colony-stimulating factor receptor at phosphotyrosine ligands 704 and 744

G-CSFR cytoplasmic tyrosine (Y) residues (Y704, Y729, Y744, and Y764) become phosphorylated upon ligand binding and recruit specific Src homology 2 domain-containing proteins that link to distinct yet overlapping programs for myeloid cell survival, differentiation, proliferation, and activation. The structural basis for recruitment specificity is poorly understood but could be exploited to selectively target deleterious G-CSFR-mediated signaling events such as aberrant Stat3 activation demonstrated in a subset of acute myeloid leukemia patients with poor prognosis. Recombinant Stat3 bound to G-CSFR phosphotyrosine peptide ligands pY704VLQ and pY744LRC with similar kinetics. Testing of three models for Stat3 Src homology 2-pY ligand binding in vitro and in vivo revealed unique determinants for Stat3 recruitment and activation by the G-CSFR, the side chain of Stat3 R609, which interacts with the pY ligand phosphate group, and the peptide amide hydrogen of E638, which bonds with oxygen/sulfur within the + 3 Q/C side chain of the pY ligand when it assumes a beta turn. Thus, our findings identify for the first time the structural basis for recruitment and activation of Stat3 by the G-CSFR and reveal unique features of this interaction that can be exploited to target Stat3 activation for the treatment of a subset of acute myeloid leukemia patients.     

19F NMR studies of solvent exposure and peptide binding to an SH3 domain

(19)F NMR was used to study topological features of the SH3 domain of Fyn tyrosine kinase for both the free protein and a complex formed with a binding peptide. Metafluorinated tyrosine was biosynthetically incorporated into each of 5 residues of the G48M mutant of the SH3 domain (i.e. residues 8, 10, 49 and 54 in addition to a single residue in the linker region to the C-terminal polyhistidine tag). Distinct (19)F NMR resonances were observed and subsequently assigned after separately introducing single phenylalanine mutations. (19)F NMR chemical shifts were dependent on protein concentration above 0.6 mM, suggestive of dimerization via the binding site in the vicinity of the tyrosine side chains. (19)F NMR spectra of Fyn SH3 were also obtained as a function of concentration of a small peptide (2-hydroxynicotinic-NH)-Arg-Ala-Leu-Pro-Pro-Leu-Pro-diaminopropionic acid -NH(2), known to interact with the canonical polyproline II (PPII) helix binding site of the SH3 domain. Based on the (19)F chemical shifts of Tyr8, Tyr49, and Tyr54, as a function of peptide concentration, an equilibrium dissociation constant of 18 +/- 4 microM was obtained. Analysis of the line widths suggested an average exchange rate, k(ex), associated with the peptide-protein two-site exchange, of 5200 +/- 600 s(-1) at a peptide concentration where 96% of the FynSH3 protein was assumed to be bound. The extent of solvent exposure of the fluorine labels was studied by a combination of solvent isotope shifts and paramagnetic effects from dissolved oxygen. Tyr54, Tyr49, Tyr10, and Tyr8, in addition to the Tyr on the C-terminal tag, appear to be fully exposed to the solvent at the metafluoro position in the absence of binding peptide. Tyr54 and, to some extent, Tyr10 become protected from the solvent in the peptide bound state, consistent with known structural data on SH3-domain peptide complexes. These results show the potential utility of (19)F-metafluorotyrosine to probe protein-protein interactions in conjunction with paramagnetic contrast agents.     

Long-range effects in protein--ligand interactions mediate peptide specificity in the human major histocompatibilty antigen HLA-B27 (B*2701).

