Analysis of interactions of signaling proteins with phage-displayed ligands by fluorescence correlation spectroscopy

Fluorescent correlation spectroscopy (FCS) was used to measure binding affinities of ligands to ligates that are expressed by phage-display technology. Using this method we have quantified the binding of the 14-3-3 signaling protein to artificial peptide ligand. As a ligand we used the R18 artificial peptide expressed as a fusion in the cpIII coat protein that is present in 3 to 5 copies in an M13 phage. Comparisons of binding affinities were made with free R18 ligands using FCS. The result showed a relatively high binding affinity for the phage-displayed R18 peptide compared with binding to free fluorescently labeled R18. Quantification was supported by titration of the phage numbers using atomic force microscopy (AFM). AFM was shown to accurately determine phage numbers in solution as a good alternative for electron microscopy. It was shown to give reliable data that correlated perfectly with those of the viable phage numbers determined by classical bacterial infection studies. In conclusion, a very fast and sensitive method for the selection of new peptide ligands or ligates based on a quantitative assay in solution has been developed.     

Selection of staphylococcal enterotoxin B (SEB)-binding peptide using phage display technology

In this study, peptides were selected to recognize staphylococcal enterotoxin B (SEB) which cause food intoxication and can be used as a biological war agent. By using commercial M13 phage library, single plaque isolation of 38 phages was done and binding affinities were investigated with phage-ELISA. The specificities of the selected phage clones showing high affinity to SEB were checked by using different protein molecules which can be found in food samples. Furthermore, the affinities of three selected phage clones were determined by using surface plasmon resonance (SPR) sensors. Sequence analysis was realized for three peptides showing high binding affinity to SEB and WWRPLTPESPPA, MNLHDYHRLFWY, and QHPQINQTLYRM amino acid sequences were obtained. The peptide sequence with highest affinity to SEB was synthesized with solid phase peptide synthesis technique and thermodynamic constants of the peptide-SEB interaction were determined by using isothermal titration calorimetry (ITC) and compared with those of antibody-SEB interaction. The binding constant of the peptide was determined as 4.2 ± 0.7 × 10⁵ M⁻¹ which indicates a strong binding close to that of antibody.     

A G protein gamma subunit peptide stabilizes a novel muscarinic receptor state

A prenylated peptide specific to the C terminal tail of a G protein gamma subunit type, gamma5, inhibits activation of a G protein by the M2 muscarinic receptor. The gamma5 peptide was tested for direct effects on the M2 receptor's properties. The wild type gamma5 peptide reduced the affinity of M2 for the agonist, carbachol, more than 5-fold in an antagonist displacement assay. The peptide was inactive when its amino acid sequence was scrambled or when it was unprenylated. Although the wild type peptide reduced the affinity of M2 for the antagonist QNB, it had no effect on the antagonists NMS or atropine. These results suggest that in the presence of the peptide the M2 receptor adopts a novel conformational state that affects the ligand binding surface. The results also suggest that the G protein gamma5 subunit tail interacts with a receptor.     

Identification of a RON tyrosine kinase receptor binding peptide using phage display technique and computational modeling of its binding mode

RON (Recepteur d’Origine Nantais) tyrosine kinase receptor is a promising target for therapeutic intervention in cancer therapy. The aim of this work was identification of RON-binding peptides using phage display and computational modeling their mode of binding. A 12-mer peptide phage library was utilized to perform biopanning against RON. The RON-binding ability of the selected peptide-displaying phage and their possible binding sites were examined by ELISA. Binding modes and affinities were also predicted by docking and molecular dynamics (MD) simulation. The results of ELISA experiment showed that P6 peptide displaying phage has higher affinity for RON compared to others and its binding site is located out of ligand binding site. Docking and MD simulation results also indicated higher affinity of P6 to RON as well as its exosite-binding feature. Taken together, our data suggest a capacity for P6 peptide (FEHSLYKEMTHL) to be utilized as RON binding agent, and hence be used for various purposes, including design of drug delivery systems for transferring cytotoxic agents to RON-positive cancer cells, interfering with RON signaling, peptidomimetics design, and diagnostic imaging.     

