Characterization of two peptide epitopes on Mdm2 oncoprotein that affect p53 degradation.

Phosphorylation of Mdm2, in response to DNA damage, resulted in prevention of p53 degradation in the cytoplasm as well as reduction of its binding with monoclonal antibody (mAb) 2A10. Using a 15-mer phage-peptide library, we identified two 2A10-epitopes on human Mdm2 (hdm2): at positions 255-266 (LDSEDYSLSEEG) and 389-400 (QESDDYSQPSTS). Synthetic peptides corresponding to the above sites, inhibit the binding of mAb2A10 to Mdm2 with high (4.5 x 10(-9)M) and moderate affinity (1.1 x 10(-7)M), respectively. Phospho-derivatives of these peptides, and of single human Mdm2 mutations S260D or S395D resulted in a considerable reduction in their binding with mAb2A10. These results provide a molecular explanation for the observation that reactivity of Mdm2 with mAb2A10 is inhibited by phosphorylation.

The binding site of acetylcholine receptor as visualized in the X-Ray structure of a complex between alpha-bungarotoxin and a mimotope peptide.

We have determined the crystal structure at 1.8 A resolution of a complex of alpha-bungarotoxin with a high affinity 13-residue peptide that is homologous to the binding region of the alpha subunit of acetylcholine receptor. The peptide fits snugly to the toxin and adopts a beta hairpin conformation. The structures of the bound peptide and the homologous loop of acetylcholine binding protein, a soluble analog of the extracellular domain of acetylcholine receptor, are remarkably similar. Their superposition indicates that the toxin wraps around the receptor binding site loop, and in addition, binds tightly at the interface of two of the receptor subunits where it inserts a finger into the ligand binding site, thus blocking access to the acetylcholine binding site and explaining its strong antagonistic activity.

A beta -hairpin structure in a 13-mer peptide that binds alpha -bungarotoxin with high affinity and neutralizes its toxicity.

Snake-venom alpha-bungarotoxin is a member of the alpha-neurotoxin family that binds with very high affinity to the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. The structure of the complex between alpha-bungarotoxin and a 13-mer peptide (WRYYESSLEPYPD) that binds the toxin with high affinity, thus inhibiting its interactions with AChR with an IC(50) of 2 nM, has been solved by (1)H-NMR spectroscopy. The bound peptide folds into a beta-hairpin structure created by two antiparallel beta-strands, which combine with the already existing triple-stranded beta-sheet of the toxin to form a five-stranded intermolecular, antiparallel beta-sheet. Peptide residues Y3(P), E5(P), and L8(P) have the highest intermolecular contact area, indicating their importance in the binding of alpha-bungarotoxin; W1(P), R2(P), and Y4(P) also contribute significantly to the binding. A large number of characteristic hydrogen bonds and electrostatic and hydrophobic interactions are observed in the complex. The high-affinity peptide exhibits inhibitory potency that is better than any known peptide derived from AChR, and is equal to that of the whole alpha-subunit of AChR. The high degree of sequence similarity between the peptide and various types of AChRs implies that the binding mode found within the complex might possibly mimic the receptor binding to the toxin. The design of the high-affinity peptide was based on our previous findings: (i) the detection of a lead peptide (MRYYESSLKSYPD) that binds alpha-bungarotoxin, using a phage-display peptide library, (ii) the information about the three-dimensional structure of alpha-bungarotoxin/lead-peptide complex, and (iii) the amino acid sequence analysis of different AChRs.

Design and synthesis of peptides that bind alpha-bungarotoxin with high affinity.

BACKGROUND: Alpha-bungarotoxin (alpha-BTX) is a highly toxic snake venom alpha-neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. We describe the design and synthesis of peptides that bind alpha-BTX with high affinity, and inhibit its interaction with AChR with an IC(50) of 2 nM. The design of these peptides was based on a lead peptide with an IC(50) of 3x10(-7) M, previously identified by us [M. Balass et al., Proc. Natl. Acad. Sci. USA 94 (1997) 6054] using a phage-display peptide library. RESULTS: Employing nuclear magnetic resonance-derived structural information [T. Scherf et al., Proc. Natl. Acad. Sci. USA 94 (1997) 6059] of the complex of alpha-BTX with the lead peptide, as well as structure-function analysis of the ligand-binding site of AChR, a systematic residue replacement of the lead peptide, one position at a time, yielded 45 different 13-mer peptides. Of these, two peptides exhibited a one order of magnitude increase in inhibitory potency in comparison to the lead peptide. The design of additional peptides, with two or three replacements, resulted in peptides that exhibited a further increase in inhibitory potency (IC(50) values of 2 nM), that is more than two orders of magnitude better than that of the original lead peptide, and better than that of any known peptide derived from AChR sequence. The high affinity peptides had a protective effect on mice against alpha-BTX lethality. CONCLUSIONS: Synthetic peptides with high affinity to alpha-BTX may be used as potential lead compounds for developing effective antidotes against alpha-BTX poisoning. Moreover, the procedure employed in this study may serve as a general approach for the design and synthesis of peptides that interact with high affinity with any desired biological target.