B*2701 differs from all other HLA-B27 subtypes of known peptide specificity in that, among its natural peptide ligands, arginine is not the only allowed residue at peptide position 2. Indeed, B*2701 is unique in binding many peptides with Gln2 in vivo. However, the mutation (Asp74Tyr) responsible for altered selectivity is far away from the B pocket of the peptide binding site to which Gln/Arg2 binds. Here, we present a model that explains this effect. It is proposed that a new rotameric state of the conserved Lys70 is responsible for the unique B*2701 binding motif. This side chain should be either kept away from pocket B through its interaction with Asp74 in most HLA-B27 subtypes, or switched to this pocket if residue 74 is Tyr as in B*2701. Involvement of Lys70 in pocket B would thus allow binding of peptides with Gln2. Binding of Arg2-containing peptides to B*2701 is also possible because Lys70 could adopt another conformation, H-bonded to Asn97, which preserves the same binding mode of Arg2 as in B*2705. This model was experimentally validated by mutating Lys70 into Ala in B*2701. Edman sequencing of the B*2701(K70A) peptide pool showed only Arg2, characteristic of HLA-B27-bound peptides, and no evidence for Gln2. This supports the computational model and demonstrates that allowance of B*2701 for peptides with Gln2 is due to the long-range effect of the polymorphic residue 74 of HLA-B27, by inducing a conformational switch of the conserved Lys70.     

Mutational analysis and membrane-interactions of the beta-sheet-like N-terminal domain of the pediocin-like antimicrobial peptide sakacin P

To gain insight into how the N-terminal three-stranded beta-sheet-like domain in pediocin-like antimicrobial peptides positions itself on membranes, residues in the well-conserved (Y)YGNGV-motif in the domain were substituted and the effect of the substitutions on antimicrobial activity and binding of peptides to liposomes was determined. Peptide-liposome interactions were detected by measuring tryptophan-fluorescence upon exposing liposomes to peptides in which a tryptophan residue had been introduced in the N-terminal domain. The results revealed that the N-terminal domain associates readily with anionic liposomes, but not with neutral liposomes. The electrostatic interactions between peptides and liposomes facilitated the penetration of some of the peptide residues into the liposomes. Measuring the antimicrobial activity of the mutated peptides revealed that the Tyr2Leu and Tyr3Leu mutations resulted in about a 10-fold reduction in activity, whereas the Tyr2Trp, Tyr2Phe, Tyr3Trp and Tyr3Phe mutations were tolerated fairly well, especially the mutations in position 3. The Val7Ile mutation did not have a marked detrimental effect on the activity. The Gly6Ala mutation was highly detrimental, consistent with Gly6 being in one of the turns in the beta-sheet-like N-terminal domain, whereas the Gly4Ala mutation was tolerated fairly well. All mutations involving Asn5, including the conservative mutations Asn5Gln and Asn5Asp, were very deleterious. Thus, both the polar amide group on the side chain of Asn5 and its exact position in space were crucial for the peptides to be fully active. Taken together, the results are consistent with Val7 positioning itself in the hydrophobic core of target membranes, thus forcing most of the other residues in the N-terminal domain into the membrane interface region: Tyr3 and Asn5 in the lower half with their side chains pointing downward and approaching the hydrophobic core, Tyr2, Gly4 and His8 and 12 in the upper half, Lys1 near the middle of the interface region, and the side chain of Lys11 pointing out toward the membrane surface.     

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.     

Structure characterization of the central repetitive domain of high molecular weight gluten proteins. I. Model studies using cyclic and linear peptides.

The high molecular weight (HMW) proteins from wheat contain a repetitive domain that forms 60-80% of their sequence. The consensus peptides PGQGQQ and GYYPTSPQQ form more than 90% of the domain; both are predicted to adopt beta-turn structure. This paper describes the structural characterization of these consensus peptides and forms the basis for the structural characterization of the repetitive HMW domain, described in the companion paper. The cyclic peptides cyclo-[PGQGQQPGQGQQ] (peptide 1), cyclo-[GYYPTSPQQGA] (peptide 2), and cyclo-[PGQGQQGYYPTSPQQ] (peptide 3) were prepared using a novel synthesis route. In addition, the linear peptides (PGQGQQ)n (n = 1, 3, 5) were prepared. CD, FTIR, and NMR data demonstrated a type II beta-turn structure at QPGQ in the cyclic peptide 1 that was also observed in the linear peptides 9PGQGQQ)n. A type I beta-turn was observed at YPTS and SPQQ in peptides 2 and 3, with additional beta-turns of either type I or II at GAGY (peptide 2) and QQGY (peptide 3). The proline in YPTS showed considerable cis/trans isomerization, with up to 50% of the population in the cis-conformation; the other prolines were more than 90% in the trans conformation. The conversion from trans to cis destroys the type I beta-turn at YPTS, but leads to an increase in turn character at SPQQ and GAGY (peptide 2) or QQGY (peptide 3).     