Purification of anti-MUC1 antibodies by peptide mimotope affinity chromatography using peptides derived from a polyvalent phage display library

A polyvalent, lytic phage display system (T7Select415-1b) displaying a random peptide library has been investigated for its ability to discover novel mimotopes reactive with the therapeutic monoclonal antibody C595. Sequence analysis of enriched phage lead to the identification of a predominant sequence RNREAPRGKICS, and two other consensus sequences RXXP and RXP. The novel synthetic peptide RNREAPRGKICS was linked to beaded agarose and the performance as a mimotope affinity chromatography matrix evaluated. Antibody purified using the novel matrix was found to be of higher specific reactivity than antibody purified using the conventional epitope matrix (peptide APDTRPAPG). The RNREAPRGKICS peptide binding to C595 demonstrated a higher equilibrium association constant (K(A)=0.75 x 10(6)) than the epitope peptide (K(A)=0.16 x 10(6)). Circular dichroism showed that the novel peptide had a more highly ordered structure at 4 degrees C and room temperature, than the epitope peptide.     

The predicted binding site and dynamics of peptide inhibitors to the Methuselah GPCR from Drosophila melanogaster

Peptide inhibitors of Methuselah (Mth), a G protein-coupled receptor (GPCR), were reported that can extend the life span of Drosophila melanogaster. Mth is a class B GPCR, which is characterized by a large, N-terminal ectodomain that is often involved with ligand recognition. The crystal structure of the Mth ectodomain, which binds to the peptide inhibitors with high affinity, was previously determined. Here we report the predicted structures for RWR motif peptides in complex with the Mth ectodomain. We studied representatives of both Pro-class and Arg-class RWR motif peptides and identified ectodomain residues Asp139, Phe130, Asp127, and Asp78 as critical in ligand binding. To validate these structures, we predicted the effects of various ligand mutations on the structure and binding to Mth. The binding of five mutant peptides to Mth was characterized experimentally by surface plasmon resonance, revealing measured affinities that are consistent with predictions. The electron density map calculated from our MD structure compares well with the experimental map of a previously determined peptide/Mth crystal structure and could be useful in refining the current low-resolution data. The elucidation of the ligand binding site may be useful in analyzing likely binding sites in other class B GPCRs.     

Evaluation of Phage Display Discovered Peptides as Ligands for Prostate-Specific Membrane Antigen (PSMA)

The aim of this study was to identify potential ligands of PSMA suitable for further development as novel PSMA-targeted peptides using phage display technology. The human PSMA protein was immobilized as a target followed by incubation with a 15-mer phage display random peptide library. After one round of prescreening and two rounds of screening, high-stringency screening at the third round of panning was performed to identify the highest affinity binders. Phages which had a specific binding activity to PSMA in human prostate cancer cells were isolated and the DNA corresponding to the 15-mers were sequenced to provide three consensus sequences: GDHSPFT, SHFSVGS and EVPRLSLLAVFL as well as other sequences that did not display consensus. Two of the peptide sequences deduced from DNA sequencing of binding phages, SHSFSVGSGDHSPFT and GRFLTGGTGRLLRIS were labeled with 5-carboxyfluorescein and shown to bind and co-internalize with PSMA on human prostate cancer cells by fluorescence microscopy. The high stringency requirements yielded peptides with affinities K_D∼1 µM or greater which are suitable starting points for affinity maturation. While these values were less than anticipated, the high stringency did yield peptide sequences that apparently bound to different surfaces on PSMA. These peptide sequences could be the basis for further development of peptides for prostate cancer tumor imaging and therapy.     

Further evidence that the serotonin receptor in the rat stomach fundus is not 5HT1A or 5HT1B

The nature of the receptor mediating serotonin contraction in the rat stomach fundus has not been clearly associated with either 5HT1 or 5HT2 receptors. We have explored the possibility that such receptors in the rat fundus may better correlate with 5HT1A or 5HT1B receptor subtypes as defined by radiolabeled ligand binding studies with brain cortical membranes. Meta chlorophenylpiperazine (CPP) and meta trifluoromethylphenylpiperazine (TFMPP), selective ligands for the 5HT1B receptor and LY165163, a selective ligand for the 5HT1A receptor, have been evaluated for their agonist and antagonist activity at serotonin receptors in the rat stomach fundus. CPP and TFMPP were partial agonists in the rat stomach fundus whereas LY165163 showed no agonist activity in this smooth muscle in concentrations up to 10(-4)M. All three phenylpiperazines antagonized serotonin-induced contractions in the rat stomach fundus. The affinity for serotonin receptors in the rat fundus was similar for all three phenylpiperazines in spite of the reported selectivity of MCPP and TFMPP for 5HT1B and of LY165163 for 5HT1A receptors. Furthermore, the affinity of these agents for serotonin receptors in the rat stomach fundus did not agree with their reported affinity for either 5HT1A or 5HT1B binding sites in rat cortical membranes. Thus, the similarity in affinities of these phenylpiperazine derivatives for serotonin receptors mediating contraction in the rat fundus along with their different affinities for 5HT1A and 5HT1B binding sites argues against the possibility that the serotonin receptor in the rat fundus is of the 5HT1A or 5HT1B subtype of serotonin receptor.     

