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PubMed1998

Stimulation of mitogenesis by a cell-permeable PI 3-kinase binding peptide.

The binding of small phosphopeptides to the SH2 domains of the p85 regulatory subunit of PI 3-kinase can activate the enzyme in vitro. In the present study a cell-permeable peptide that binds specifically to the SH2 domains of p85 has been evaluated for its ability to stimulate a mitogenic response in the C2 muscle cell line. This peptide, in contrast to four other SH2-binding peptides, was as effective as serum, EGF, and FGF at stimulating entry into S-phase. The response to the p85 binding peptide, but not FGF, was inhibited by wortmannin and rapamycin, indicating that the peptide activates the PI 3-kinase/S6 kinase signalling pathway. The peptide response was not inhibited by the MEK inhibitor (PD098059) and did not stimulate Erk phosphorylation. Thus, there would appear to be no direct cross-talk between the pathway activated by the p85 binding peptide and the p42/p44 MAPK cascade.

3T3 Cells↗

PubMed1996

Probing HLA-B7 conformational shifts induced by peptide-binding groove mutations and bound peptide with anti-HLA monoclonal antibodies.

To determine the influence of peptide-binding groove residues and MHC-bound peptide on HLA-B7 conformation, we investigated the binding sites of nine locus- or allele-specific mAbs using a panel of 82 HLA-B7 variants. The functional mAb epitopes encircle the HLA-B7 peptide-binding groove. Three mAbs are affected by mutations at solvent-accessible peptide-binding groove mutations. Mutations in peptide-binding groove residues 45, 63, and 150 affect multiple nonoverlapping mAb epitopes, probably by interaction with other MHC residues or bound peptide. However, 18 of 24 peptide-binding groove mutations do not affect mAb binding, indicating that the conformation of solvent-accessible HLA-B7 structures is largely dissociated from changes in the peptide-binding groove. To test whether bound peptides alter HLA-B7 conformation, we loaded HLA-B7 heavy chains on acid-stripped cells with beta2-microglobulin and 20 individual synthetic peptides. Two of eight mAbs are sensitive to HLA-B7-bound peptides. A likely interpretation of these data is that the conformational flexibility of HLA-B7 is due to peptide-induced conformational shifts in MHC side chains, rather than major shifts in the MHC main chain. These results suggest that HLA-B7 conformation is largely maintained in the context of different bound peptides and different peptide-binding grooves.

Antibodies, Monoclonal↗

PubMed2006

A high efficiency strategy for binding property characterization of peptide-binding domains.

A large proportion of protein-protein interactions is mediated by families of peptide-binding domains. Comprehensive characterization of each of these domains is critical for understanding the mechanisms and networks of protein interaction at the domain level. However, existing methods are all based on large scale screenings for each domain that are inefficient to deal with hundreds of members in major domain families. We developed a systematic strategy for efficient binding property characterization of peptide-binding domains based on high throughput validation screening of a specialized candidate ligand library using yeast two-hybrid mating array. Its outstanding feature is that the overall efficiency is dramatically improved compared with that of traditional screening, and it will be higher as the system cycles. PDZ domain family was first used to test the strategy. Five PDZ domains were rapidly characterized. Broader binding properties were identified compared with other methods, including novel recognition specificities that provided the basis for major revision of conventional PDZ classification. Several novel interactions were discovered, serving as significant clues for further functional investigation. This strategy can be easily extended to a variety of peptide-binding domains as a powerful tool for comprehensive analysis of domain binding property in proteomic scale.

Animals↗

PubMed1991

Effect of natural polymorphism at residue 86 of the HLA-DR beta chain on peptide binding.