Characterization of the increased biological potency in BALB/C 3T3 cells of two analogs of human insulinlike growth factor I which have reduced affinity for the 28 K cell-derived binding protein

We have characterized the biological activity of two analogs of insulinlike growth factor I (IGF I) which have significantly reduced affinity for the soluble 28 K binding proteins which are secreted by various cell types. The analogs, which were made by site-directed mutagenesis of a synthetic gene encoding for IGF I, are [Gln 3, Ala 4, Tyr 15, Leu 16] IGF I and an analog in which the first 16 amino acids of IGF I were replaced with the first 17 amino acids of insulin (B-chain mutant). These two peptides have 100-fold and greater than 1,000-fold lower affinity, respectively, than IGF I for the 28 K binding protein present in the conditioned medium of two cell types, the clonal rat vascular smooth muscle line A10, and BALB/C 3T3 cells. The 28 K protein secreted by BALB/C 3T3 cells has fivefold-lower apparent affinity for both IGF I and [Gln 3, Ala 4, Tyr 15, Leu 16] IGF I than does the 28 K protein secreted by A 10 cells. Conditioned medium from these two cell types has similar amounts of unoccupied 28 K protein as evidenced by the ability of 125I-IGF I to specifically bind to and be covalently bound to the protein after treatment with the bifunctional cross-linking reagent disuccinimidyl suberate. In the presence of 0.1% calf serum, IGF I and [Gln 3, Ala 4, Tyr 15, Leu 16] IGF I stimulate DNA synthesis in A10 cells with ED50 = 0.4 nM, and in BALB/C 3T3 cells with ED50 = 10 nM and 1.3 nM, respectively. Thus, these peptides are equipotent in A10 cells, but the mutant peptide is ten times more active than IGF I in BALB/C 3T3 cells. A10 cells can be made ten times less sensitive to IGF I by performing the incubation in the presence of conditioned media from BALB/C 3T3 cells but not from A10 cells. The activity of [Gln 3, Ala 4, Tyr 15, Leu 16] IGF I is not altered under these conditions. Thus, the conditioned media, which contain 28 K proteins secreted by A10 cells and BALB/C 3T3 cells, have different effects on the biological action of IGF I. These data suggest that the 28 K binding proteins can have important effects on the sensitivity of tissues to IGF I and that the B-chain mutant and [Gln 3, Ala 4, Tyr 15, Leu 16] IGF I will be useful in assessing the biological role of these proteins.     

Targeting Stat3 with G-quartet oligodeoxynucleotides in human cancer cells

Stat3 is an oncogene that is activated in many human cancer cells. Genetic approaches that disrupt Stat3 activity result in inhibition of cancer cell growth and enhanced cell apoptosis supporting the development of novel drugs targeting Stat3 for cancer therapy. G-quartet oligodeoxynucleotides (ODNs) were demonstrated to be potent inhibitors of Stat3 DNA binding activity in vitro with the G-quartet ODN, T40214, having an IC(50) of 7 microM. Computer-simulated docking studies indicated that G-quartet ODNs mainly interacted with the SH2 domain of Stat3 and were capable of inserting between the SH2 domains of Stat3 dimers bound to DNA. We demonstrated that the G-rich ODN T40214, which forms a G-quartet structure at intracellular but not extracellular K+ ion concentrations, is delivered efficiently into the cytoplasm and nucleus of cancer cells where it inhibited IL-6-stimulated Stat3 activation and suppressed Stat3-mediated upregulation of bcl-x and mcl-1 gene expression. Thus, G-quartet represents a new class of drug for targeting of Stat3 within cancer cells.     