Development of p-benzoylbenzoylated [N,C,rANP(1-28)]pBNP32 (pBNP1) derivatives and affinity photolabeling of the bovine NPR-A receptor

Two Nalpha-benzophenone-substituted photoprobes, derived from the high affinity NPR-A chimeric agonist [N, C, rANP(1-28)]pBNP32 (pBNP1) were assembled by solid-phase peptide synthesis. [Nalpha-p-benzoylbenzoyl, Tyr2]pBNP1 (probe A), and [Nalpha-p-benzoylbenzoyl, Tyr18]pBNP1 (probe B) were synthesized and their affinity was tested on bovine zona glomerulosa membrane preparations. Both were found to exert ANP-type high affinities (Kd = 20 pM) with Kd of 10 pM and 30 pM for probe A, and probe B, respectively. Photolabeling of NPR-A with both analogs cross-linked specifically the 130 kDa monomeric NPR-A. The maximal irreversible ligand incorporations were estimated at 18% and 41% for probe A, and probe B, respectively. These results show that the N-terminus of the chimeric compound can be acylated with a large chemical function, such as the benzophenone moiety, without loosing its affinity for the NPR-A receptor. Furthermore, Leu2 or Leu18 can be substituted with tyrosine without disturbing the binding capacity of the ligand. Finally, it appears that the pBNP1 N-terminus is close to the receptor structure as irreversible incorporation is observed after photolabeling.     

New highly specific agonistic peptides for human melanocortin MC(1) receptor

A peptide with very high specificity for the human melanocortin MC(1) receptor identified by phage display was used as a lead for the design of new peptides. Two new peptides, MS05 and MS09, were synthesized and found to bind with sub-nanomolar affinities to the MC(1) receptor. Both these peptides showed strong agonistic activity at the MC(1) receptor. The MS05 was the most MC(1) receptor selective as it showed virtually no binding affinity for the MC(4) and MC(5) receptors and only micromolar affinity for the MC(3) receptor. The selectivity and potency of the new peptides make them potent tools for studies of MC(1) receptors, as well as novel potential candidate drugs for the treatment of inflammatory conditions.     

Molecular modeling and dynamic simulations of agglutinin-like family members from Candida albicans: New insights into potential targets for the treatment of candidiasis

Infections by Candida albicans in immune compromised patients cause significant morbidity and mortality. In the search for potential molecular targets for drug development, the family of agglutinin-like proteins (Als) in C. albicans have been identified due to numerous attributes associated with high virulence, most prominently due to their role in adherence. Here, molecular models of individual members of the Als family illustrated common and unique structure features. Additionally, dynamic simulations were performed to display regions of high mobility. The results showed variations between Als members in the fluctuation of the A1B1 protein loop, which is located at the entrance to the peptide binding cavity, suggesting that this feature may be a factor contributing to observed differences in affinities to ligands and adhesion properties. Molecular docking results further suggested that ligand affinity could be influenced by movements in the A1B1 loop. In addition, a new site was identified in Als in an area adjacent to the peptide binding cavity that could serve as a new binding site for the design of future anti-adhesion ligands that provide increased specificity inhibiting Als proteins from C. albicans.     

Insights into human Lck SH3 domain binding specificity: different binding modes of artificial and native ligands