A cyclic peptide with high affinity to alpha-bungarotoxin protects mice from the lethal effect of the toxin.

Employing a combinatorial phage-peptide library, we previously identified the peptide MRYYESSLKSYPD (designated, library-peptide) that binds the snake toxin alpha-bungarotoxin (alpha-BTX) with a moderate binding constant of 10(-6)M (Balass et al., 1997. Proc. Natl. Acad. Sci. USA 94, 6054-6058). Under the experimental conditions employed, we found that the library-peptide did not protect mice from alpha-BTX lethality when injected concomitantly with the toxin. In order to improve the affinity of the peptide to alpha-BTX, we designed and synthesized the peptide CRYYESSLKSYCD (Met1 and Pro12 were replaced by cysteines), which following oxidation creates a single disulfide bond and forms a cyclic structure. The design of the cyclic peptide was based on our previous NMR analysis of the library-peptide/alpha-BTX complex (Scherf et al., 1997. Proc. Natl. Acad. Sci. USA 94, 6059-6064). The cyclic peptide binds alpha-BTX with affinity two orders of magnitude higher than that of the linear library selected peptide. Whereas the library peptide was ineffective, the cyclic peptide conferred protection from alpha-BTX lethality in mice, even when given 1h after the toxin injection. The cyclic peptide conferred complete protection from alpha-BTX lethality in mice when administered 40min prior to toxin injection. However, experiments with the whole venom of the snake Bungarus multicinctus showed that protection could be achieved only when the cyclic peptide was administered concomitantly with the venom.

Sorting polyclonal antibodies into functionally distinct fractions using peptide phage display: 'a library on top of a library'.

A general approach for sorting antibodies (Abs) to a restricted protein domain was developed using phage-displayed peptide libraries. The method is demonstrated by fractionating polyclonal antibodies (pAbs), raised against a short peptide derived from the extracellular, juxtamembrane region of fibroblast growth factor receptor 1 (FGFR1) into fractions with distinct chemical and biological characteristics. Screening two combinatorial peptide libraries, with the pAb, several sequences, homologous to different regions within the original peptide, were identified. Four of the corresponding peptides were synthesized and used as peptide-conjugated affinity columns for the fractionation of the pAbs. The fractions obtained were unique in their recognition patterns and in their capacity to immunoprecipitate and immunoblot, as well as to modulate the activity of FGFR1. This technique is, therefore, highly sufficient in separating pAbs to monospecific fractions and may also be used for fine mapping of different, even overlapping, sequences within a restricted peptide or protein domain.

Prevention of passively transferred experimental autoimmune myasthenia gravis by a phage library-derived cyclic peptide.

Many pathogenic antibodies in myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are directed against the main immunogenic region (MIR) of the acetylcholine receptor (AcChoR). These antibodies are highly conformation dependent; hence, linear peptides derived from native receptor sequences are poor candidates for their immunoneutralization. We employed a phage-epitope library to identify peptide-mimotopes capable of preventing the pathogenicity of the anti-MIR mAb 198. We identified a 15-mer peptide (PMTLPENYFSERPYH) that binds specifically to mAb 198 and inhibits its binding to AcChoR. A 10-fold increase in the affinity of this peptide was achieved by incorporating flanking amino acid residues from the coat protein as present in the original phage library. This extended peptide (AEPMTLPENYFSERPYHPPPP) was constrained by the addition of cysteine residues on both ends of the peptide, thus generating a cyclic peptide that inhibited the binding of mAb 198 to AcChoR with a potency that is three orders of magnitude higher when compared with the parent library peptide. This cyclic peptide inhibited the in vitro binding of mAb 198 to AcChoR and prevented the antigenic modulation of AcChoR caused by mAb 198 in human muscle cell cultures. The cyclic peptide also reacted with several other anti-MIR mAbs and the sera of EAMG rats. In addition, this peptide blocked the ability of mAb 198 to passively transfer EAMG in rats. Further derivatization of the cyclic peptide may aid in the design of suitable synthetic mimotopes for modulation of MG.