Discovery of the catechol structural moiety as a Stat3 SH2 domain inhibitor by virtual screening

The Stat3 SH2 domain is essential for its activation, and development of a potent SH2 inhibitor will be therapeutically valuable in treating cancers with constant Stat3 activation. We report here the identification of the catechol (1,2-dihydroxybenzene) structural moiety by virtual screening as a Stat3 SH2 inhibitor. The catechol compound docked to the Stat3 SH2 domain in computer modeling forms hydrogen bonds with the conserved pTyr-interacting amino acids. In the biochemical assay, a catechol-containing compound, but not the hydroxyl group-acetalized analogue, was able to inhibit Stat3 DNA-binding activity. Furthermore, the catechol compound was demonstrated to compete with pTyr peptides in binding to the Stat3 SH2 domain, suggesting that the catechol moiety is a pTyr bioisostere and may potentially be used for designing cell-permeable SH2 inhibitors. In our preliminary effort, we also demonstrated that the potency of catechol compound as Stat3 SH2 inhibitors could be improved by modifying the non-catechol part of the compound structure.     

Interactions of phosphatidylinositol 3-kinase Src homology 3 domain with its ligand peptide studied by absorption, circular dichroism, and UV resonance raman spectroscopies

Absorption, circular dichroism (CD), and UV resonance Raman (UVRR) spectroscopies were applied to selectively examine the environmental and structural changes of Trp and Tyr residues in the phosphatidylinositol 3-kinase (PI3K) SH3 domain induced by ligand association. Comparison of the spectra of PI3K SH3 in the presence or absence of its ligand peptide RLP1 (RKLPPRPSK) indicated that RLP1 binding changed the environment of Trp55 of the SH3 to be more hydrophilic and its H bonding weaker and that of Tyr residues to be more hydrophobic. The D21N mutant (Asp21 --> Asn) of the SH3 yielded a UV CD distinct from that of the wild type, and its spectral changes induced by RLP1 binding were smaller and different from those of the wild type in absorption, CD, and UVRR spectra, suggesting that the mutation of conserved Asp21 affected the conformation of the ligand binding cleft and thus might lead to the decrease in the ligand affinity. These data provide direct evidence for the occurrence of environmental and structural changes of PI3K SH3 by the association of a ligand and the D21N mutation.     

Proteomic screening method for phosphopeptide motif binding proteins using peptide libraries

Phosphopeptide binding domains mediate the directed and localized assembly of protein complexes essential to intracellular kinase signaling. To identify phosphopeptide binding proteins, we developed a proteomic screening method using immobilized partially degenerate phosphopeptide mixtures combined with SILAC and microcapillary LC-MS/MS. The method was used to identify proteins that specifically bound to phosphorylated peptide library affinity matrices, including pTyr, and the motifs pSer/pThr-Pro, pSer/pThr-X-X-X-pSer/pThr, pSer/pThr-Glu/Asp, or pSer/pThr-pSer/pThr in degenerate sequence contexts. Heavy and light SILAC lysates were applied to columns containing these phosphorylated and nonphosphorylated (control) peptide libraries respectively, and bound proteins were eluted, combined, digested, and analyzed by LC-MS/MS using a hybrid quadrupole-TOF mass spectrometer. Heavy/light peptide ion ratios were calculated, and peptides that yielded ratios greater than ～3:1 were considered as being from potential phosphopeptide binding proteins since this ratio represents the lowest ratio from a known positive control. Many of those identified were known phosphopeptide-binding proteins, including the SH2 domain containing p85 subunit of PI3K bound to pTyr, 14-3-3 bound to pSer/pThr-Asp/Glu, polo-box domain containing PLK1 and Pin1 bound to pSer/pThr-Pro, and pyruvate kinase M2 binding to pTyr. Approximately half of the hits identified by the peptide library screens were novel. Protein domain enrichment analysis revealed that most pTyr hits contain SH2 domains, as expected, and to a lesser extent SH3, C1, STAT, Tyr phosphatase, Pkinase, C2, and PH domains; however, pSer/pThr motifs did not reveal enriched domains across hits.