We analyzed the ligand binding specificity of the lymphocyte specific kinase (Lck) SH3 domain. We identified artificial Lck SH3 ligands using phage display. In addition, we analyzed Lck SH3 binding sites within known natural Lck SH3 binding proteins using an Lck specific binding assay on membrane-immobilized synthetic peptides. On one hand, from the phage-selected peptides, representing mostly special class I' ligands, a well-defined consensus sequence was obtained. Interestingly, a histidine outside the central polyproline motif contributes significantly to Lck SH3 binding affinity and specificity. On the other hand, we confirmed previously mapped Lck SH3 binding sites in ADAM15, HS1, SLP76, and NS5A, and identified putative Lck SH3 binding sites of Sam68, FasL, c-Cbl, and Cbl-b. Without exception, the comparatively diverse Lck SH3 binding sites of all analyzed natural Lck SH3 binding proteins emerged as class II proteins. Possible explanations for the observed variations between artificial and native ligands-which are not due to significant K(D) value differences as shown by calculating Lck SH3 affinities of artificial peptide PD1-Y(-3)R as well as for peptides comprising putative Lck SH3 binding sites of NS5A, Sos, and Sam68-are discussed. Our data suggest that phage display, a popular tool for determining SH3 binding specificity, must-at least in the case of Lck-not irrevocably mirror physiologically relevant protein-ligand interactions.     

Application of peptide probe for evaluating affinity properties of proteins using quartz crystal microbalance

This study proved a possibility of a peptide probe for evaluating affinity properties of proteins. We have designed and synthesized three different peptide probes, H-Ala3-(Gly-Pro5)3-Gly-OH (peptide A), H-Ala3-(Gly-Pro5)-Gly-OH (peptide B) and H-Ala3-Gly-OH (peptide C) for testing their affinities to profilin. Each peptide probe was immobilized on a quartz crystal microbalance (QCM) sensor. The QCM sensor with the peptide A showed a 93 Hz decrease of resonant frequency which indicated profilin bound to the QCM sensor in a single layer. In a successive reaction with actin, the QCM analysis resulted in a 123 Hz decrease of resonant frequency which showed actin bound to the QCM sensor. A fluorescence microscope image of the sensor surface exhibited clear fluorescence after binding a rhodamine labeled actin on the sensor surface. These results supported stepwise reactions of profilin binding to the peptide A and actin binding to profilin. In the three peptide probes, the peptide A showed the highest affinity to profilin, i.e., sequence dependent affinity was confirmed.     

Thermodynamic study of ligand binding to protein-tyrosine phosphatase 1B and its substrate-trapping mutants

The binding of several phosphonodifluoromethyl phenylalanine (F(2)Pmp)-containing peptides to protein-tyrosine phosphatase 1B (PTP1B) and its substrate-trapping mutants (C215S and D181A) has been studied using isothermal titration calorimetry. The binding of a high affinity ligand, Ac-Asp-Ala-Asp-Glu-F(2)Pmp-Leu-NH(2), to PTP1B (K(d) = 0.24 microm) is favored by both enthalpic and entropic contributions. Disruption of ionic interactions between the side chain of Arg-47 and the N-terminal acidic residues reduces the binding affinity primarily through the reduction of the TDeltaS term. The role of Arg-47 may be to maximize surface contact between PTP1B and the peptide, which contributes to high affinity binding. The active site Cys-215 --> Ser mutant PTP1B binds ligands with the same affinity as the wild-type enzyme. However, unlike wild-type PTP1B, peptide binding to C215S is predominantly driven by enthalpy change, which likely results from the elimination of the electrostatic repulsion between the thiolate anion and the phosphonate group. The increased enthalpic contribution is offset by reduction in the binding entropy, which may be the result of increased entropy of the unbound protein caused by this mutation. The general acid-deficient mutant D181A binds the peptide 5-fold tighter than the C215S mutant, consistent with the observation that the Asp to Ala mutant is a better "substrate-trapping" reagent than C215S. The increased binding affinity for D181A as compared with the wild-type PTP1B results primarily from an increase in the DeltaH of binding in the mutant, which may be related to decreased electrostatic repulsion between the phosphate moiety and PTP1B. These results have important implications for the design of high affinity PTP1B inhibitors.     

Toward the identification of selective modulators of protein kinase C (PKC) isozymes: establishment of a binding assay for PKC isozymes using synthetic C1 peptide receptors and identification of the critical residues involved in the phorbol ester binding

