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Estrogen has a key role in the regulation of skeletal growth and maintenance of bone mass. Recently, we developed peptides having estrogen-like activity as potential estrogen-based new drugs. The aim of the present study was to evaluate the influence of long-term administration of the most efficacious of these peptides, the hexapeptide EMP-1 (VSWFFE), on bone mass and development. EMP-1 was injected daily to ovariectomized (OVX) and intact young, sexually mature female mice for 10 weeks. Whole femora, including the cartilaginous growth plates were analyzed by micro-computed tomography (microCT). We found that peptide EMP-1 restrains bone growth in OVX mice: it inhibited dramatically bone longitudinal growth (40%), and decreased femoral diaphyseal diameter. Peptide EMP-1 had no effect on bone growth in normal mice, and did not influence the OVX-induced bone loss. We then developed a new microCT methodology to evaluate uncalcified and calcified growth plate parameters. In the OVX mice, peptide EMP-1 reduced volume and thickness of the uncalcified growth plate, a possible cause for the inhibition of bone longitudinal growth. Peptide EMP-1 may be used as a lead compound for the development of drugs to treat acromegalic patients.

Some time ago Dr Whelan informed me that there would be a 50th anniversary celebration of IUB's admission into ICSU, and IUBMB Life would publish a special issue to coincide with the IUMB-FEBS Conference in Budapest next July. While checking the past history of IUB's Congresses, Dr Whelan noticed that I was the only person who had ever been asked to deliver two plenary lectures at these Congresses (1964 and 1979). He invited me to contribute my reminiscences concerning these two lectures, and I promised to explain how this had happened. IUBMB Life, 57: 239-242, 2005

Our group has been employing short synthetic peptides, encompassing sequences from the acetylcholine receptor (AChR) alpha-subunit for the analysis of the binding site of the AChR. A 13-mer peptide mimotope, with similar structural motifs to the AChR binding region, was selected by alpha-bungarotoxin (alpha-BTX) from a phage-display peptide library. The solution structure of a complex between this library-lead peptide and alpha-BTX was solved by NMR spectroscopy. On the basis of this NMR study and on structure-function analysis of the AChR binding site, and in order to obtain peptides with higher affinity to alpha-BTX, additional peptides resulting from systematic residue replacement in the lead peptide were designed and characterized. Of these, four peptides, designated high-affinity peptides (HAPs), homologous to the binding region of the AChR, inhibited the binding of alpha-BTX to the AChR with an IC(50) of 2 nM. The solution and crystal structures of complexes of alpha-BTX with HAP were solved, demonstrating that the HAP fits snugly to alpha-BTX and adopts a beta-hairpin conformation. The X-ray structures of the bound HAP and the homologous loop of the acetylcholine binding protein (AChBP) are remarkably similar. Their superposition results in a model indicating that alpha-BTX 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. Our proposed model explains the strong antagonistic activity of alpha-BTX and accommodates much of the biochemical data on the mode of interaction of alpha-BTX with the AChR.

a-Bungarotoxin (a-BTX) is a highly toxic snake neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. In the following we review multi-phase research of the design, synthesis and structure analysis of peptides that bind a-BTX and inhibit its binding to AChR. Structure- based design concomitant with biological information of the a-BTXyAChR system yielded 13-mer peptides that bind to a-BTX with high affinity and are potent inhibitors of a-BTX binding to AChR (IC of 2 nM). X-Ray and NMR 50 spectroscopy reveal that the high-affinity peptides fold into an anti-parallel b-hairpin structure when bound to a- BTX. The structures of the bound peptides and the homologous loop of acetylcholine binding protein, a soluble analog of AChR, are remarkably similar. Their superposition indicates that the toxin wraps around the binding-site loop, and in addition, binds tightly at the interface of two of the receptor subunits and blocks access of acetylcholine to its binding site. The procedure described in this article may serve as a paradigm for obtaining high-affinity peptides in biochemical systems that contain a ligand and a receptor molecule. 2002 Elsevier Science B.V. All rights reserved.

Background: α-Bungarotoxin (α-BTX) is a highly toxic snake venom α-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 α-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 3×10 −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 α-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 α-BTX lethality. Conclusions: Synthetic peptides with high affinity to α-BTX may be used as potential lead compounds for developing effective antidotes against α-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 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.

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 thein vitrobinding 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.

The structures of two binary complexes (the TEM-1/BLIP complex and the trypsin/amiloride complex) were predicted prior to their experimental determination and compared to the corresponding experimental structures when these became available. In both predictions the rigid-body geometric docking algorithm ranked the correct solution among the top ones. Additional information concerning the structure and chemical character of the binding site of one of the molecules in the complex was used to single out the correct solution. The results indicate that the combination of geometric surface matching with biochemical information produces a useful tool for structure prediction.