Class I and class II MHC glycoproteins are highly polymorphic molecules that bind antigenic peptides and present them on cell surfaces for recognition by T lymphocytes. Even though MHC polymorphism has long been known to affect both peptide binding and recognition by the TCR, the role of individual amino acids of MHC proteins in these interactions is poorly understood. To examine the effect of a small number of amino acid residues on T cell stimulation, B lymphoblastoid cell lines homozygous for the closely related DR1 subtypes, Dw1 and Dw20, and the DR4 subtypes, Dw4 and Dw14, were compared for their ability to present an immunogenic influenza hemagglutinin peptide (HA307-319) to an Ag-specific, DR1,4-restricted T cell clone. B cell lines expressing DR1 Dw20 and DR4 Dw14 presented HA307-319 much less efficiently than DR1 Dw1 and DR4 Dw4 and bound a biotinylated analogue of the same peptide less well. Analysis of DRB1 gene sequences suggested that polymorphism at residue 86 had a major effect on peptide binding. Differences in binding of a set of HA307-319 analogues biotinylated at each residue to cells expressing DR1 Dw1 and DR1 Dw20 suggested that the polymorphism affected the interactions of many peptide residues with the class II molecule. In inhibition assays, DR1 Dw1 and DR4 Dw4 were shown to differ from DR1 Dw20 and DR4 Dw14 in their length requirements for peptide binding. Using a larger panel of homozygous B cell lines expressing many class II haplotypes, a Ser-309 substituted HA307-319 analogue was shown to bind to most B cell lines expressing Val-86 containing alleles (including DR1 Dw20 and DR4 Dw14) but failed to bind most B cell lines expressing Gly-86 alleles (including DR1 Dw1 and DR4 Dw4). The results indicated that polymorphism at residue 86 influenced the specificity and affinity of peptide binding and affected the conformation of peptide-DR protein complexes without completely eliminating T cell recognition.

Alleles↗

PubMed1996

HLA-DQ-binding peptide motifs. 1. Comparative binding analysis of type II collagen-derived peptides to DR and DQ molecules of rheumatoid arthritis-susceptible and non-susceptible haplotypes.

The frequency of the HLA-DR4-DQ4 haplotype (DRB1(*)0405-DQA1(*)0302-DQB1(*)0401) is significantly increased in Japanese patients with rheumatoid arthritis (RA) and DRB1(*)0405-binding peptide motifs were identified in our previous studies. To clarify the DQ4-binding peptide motifs, the primary structure of DQ4-binding peptides was determined by affinity-based selection of a phage random peptide library. Analog peptides of a high-affinity DQ4 binder revealed that two major anchors (VxxxxxxxR; where x is any amino acid) play an essential role in binding to DQ4. The affinity of synthetic VAAAAAAAR-based analog peptides showed that substituting V to W, G, L, I, M, P, F, Y or A and R to H, M, L, I or V allows binding. The involvement of the ninth residue of the peptides, especially Arg, was critical for high-affinity binding. In comparison with other class II-binding peptide motifs reported to date, peptide motifs for DQ4 were unique, in that Gly and Pro are allowed as low-affinity N-terminal anchors. Interestingly, 94 putative DQ4-binding motifs were detected in the human type II collagen molecule, since it is composed of (Gly-X1-X2)n and is rich in R and P at positions X2. However, no significant differences were observed between the affinities of the collagen-derived peptides with DR or DQ molecules of RA-susceptible DR4-DQ4 and with those of non-susceptible DR4-DQ8 (DRB1(*)0406-DQA1(*)0301-DQB1(*)0302) haplotypes, indicating that the susceptibility to RA is not a simple immune response gene phenomenon specific to collagen. The immunogenetic implications of the unique peptide motifs for DQ are discussed.

Amino Acid Sequence↗

PubMed2005

Isolation of a mycoplasma-specific binding peptide from an unbiased phage-displayed peptide library.