High affinity binding of monoclonal antibodies to the sequential epitope EFRH of beta-amyloid peptide is essential for modulation of fibrillar aggregation.

Monoclonal antibodies raised against the N-terminal of Alzheimer's beta-amyloid peptide (betaAP) were found to modulate its fibrillar aggregation. While mAbs 6C6 and 10D5 inhibit the formation of beta-amyloid fibrils, trigger disaggregation and reversal to its non-toxic form, mAb 2H3 is devoid of these properties. MAb 2H3 binds the sequence DAEFRHD, corresponding to position 1-7 of the betaAP with high affinity (2 x 10(-9) M) similar to its binding with the whole betaAP. The EFRH peptide strongly inhibits binding of mAbs 6C6 and 10D5 to betaAP, whereas it inhibits weakly the interaction of 2H3 with betaAP. Low affinity binding of mAb 2H3 to EFRH might explain its failure in prevention of beta-amyloid formation.

Prevention of experimental antiphospholipid syndrome and endothelial cell activation by synthetic peptides.

Antiphospholipid syndrome (APS) is characterized by recurrent fetal loss, repeated thromboembolic phenomena, and thrombocytopenia. The syndrome is believed to be caused by antiphospholipid beta-2-glycoprotein-I (beta2GPI)-dependent Abs or anti-beta2GPI Abs by themselves. Using a hexapeptide phage display library, we identified three hexapeptides that react specifically with the anti-beta2GPI mAbs ILA-1, ILA-3, and H-3, which cause endothelial cell activation and induce experimental APS. To enhance the binding of the peptides to the corresponding mAbs, the peptides were lengthened to correspond with the site of the beta2GPI epitope being recognized by these mAbs. As a result, the following three peptides were prepared: A, NTLKTPRVGGC, which binds to ILA-1 mAb; B, KDKATFGCHDGC, which binds to ILA-3 mAb; and C, CATLRVYKGG, which binds to H-3 mAb. Peptides A, B, and C specifically inhibit both in vitro and in vivo the biological functions of the corresponding anti-beta2GPI mAbs. Exposure of endothelial cells to anti-beta2GPI mAbs and their corresponding peptides led to the inhibition of endothelial cell activation, as shown by decreased expression of adhesion molecules (E-selectin, ICAM-1, VCAM-1) and monocyte adhesion. In vivo infusion of each of the anti-beta2GPI mAbs into BALB/c mice, followed by administration of the corresponding specific peptides, prevented the peptide-treated mice from developing experimental APS. The use of synthetic peptides that focus on neutralization of pathogenic anti-beta2GPI Abs represents a possible new therapeutic approach to APS.

Synthesis, structure and function of poly-alpha-amino acids--the simplest of protein models.

During the 1950s, linear and multichain poly-alpha-amino acids were synthesized by polymerization of the corresponding N-carboxyamino acid anhydrides in solution in the presence of suitable catalysts. The resulting homo- and heteropolymers have since been widely employed as simple protein models. Under appropriate conditions, poly-alpha-amino acids, in the solid state and in solution, were found to acquire conformations of an alpha-helix and of beta-parallel and antiparallel pleated sheets, or to exist as random coils. Their use in experimental and theoretical investigations of helix-coil transitions helped to shed new light on the mechanisms involved in protein denaturation. Conformational fluctuations of peptides in solution were analysed theoretically and studied experimentally by nonradiative energy-transfer techniques. Poly-alpha-amino acids played an important role in the deciphering of the genetic code. In addition, analysis of the antigenicity of poly-alpha-amino acids led to the elucidation of the factors determining the antigenicity of proteins and peptides. The synthetic procedures developed made possible the preparation of immobilized enzymes which were shown to be of considerable use as heterogeneous biocatalysts in the chemical and pharmaceutical industry. Interest in the biological and physicochemical characteristics of poly-alpha-amino acids was recently renewed because of the reported novel findings that some copolymers of amino acids are effective as drugs in multiple sclerosis, and that glutamine repeats and reiteration of other amino acids occur in inherited neurodegenerative diseases.