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 α-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 α-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.

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.

Ephraim Katchalski-Katzir grew up in Israel at a time when the State was coming into being, and like many of those of his generation, participated in its creation and defense. He received his scientific education at the Hebrew University of Jerusalem and thereafter at the Polytechnic Institute of Brooklyn, Columbia University, and Harvard University. During and after the establishment of the State, he acted as scientific consultant to the defense leadership, and continued throughout his career to advise the Government of Israel on research and development. His public activities culminated in his election in 1973 as President of the State of Israel. As one of the founders of the Weizmann Institute of Science, he headed the Department of Biophysics for many years. His research included the study of poly-alpha-amino acids as protein models, immobilized enzymes, and polymers as chemical reagents. He also carried out theoretical and experimental work on the determination of distance distributions and conformational fluctuations in proteins by nonradiative energy-transfer techniques.

Antiphospholipid syndrome (APS) is characterized by thromboembolic phenomena and recurrent fetal loss associated with elevated circulating anti-phospholipid/beta2glycoprotein-I(β2GPI)-binding-antibodies(Abs). Individual APS patients harbor diverse clusters of circulating anti-β2GPI Abs, targeting different epitopes on the β2GPI molecule. Our novel approach was to construct a peptide composed of β2GPI-ECs-binding-site (phospholipids-membrane), named "EMBI". EMBI exert dual activities: a) At first EMBI prevented β2GPI ECs binding, thus reduced by 89% the binding of β2GPI/anti-β2GPI to the cells in comparison with 9.3% inhibition by EMBI scrambled form (scEMBI). b) Longer exposure of ECs to EMBI resulted in intracellular EMBI penetration which did not prevent β2GPI/anti-β2GPI binding to HUVEC. Surprisingly, β2GPI/anti-β2GPI did not activate ECs harboring EMBI, illustrated by prevention of E-selectin and tissue factor (TF) expression. The inhibition of TF mRNA transcription was illustrated by quantitative RT-PCR. EMBI decreased the expression of phosphorylated JNK1/2, p38, HSP27 and enhanced phosphorylation of glycogen synthase kinase-3β (pGSK3β). Knocking down the GSK3β expression by siRNA-GSK3β, reduced the TF expression by β2GPI/anti-β2GPI-exposed-HUVEC. In-vivo, EMBI significantly decreased the percentage of fetal loss in naïve mice infused with anti-β2GPI Abs, p0.04. Thus, the dual activity of EMBI may introduce EMBI as a potential novel candidate peptide, to treat patients with APS.

Basic fibroblast growth factor (bFGF) is known to bind to its cell-surface receptors with high affinity and in a heparin-dependent manner. In an attempt to predict the receptor recognition site on bFGF we screened phage-epitope libraries with monoclonal antibodies DG2 and DE6, which inhibit bFGF binding to its receptor. On the affinity-isolated phages, we identified several peptide sequences as the putative antibody-binding epitopes on bFGF. The identified library epitopes shared the consensus sequence Pro-(Pro/Ser)-Gly-His-(Tyr/Phe)-Lys, corresponding to two continuous protein sequences of bFGF: Pro-Pro-Gly-His-Phe-Lys and Arg-Thr-Gly-Gln-Tyr-Lys at amino acids 13-18 and 120-125 of bFGF, respectively. Synthetic peptides of the corresponding phage epitopes or of the above bFGF sequences specifically inhibited binding of the antibodies to bFGF, blocked binding of bFGF to its high-affinity receptor, and inhibited basal and bFGF-induced proliferation of vascular endothelial cells at submicromolar peptide concentrations. The potent inhibition of bFGF binding and biological activity by peptides recognized by the antibodies suggests that these sequences are functionally involved in receptor binding and may constitute part of the receptor-binding determinants on bFGF.

Monoclonal antibody (mAb) 5.5 is directed against the ligand-binding site of the nicotinic acetylcholine receptor. The epitope for this antibody is conformation-dependent, and the antibody does not react with synthetic peptides derived from the receptor sequence. We have identified a ligand peptide that mimics this conformation-dependent epitope from a phage-epitope library composed of filamentous phage displaying random hexapeptides. Among 38 positive phage clones, individually selected from the library, 34 positive clones carried the sequence Asp-Leu-Val-Trp-Leu-Leu (DLVWLL), 1 positive clone had the sequence Asp-Ile-Val-Trp-Leu-Leu (DIVWLL), and 3 positive clones expressed the sequence Leu-Ile-Glu-Trp-Leu-Leu (LIEWLL), none of which are significantly homologous with the nicotinic acetylcholine receptor alpha subunit sequence. All of these phages bind specifically to mAb 5.5. The synthetic peptide DLVWLL inhibits binding of mAb 5.5 to the related peptide-presenting phage and to the nicotinic acetylcholine receptor in a concentration-dependent manner; the IC50 value is of the order of 10(-4) M. Bioactivity of the peptide "mimotope" DLVWLL was demonstrated in vivo in hatched chickens by inhibition of the mAb 5.5 effect by the peptide. The neuromuscular block and myasthenia gravis-like symptoms that are induced in chicken by passive transfer of mAb 5.5 were specifically abolished by DLVWLL. This study shows the potential of a random peptide phage-epitope library for selecting a mimotope for an antibody that recognizes a folded form of the protein, where peptides from the linear amino acid sequence of the protein are not applicable.