An important goal in medicine is the development of methods for cell-specific targeting of therapeutic molecules to pathogens or pathogen-infected cells. However, little progress has been made in cell-specific targeting of bacterially infected cells. Using a phage display approach, we have isolated a 20-mer peptide that binds to Mycoplasma arginini infected pancreatic beta-cells in tissue culture. This peptide binds to M. arginini infected beta-cells 200 times better than a control phage and is specific for the infected cells. Furthermore, transferring the M. arginini contamination to another cell line renders the newly infected cell line susceptible to peptide binding. Immunolocalization experiments suggest that the peptide is binding to M. arginini adhered to the cell surface. The free synthetic peptide retains its binding in the absence of the phage vehicle and tetramerization of the peptide increases its affinity for the infected cells. Efforts have been made to use this peptide to eliminate Mycoplasma from infected cell lines using ferromagnetic beads coated with the selected peptide. A ten-fold reduction of infection was accomplished with one fractionation via this approach. Our results suggest that this peptide, isolated from an unbiased selection, may be of utility for the detection and reduction of Mycoplasma infection in cultured cells. Furthermore, a general implication of our findings is that phage display methods may be useful for identifying peptides that target a broad array of other biological pathogens in a specific fashion.

Bacteriophages↗

PubMed1999

In vivo selection of RNA-binding peptides from combinatorial libraries.

We have used a two step procedure to identify peptides that bind strongly to the Rev-response element (RRE) of HIV. In the first step, RRE-binding peptides were screened from a combinatorial peptide library generated by "randomization" using a small subset of the 20 amino acids. In the second step, one such RRE-binding peptide, RSG-1, was "evolved" into an even stronger RRE-binding peptide using a codon-based mutagenesis procedure. After 2 rounds of evolution, RSG-1.2 bound the RRE with 7-fold higher affinity than wild-type Rev peptide.

Amino Acid Sequence↗

PubMed2001

Predicting sequences and structures of MHC-binding peptides: a computational combinatorial approach.

Peptides bound to MHC molecules on the surface of cells convey critical information about the cellular milieu to immune system T cells. Predicting which peptides can bind an MHC molecule, and understanding their modes of binding, are important in order to design better diagnostic and therapeutic agents for infectious and autoimmune diseases. Due to the difficulty of obtaining sufficient experimental binding data for each human MHC molecule, computational modeling of MHC peptide-binding properties is necessary. This paper describes a computational combinatorial design approach to the prediction of peptides that bind an MHC molecule of known X-ray crystallographic or NMR-determined structure. The procedure uses chemical fragments as models for amino acid residues and produces a set of sequences for peptides predicted to bind in the MHC peptide-binding groove. The probabilities for specific amino acids occurring at each position of the peptide are calculated based on these sequences, and these probabilities show a good agreement with amino acid distributions derived from a MHC-binding peptide database. The method also enables prediction of the three-dimensional structure of MHC-peptide complexes. Docking, linking, and optimization procedures were performed with the XPLOR program [1].

Amino Acid Sequence↗

PubMed1998

Several common HLA-DR types share largely overlapping peptide binding repertoires.

The peptide binding specificities of HLA-DRB1*0401, DRB1*0101, and DRB1*0701 have been analyzed by the use of large collections of synthetic peptides corresponding to naturally occurring sequences. The results demonstrated that nearly all peptides binding to these DR molecules bear a motif characterized by a large aromatic or hydrophobic residue in position 1 (Y, F, W, L, I, V, M) and a small, noncharged residue in position 6 (S, T, C, A, P, V, I, L, M). In addition, allele-specific secondary effects and secondary anchors were defined, and these parameters were utilized to derive allele-specific motifs and algorithms. By the combined use of such algorithms, peptides capable of degenerate DRB1*0101, DRB1*0401, and DRB1*0701 binding were identified. Additional experiments utilizing a panel of quantitative assays specific for nine additional common DR molecules identified a large set of DR molecules, which includes at least the DRB1*0101, DRB1*0401, DRB1*0701, DRB5*0101, DRB1*1501, DRB1*0901, and DRB1*1302 allelic products, characterized by overlapping peptide-binding repertoires. These results have implications for understanding the molecular interactions involved in peptide-DR binding, as well as the genetic and structural basis of MHC polymorphism. These results also have potential practical implications for the development of epitope-based prophylactic and therapeutic vaccines.

Algorithms↗

PubMed2003

Plasmodium falciparum normocyte binding protein (PfNBP-1) peptides bind specifically to human erythrocytes.