The alpha-bungarotoxin binding site on the nicotinic acetylcholine receptor: analysis using a phage-epitope library.

The nicotinic acetylcholine receptor (AcChoR) is a ligand-gated ion channel that is activated upon binding of acetylcholine. alpha-Neurotoxins, in particular alpha-bungarotoxin (alpha-BTX), bind specifically and with high affinity to the AcChoR and compete with binding of the natural ligand. We employed a 15-mer phage-display peptide library to select epitopes reacting with alpha-BTX. Phages bearing the motif YYXSSL as a consensus sequence were found to bind with high affinity to alpha-BTX. The library-derived peptide (MRYYESSLKSYPD) bears amino acid sequence similarities to a region of the alpha-subunit of the Torpedo muscle AcChoR, as well as of other muscle and neuronal AcChoRs that bind alpha-BTX. The library-derived peptide and the corresponding peptides containing residues 187-199 of the Torpedo AcChoR alpha-subunit (WVYYTCCPDTPYL), as well as peptides analogous to the above region in the neuronal AcChoR (e.g., human alpha7; ERFYECCKEPYPD) that binds alpha-BTX, inhibit the binding of alpha-BTX to the intact Torpedo AcChoR with IC50 values of 10(-6) M. A synthetic peptide from a neuronal AcChoR that does not bind alpha-BTX (e.g., human alpha2; ERKYECCKEPYPD) which differs by just one amino acid from the homologous peptide from the alpha-BTX-binding protein (alpha7)-i.e., Lys in alpha2 and Tyr in alpha7-does not inhibit the binding of alpha-BTX to Torpedo AcChoR. These results indicate the requirement for two adjacent aromatic amino acid residues for binding to alpha-BTX.

Three-dimensional solution structure of the complex of alpha-bungarotoxin with a library-derived peptide.

The solution structure of the complex between alpha-bungarotoxin (alpha-BTX) and a 13-residue library-derived peptide (MRYYESSLKSYPD) has been solved using two-dimensional proton-NMR spectroscopy. The bound peptide adopts an almost-globular conformation resulting from three turns that surround a hydrophobic core formed by Tyr-11 of the peptide. The peptide fills an alpha-BTX pocket made of residues located at fingers I and II, as well as at the C-terminal region. Of the peptide residues, the largest contact area is formed by Tyr-3 and Tyr-4. These findings are in accord with the previous data in which it had been shown that substitution of these aromatic residues by aliphatic amino acids leads to loss of binding of the modified peptide with alpha-BTX. Glu-5 and Leu-8, which also remarkably contribute to the contact area with the toxin, are present in all the library-derived peptides that bind strongly to alpha-BTX. The structure of the complex may explain the fact that the library-derived peptide binds alpha-BTX with a 15-fold higher affinity than that shown by the acetylcholine receptor peptide (alpha185-196). Although both peptides bind to similar sites on alpha-BTX, the latter adopts an extended conformation when bound to the toxin [Basus, V., Song, G. & Hawrot, E. (1993) Biochemistry 32, 12290-12298], whereas the library peptide is nearly globular and occupies a larger surface area of alpha-BTX binding site.

Modeling supra-molecular helices: extension of the molecular surface recognition algorithm and application to the protein coat of the tobacco mosaic virus.

Geometric matching of molecular surfaces appears to be essential for the formation of binary molecular complexes and of supra-molecular aggregates. The structure of a binary complex is characterized by the best geometric match, whereas the structure of an aggregate is characterized by the best combined match, i.e. the sum of all the internal matches in the system. We describe a method to identify and quantify the binary matches between molecules and then use them to form the supra-molecular helices and evaluate them. This method is applied to the single protein subunit of tobacco mosaic virus. It successfully predicts the structure of the helical protein coat of the virus and the structure of the disk that is formed as the initial step in the virus assembly process. It also predicts structural intermediates, between disk and helix, which explain how the disk can transform into a helix without dissociating into subunits.

Recovery of high-affinity phage from a nitrostreptavidin matrix in phage-display technology.