The use of immobilized enzymes in the food, pharmaceutial and chemical industries has increased steadily during the past decade. Further research and development in enzyme technology is expected to expand their use in the synthesis of chiral drugs, complex organic compounds and fine chemicals, and lead to the contruction of novel, specific biosensors.

A geometric recognition algorithm was developed to identify molecular surface complementarity. It is based on a purely geometric approach and takes advantage of techniques applied in the field of pattern recognition. The algorithm involves an automated procedure including (i) a digital representation of the molecules (derived from atomic coordinates) by three-dimensional discrete functions that distinguishes between the surface and the interior; (ii) the calculation, using Fourier transformation, of a correlation function that assesses the degree of molecular surface overlap and penetration upon relative shifts of the molecules in three dimensions; and (iii) a scan of the relative orientations of the molecules in three dimensions. The algorithm provides a list of correlation values indicating the extent of geometric match between the surfaces of the molecules; each of these values is associated with six numbers describing the relative position (translation and rotation) of the molecules. The procedure is thus equivalent to a six-dimensional search but much faster by design, and the computation time is only moderately dependent on molecular size. The procedure was tested and validated by using five known complexes for which the correct relative position of the molecules in the respective adducts was successfully predicted. The molecular pairs were deoxyhemoglobin and methemoglobin, tRNA synthetase-tyrosinyl adenylate, aspartic proteinase-peptide inhibitor, and trypsin-trypsin inhibitor. A more realistic test was performed with the last two pairs by using the structures of uncomplexed aspartic proteinase and trypsin inhibitor, respectively. The results are indicative of the extent of conformational changes in the molecules tolerated by the algorithm.

Currently used antiestrogenic drugs against hormone-dependent breast cancer, and estrogenic drugs used in treatment of osteoporosis, are associated with risk factors. Therefore, there is a strong need to develop selective estrogen receptor modulators with better tissue selectivity. In a recent study (Peptides, 2002, Vol. 3, 573-580), we used a monoclonal antibody to estradiol (mAb-E2) to screen a phage-display peptide library. We identified a 15-mer peptide (peptide H5) that recognizes mAb-E2 (IC(50) 1 microM) and estrogen receptor (ER)alpha (IC(50) 500 microM) but not ERbeta, and displays estrogen-like activity in vitro and in vivo. In this study, we designed and prepared peptides based on peptide H5, which possess improved estrogenic activity, by evaluating their binding to mAb-E2 and to ERs. Initially, we determined the minimal binding sequence of peptide H5 capable of binding mAb-E2 and ER. Subsequently, systematic single-residue replacements of the minimal sequence, followed by multiple-residue replacements, yielded hexa- and heptapeptides with increased affinities to mAb-E2 and to ER. The most promising peptides, VSWFFE (EMP-1) and VSWFFED (EMP-2) (EMP: estrogen-mimetic peptide), bind mAb-E2 with high affinity (IC(50) of 6 and 30 nM, respectively), recognize ERs with increased affinity (IC(50) of 100 microM for ERalpha, and 100-250 microM for ERbeta), and possess estrogenic activity in vivo. The short peptides described in this study may be used as potential lead compounds for developing new ER ligands.

The activity of a living cell can be portrayed as a network of interactions involving proteins and nucleic acids that transfer biological information. Intervention in cellular processes requires thorough understanding of the interactions between the molecules, which can be provided by docking techniques. Docking methods attempt to predict the structures of complexes given the structures of the component molecules. We focus hereby on protein-protein docking procedures that employ grid representations of the molecules, and use correlation for searching the solution space and evaluating putative complexes. Geometric surface complementarity is the dominant descriptor in docking. Inclusion of electrostatics often improves the results of geometric docking for soluble proteins, whereas hydrophobic complementarity is more important in construction of oligomers. Using binding-site information in the scan or as a filter helps to identify and up-rank nearly correct solutions.