Plasmodium falciparum normocyte binding protein-1 (PfNBP-1), a Plasmodium vivax RBP-1 orthologue is expressed in the apical merozoite area. PfNBP-1 binds directly to human erythrocyte membrane in a sialic acid-dependent but trypsin-resistant way. Erythrocyte binding assays were done with synthetic peptides covering the sequence reported as PfNBP-1. Two specific erythrocyte high activity binding peptides were found: 101VFINDLDTYQYEYFYEWNQ(120), peptide 26332, and 181NTKETYLKELNKKKMLQNKK(200), peptide 26336. These two peptides' binding was saturable and presenting nanomolar affinity constants. The critical binding residues (those residues underlined and highlighted in bold) were determined by competition assays with glycine-scan analogue peptides. These peptides were able to block merozoite in vitro invasion of erythrocytes.

Amino Acid Sequence↗

PubMed1993

Characterization of cytotoxic T lymphocyte epitopes of a self-protein, p53, and a non-self-protein, influenza matrix: relationship between major histocompatibility complex peptide binding affinity and immune responsiveness to peptides.

We previously described a motif prediction of major histocompatibility complex allele-specific peptides and an in vitro assay for actual measurement of peptide binding to human leukocyte antigen HLA-A2.1 molecules. Using this method we have identified candidate cytotoxic T lymphocyte (CTL) epitopes derived from a non-self-protein (influenza matrix) and self-protein (p53). We now show that results of binding assays performed over a range of peptide concentrations indicate that distinct differences in HLA-A2.1 peptide binding affinities exist between the influenza matrix and p53 protein. The results for the influenza matrix protein indicate that the peptide that shows the highest binding affinity to HLA-A2.1 is identical to the known immunodominant peptide recognized by influenza virus-specific CTLs. The results for p53 indicate that one of the peptides with a low binding affinity is capable of inducing specific CTL responses, but CTLs recognizing the highest affinity binding peptides were not obtained. These findings are discussed in terms of the distinct implications for induction of cellular immune responses directed against peptides with different binding affinities for HLA-A2.1 of proteins that constitute attractive targets for tumor immunotherapy.

Amino Acid Sequence↗

PubMed2002

Empirical evaluation of a dynamic experiment design method for prediction of MHC class I-binding peptides.

The ability to predict MHC-binding peptides remains limited despite ever expanding demands for specific immunotherapy against cancers, infectious diseases, and autoimmune disorders. Previous analyses revealed position-specific preference of amino acids but failed to detect sequence patterns. Efforts to use computational analysis to identify sequence patterns have been hampered by the insufficiency of the number/quality of the peptide binding data. We propose here a dynamic experiment design to search for sequence patterns that are common to the MHC class I-binding peptides. The method is based on a committee-based framework of query learning using hidden Markov models as its component algorithm. It enables a comprehensive search of a large variety (20(9)) of peptides with a small number of experiments. The learning was conducted in seven rounds of feedback loops, in which our computational method was used to determine the next set of peptides to be analyzed based on the results of the earlier iterations. After these training cycles, the algorithm enabled a real number prediction of MHC binding peptides with an accuracy surpassing that of the hitherto best performing positional scanning method.

Algorithms↗

PubMed1996

Identification of elements of the peptide binding site of DnaK by peptide cross-linking.