Conventional and novel protein kinase C (PKC) isozymes contain two cysteine-rich C1 domains (C1A and C1B), both of which are candidate phorbol-12,13-dibutyrate (PDBu) binding sites. We previously synthesized C1 peptides (of approximately 50 residues) corresponding to all PKC isozymes and measured their PDBu binding affinity. While many of these peptide receptors exhibited PDBu affinities comparable to the respective complete isozyme, some of the C1A peptides could not be used because they undergo temperature dependent inactivation. This problem was however eliminated by 4 degrees C incubation or elongation of the 50-mer C1 peptides at both N- and C-termini to increase their folding efficiency and stability. These findings enabled us to determine the K(d)'s of PDBu for all PKC C1 peptides (except for theta-C1A) and establish the value of these peptides as readily available, stable, and easily handled surrogates of the individual isozymes. The resultant C1 peptide receptor library can be used to screen for new ligands with PKC isozyme and importantly C1 domain selectivity. Most of the C1 peptide receptors showed strong PDBu binding affinities with K(d)'s in the nanomolar range (0.45-7.4 nM). Two peptides (delta-C1A and theta-C1A) bound PDBu over 100-fold less tightly. To identify the residues that contribute to this affinity difference, several mutants of delta-C1A and theta-C1A were synthesized. Both the G9K mutant of delta-C1A and the P9K mutant of theta-C1A showed K(d)'s of 2-3 nM. This approach provides a useful procedure to determine the role of each C1 domain of the PKC isozymes by point mutation.     

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.     

Characterization of rodent liver and kidney AVP receptors: pharmacologic evidence for species differences.

Radioligand binding studies with [3H]vasopressin (AVP) were used to determine the affinities of AVP receptor agonists and antagonists for mouse liver and kidney plasma membrane preparations. Both membrane preparations exhibited one class of high-affinity binding site. AVP ligand binding inhibition studies confirmed that mouse liver binding sites belong to the V1A subtype while kidney binding sites belong to the V2 receptor subtype. The affinity of each ligand for mouse V1A receptors was very similar to that for rat V1A receptors, showing differences in Ki values of less than 3-fold. In contrast, several peptide (d(CH2)5Tyr(Me)AVP) and nonpeptide (OPC-21268 and SR 49059) ligands had different affinities for mouse and rat kidney V2 receptors, with differences in Ki values ranging from 14- to 17-fold. These results indicate that mouse and rat kidney V2 receptors show significant pharmacologic differences.     

Affinity maturation of antiHER2 monoclonal antibody MIL5 using an epitope-specific synthetic phage library by computational design

Increased affinities mainly equal to improved biological efficacy in many cases. By now, display methods including phage library are widely exploited to obtain higher affinity antibodies. Traditional library methods mainly focus on complementary determining region mutagenesis, in which extensive screening of variant combinations as well as large library capacity is required to find higher affinity clones. In this study, based on the modeling 3D complex structure of antigen (HER2)–antibody (MIL5) complex, the key residues of contact surface were predicted and identified to guide the synthetic phage library design. Then, epitope-specific site-directed mutagenesis phage library comprised of MIL5_scFv mutants was constructed, from which a higher affinity single chain antibody (M5scFv_ph) was screened out. Following experimental results showed that the novel antibody M5scFv_ph retained superimposed epitope to the parent antibody MIL5_scFv, and possessed similar tumor growth inhibitory activity in vivo on ovarian carcinoma xenografts.     

Origins of PDZ domain ligand specificity. Structure determination and mutagenesis of the Erbin PDZ domain

The LAP (leucine-rich repeat and PDZ-containing) family of proteins play a role in maintaining epithelial and neuronal cell size, and mutation of these proteins can have oncogenic consequences. The LAP protein Erbin has been implicated previously in a number of cellular activities by virtue of its PDZ domain-dependent association with the C termini of both ERB-B2 and the p120-catenins. The present work describes the NMR structure of Erbin PDZ in complex with a high affinity peptide ligand and includes a comprehensive energetic analysis of both the ligand and PDZ domain side chains responsible for binding. C-terminal phage display has been used to identify preferred ligands, whereas binding affinity measurements provide precise details of the energetic importance of each ligand side chain to binding. Alanine and homolog scanning mutagenesis (in a combinatorial phage display format) identifies Erbin side chains that make energetically important contacts with the ligand. The structure of a phage-optimized peptide (Ac-TGW(-4)ETW(-1)V; IC(50) = approximately 0.15 microm) in complex with Erbin PDZ provides a structural context to understand the binding energetics. In particular, the very favorable interactions with Trp(-1) are not Erbin side chain-mediated (and therefore may be generally applicable to many PDZ domains), whereas the beta2-beta3 loop provides a binding site for the Trp(-4) side chain (specific to Erbin because it has an unusually long loop). These results contribute to a growing appreciation for the importance of at least five ligand C-terminal side chains in determining PDZ domain binding energy and highlight the mechanisms of ligand discrimination among the several hundred PDZ domains present in the human genome.