A novel approach for the selection of high-affinity phage from phage-peptide libraries is described. The methodology employs a chemically modified form of streptavidin, termed nitrostreptavidin, which exhibits a reversible attraction for biotin. The new approach emulates conventional procedures in that a biotinylated probe, in this case biotinylated alpha-bungarotoxin, is attached to an immobilized streptavidin matrix. The phage library is introduced, and interacting phage particles are released under conventional acidic conditions (pH 2.2). At this stage, the primary peptide sequences characterizing the released phage are found to be identical with those previously known to interact with the toxin. However, other phage particles, which presumably interact more strongly than those released by acid, remain attached to the immobilized toxin. These can be released by virtue of the reversible biotin-binding properties of nitrostreptavidin. For this purpose, alkaline solutions (pH 10) or free biotin can be used. Using this approach, phage particles that recognize alpha-bungarotoxin were isolated; their peptide sequences were found to be similar to, but clearly distinct from, those collected by conventional acid elution. The affinity of the isolated phage was dramatically higher than that of phage obtained by the conventional methodology. In contrast, their synthetically prepared 15-mer peptides actually exhibited a lower affinity for the toxin than that shown by peptides prepared on the basis of the sequence obtained from conventional acid-eluted phage. This apparent discrepancy can be explained by an altered conformational state of the peptides in solution, compared to the epitopes expressed in situ on the phage surface.

Molecular docking programs successfully predict the binding of a beta-lactamase inhibitory protein to TEM-1 beta-lactamase.

Crystallization of the 1:1 molecular complex between the beta-lactamase TEM-1 and the beta-lactamase inhibitory protein BLIP has provided an opportunity to put a stringent test on current protein-docking algorithms. Prior to the successful determination of the structure of the complex, nine laboratory groups were given the refined atomic coordinates of each of the native molecules. Other than the fact that BLIP is an effective inhibitor of a number of beta-lactamase enzymes (KI for TEM-1 approximately 100 pM) no other biochemical or structural data were available to assist the practitioners in their molecular docking. In addition, it was not known whether the molecules underwent conformational changes upon association or whether the inhibition was competitive or non-competitive. All six of the groups that accepted the challenge correctly predicted the general mode of association of BLIP and TEM-1.

Synthesis, physicochemical and biological properties of poly-alpha-amino acids--the simplest of protein models.

During the 1950s, linear and multichain poly-alpha-amino acids were synthesized by polymerization of the corresponding N-carboxy-amino acid anhydrides in solution in the presence of suitable catalysts. The resulting homo- and heteropolymers have since been widely employed as simple protein models. Under appropriate conditions, poly-alpha-amino acids, in the solid state and in solution, were found to acquire conformations of an alpha-helix and of beta-parallel and antiparallel pleated sheets, or to exist as random coils. Their use in experimental and theoretical investigations of helix-coil transitions helped to shed new light on the mechanisms involved in protein denaturation. Poly-alpha-amino acids played an important role in the deciphering of the genetic code. In addition, analysis of the antigenicity of poly-alpha-amino acids led to the elucidation of the factors determining the antigenicity of proteins and peptides. Interest in the biological and physicochemical characteristics of poly-alpha-amino acids was recently renewed because of the reported novel findings that some copolymers of amino acids are effective as drugs in multiple sclerosis, and that glutamine repeats and reiteration of other amino acids occur in inherited neurodegenerative diseases. The presence of repeating sequences of amino acids in proteins, and of nucleotides in DNA, raises many interesting questions about their respective roles in determining protein structure and function, and gene performance and regulation.

Identification of epitopes within a highly immunogenic region of acetylcholine receptor by a phage epitope library.

We have employed a hexapeptide phage-epitope library to identify epitopes for a mAb (mAb 5.14), which is directed to a determinant within a highly immunogenic, cytoplasmic region of the alpha-subunit of acetylcholine receptor (AChR). We have selected two different peptide-presenting phages (SWDDIR-phage and LWILTR-phage) which interact specifically with mAb 5.14. This interaction is specifically inhibited by AChR and by synthetic peptides corresponding to the hexapeptides presented by the selected phages. Although mAb 5.14 binds to AChR in its native as well as its denatured form, the selected hexapeptides do not exist as such in the AChR molecule. However, three amino acid sequence homologies with these hexapeptides were shown to be present in the cytoplasmic region of Torpedo AChR. By extending the selected hexapeptides, at one or both ends, with amino acid residues flanking the hexapeptides in the phage, we obtained mimotopes with an up to two order of magnitude higher affinity to the Ab. These extended peptides were able to efficiently block the binding of mAb 5.14 to both peptide-presenting phages, and to AChR.