We used photocross-linking of peptides to DnaK to identify elements of the peptide binding site of DnaK. We attached a photoactivatable group (N-hydroxysuccinimidyl-4-azido-salicylic acid (NHS-ASA) or N-iodoacetamidobutyl-4-azido-salicylic acid (I-ABASA)) to different positions on peptide C of the vesicular stomatitis virus glycoprotein, 125I-radiolabeled the cross-linker, cross-linked the peptide to DnaK by UV irradiation, and then determined the amino acid residues of DnaK that were cross-linked to the peptide. Limited trypsin digestion of the DnaK-peptide complex revealed that the derivatives modified with photoactivatable cross-linker peptide C cross-linked to a C-terminal fragment of DnaK and that the N-terminal 45-kDa fragment of DnaK was not cross-linked by these modified peptides. The attachment points of the three peptide C derivatives carrying photoactivatable cross-linkers at different locations on the peptide, PepC-ASA, PepC-S7C-ABASA, and PepC-S8C-ABASA, have been identified as Arg-536, Arg-527, and His-541 of DnaK, respectively. Thus all three peptides cross-linked to amino acids located close together in a sequence that includes one end of the long alpha-helix in the NMR-based secondary structure model of the peptide binding domain of Hsp70 family (Morshauser, R., Wang, H., Flynn, G., and Zuiderweg, E. (1995) Biochemistry 34, 6261-6266).

Amino Acid Sequence↗

PubMed2000

Mitochondrial protein import motor: the ATPase domain of matrix Hsp70 is crucial for binding to Tim44, while the peptide binding domain and the carboxy-terminal segment play a stimulatory role.

The import motor for preproteins that are targeted into the mitochondrial matrix consists of the matrix heat shock protein Hsp70 (mtHsp70) and the translocase subunit Tim44 of the inner membrane. mtHsp70 interacts with Tim44 in an ATP-dependent reaction cycle, binds to preproteins in transit, and drives their translocation into the matrix. While different functional mechanisms are discussed for the mtHsp70-Tim44 machinery, little is known about the actual mode of interaction of both proteins. Here, we have addressed which of the three Hsp70 regions, the ATPase domain, the peptide binding domain, or the carboxy-terminal segment, are required for the interaction with Tim44. By two independent means, a two-hybrid system and coprecipitation of mtHsp70 constructs imported into mitochondria, we show that the ATPase domain interacts with Tim44, although with a reduced efficiency compared to the full-length mtHsp70. The interaction of the ATPase domain with Tim44 is ATP sensitive. The peptide binding domain and carboxy-terminal segment are unable to bind to Tim44 in the absence of the ATPase domain, but both regions enhance the interaction with Tim44 in the presence of the ATPase domain. We conclude that the ATPase domain of mtHsp70 is essential for and directly interacts with Tim44, clearly separating the mtHsp70-Tim44 interaction from the mtHsp70-substrate interaction.

Adenosine Triphosphatases↗

PubMed1994

Human transporters associated with antigen processing possess a promiscuous peptide-binding site.

The peptide selectivity of the human transporters associated with antigen processing (TAP) was investigated using a panel of peptides of varying length and sequence. Peptides were assayed for their ability to compete for the translocation of a labeled reporter peptide containing an N-linked glycosylation acceptor site in Streptolysin O (SLO)-permeabilized cells. We find that human TAP is very promiscuous for peptides in the 8-12 amino acid range, while showing increased selectivity and lower translocation efficiency for peptides in the 13-30 amino acid range. The minimum peptide length appears to be 8 amino acids, while the maximum length appears to be approximately 25 amino acids. Furthermore, a photoactive peptide analogue was synthesized that can photolabel TAP molecules. Using this analogue, we showed that an ATP-independent peptide-binding site exists on TAP, and that competition for translocation reflects competition for peptide binding.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

PepDist: a new framework for protein-peptide binding prediction based on learning peptide distance functions.

BACKGROUND: Many different aspects of cellular signalling, trafficking and targeting mechanisms are mediated by interactions between proteins and peptides. Representative examples are MHC-peptide complexes in the immune system. Developing computational methods for protein-peptide binding prediction is therefore an important task with applications to vaccine and drug design. METHODS: Previous learning approaches address the binding prediction problem using traditional margin based binary classifiers. In this paper we propose PepDist: a novel approach for predicting binding affinity. Our approach is based on learning peptide-peptide distance functions. Moreover, we suggest to learn a single peptide-peptide distance function over an entire family of proteins (e.g. MHC class I). This distance function can be used to compute the affinity of a novel peptide to any of the proteins in the given family. In order to learn these peptide-peptide distance functions, we formalize the problem as a semi-supervised learning problem with partial information in the form of equivalence constraints. Specifically, we propose to use DistBoost, which is a semi-supervised distance learning algorithm. RESULTS: We compare our method to various state-of-the-art binding prediction algorithms on MHC class I and MHC class II datasets. In almost all cases, our method outperforms all of its competitors. One of the major advantages of our novel approach is that it can also learn an affinity function over proteins for which only small amounts of labeled peptides exist. In these cases, our method's performance gain, when compared to other computational methods, is even more pronounced. We have recently uploaded the PepDist webserver which provides binding prediction of peptides to 35 different MHC class I alleles. The webserver which can be found at http://www.pepdist.cs.huji.ac.il is powered by a prediction engine which was trained using the framework presented in this paper. CONCLUSION: The results obtained suggest that learning a single distance function over an entire family of proteins achieves higher prediction accuracy than learning a set of binary classifiers for each of the proteins separately. We also show the importance of obtaining information on experimentally determined non-binders. Learning with real non-binders generalizes better than learning with randomly generated peptides that are assumed to be non-binders. This suggests that information about non-binding peptides should also be published and made publicly available.

Algorithms↗

PubMed1992

Characterization of penicillin-binding protein 2 of Staphylococcus aureus: deacylation reaction and identification of two penicillin-binding peptides.

Penicillin-binding protein (PBP) 2 is the major PBP of five that have been identified in susceptible strains of Staphylococcus aureus. Beta-lactam antibiotic binding to PBP 2 is important for the antibacterial effect. Antibiotic binding to PBP 2 in strain 209P was examined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis in competition assays using [3H]penicillin as the radiolabel. Clavulanic acid, which is specifically bound by PBP 2, and cefaclor, which is specific for PBP 3, were studied. Cefaclor, which alone appeared not to bind PBP 2, in combination inhibited PBP 2 binding of clavulanic acid. By varying the temperature during radiolabeling with [3H]penicillin in cefaclor competition assays and in direct radiolabeling assays with [3H]cefaclor, it was shown that cefaclor was bound by PBP 2 with high affinity (50% inhibitory concentration, less than or equal to 0.1 microgram/ml) and that the apparent low-affinity binding (50% inhibitory concentration, greater than 10 micrograms/ml) in competition assays performed at 37 degrees C was due to rapid deacylation. Two penicillin-binding peptides of PBP 2 also were identified in fluorographs of PBPs separated by nonequilibrium pH gradient gel and two-dimensional electrophoresis. Rapid deacylation for some antibiotics and the presence of two penicillin-binding peptides are two properties of PBP 2 that should be considered when correlating results of binding assays with effects of beta-lactam antibiotics on S. aureus.

Anti-Bacterial Agents↗

PubMed1999

Experimental models of protein-RNA interaction: isolation and analyses of tRNA(Phe) and U1 snRNA-binding peptides from bacteriophage display libraries.

Peptides that bind either U1 small nuclear RNA (U1 snRNA) or the anticodon stem and loop of yeast tRNA(Phe) (tRNA(ACPhe)) were selected from a random-sequence, 15-amino acid bacteriophage display library. An experimental system, including an affinity selection method, was designed to identify primary RNA-binding peptide sequences without bias to known amino acid sequences and without incorporating nonspecific binding of the anionic RNA backbone. Nitrocellulose binding assays were used to evaluate the binding of RNA by peptide-displaying bacteriophage. Amino acid sequences of RNA-binding bacteriophage were determined from the foreign insert DNA sequences, and peptides corresponding to the RNA-binding bacteriophage inserts were chemically synthesized. Peptide affinities for the RNAs (Kd approximately 0.1-5.0 microM) were analyzed successfully using fluorescence and circular dichroism spectroscopies. These methodologies demonstrate the feasibility of rapidly identifying, isolating, and initiating the analyses of small peptides that bind to RNAs in an effort to define better the chemistry, structure, and function of protein-RNA complexes.

Amino Acid Sequence↗