Our group has been employing short synthetic peptides, encompassing sequences from the acetylcholine receptor (AChR) alpha-subunit for the analysis of the binding site of the AChR. A 13-mer peptide mimotope, with similar structural motifs to the AChR binding region, was selected by alpha-bungarotoxin (alpha-BTX) from a phage-display peptide library. The solution structure of a complex between this library-lead peptide and alpha-BTX was solved by NMR spectroscopy. On the basis of this NMR study and on structure-function analysis of the AChR binding site, and in order to obtain peptides with higher affinity to alpha-BTX, additional peptides resulting from systematic residue replacement in the lead peptide were designed and characterized. Of these, four peptides, designated high-affinity peptides (HAPs), homologous to the binding region of the AChR, inhibited the binding of alpha-BTX to the AChR with an IC(50) of 2 nM. The solution and crystal structures of complexes of alpha-BTX with HAP were solved, demonstrating that the HAP fits snugly to alpha-BTX and adopts a beta-hairpin conformation. The X-ray structures of the bound HAP and the homologous loop of the acetylcholine binding protein (AChBP) are remarkably similar. Their superposition results in a model indicating that alpha-BTX 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. Our proposed model explains the strong antagonistic activity of alpha-BTX and accommodates much of the biochemical data on the mode of interaction of alpha-BTX with the AChR.

a-Bungarotoxin (a-BTX) is a highly toxic snake neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. In the following we review multi-phase research of the design, synthesis and structure analysis of peptides that bind a-BTX and inhibit its binding to AChR. Structure- based design concomitant with biological information of the a-BTXyAChR system yielded 13-mer peptides that bind to a-BTX with high affinity and are potent inhibitors of a-BTX binding to AChR (IC of 2 nM). X-Ray and NMR 50 spectroscopy reveal that the high-affinity peptides fold into an anti-parallel b-hairpin structure when bound to a- BTX. The structures of the bound peptides and the homologous loop of acetylcholine binding protein, a soluble analog of AChR, are remarkably similar. Their superposition indicates that the toxin wraps around the binding-site loop, and in addition, binds tightly at the interface of two of the receptor subunits and blocks access of acetylcholine to its binding site. The procedure described in this article may serve as a paradigm for obtaining high-affinity peptides in biochemical systems that contain a ligand and a receptor molecule. 2002 Elsevier Science B.V. All rights reserved.

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.

A novel geometric-electrostatic docking algorithm is presented, which tests and quantifies the electrostatic complementarity of the molecular surfaces together with the shape complementarity. We represent each molecule to be docked as a grid of complex numbers, storing information regarding the shape of the molecule in the real part and information regarding the electrostatic character of the molecule in the imaginary part. The electrostatic descriptors are derived from the electrostatic potential of the molecule. Thus, the electrostatic character of the molecule is represented as patches of positive, neutral, or negative values. The potential for each molecule is calculated only once and stored as potential spheres adequate for exhaustive rotation/translation scans. The geometric-electrostatic docking algorithm is applied to 17 systems, starting form the structures of the unbound molecules. The results-in terms of the complementarity scores of the nearly correct solutions, their ranking in the lists of sorted solutions, and their statistical uniqueness-are compared with those of geometric docking, showing that the inclusion of electrostatic complementarity in docking is very important, in particular in docking of unbound structures. Based on our results, we formulate several "good electrostatic docking rules": The geometric-electrostatic docking procedure is more successful than geometric docking when the potential patches are large and when the potential extends away from the molecular surface and protrudes into the solvent. In contrast, geometric docking is recommended when the electrostatic potential around the molecules to be docked appears homogeneous, that is, with a similar sign all around the molecule.

We describe a novel approach to develop peptides with estrogen like activity using a monoclonal antibody specific to estradiol (mAb E2-15) for the affinity selection of phage displayed peptides from a combinatorial peptide library. Based on the sequences of the selected phage, we synthesized a 15-mer linear peptide LPALDPTKRWFFETK which was derivatized to a 23 mer cyclic peptide CAELPALDPTKRWFFETKPPPPC. Both peptides displayed estrogen-like activity according to the following criteria:(i) in inhibiting the binding of [3H]estradiol to mAb E2-15 and to estrogen receptor (ER)alpha; (ii) in inducing transcriptional activity in MCF7 human breast cancer cells transfected with an estrogen receptor element luciferase construct and (iii) in causing an increase in creatine kinase specific activity in rat tissues in vivo. This approach can be employed to design peptide mimetic for other hormones as well.

Snake-venom α-bungarotoxin is a member of the α-neurotoxin family that binds with very high affinity to the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. The structure of the complex between α-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 β-hairpin structure created by two antiparallel β-strands, which combine with the already existing triple-stranded β-sheet of the toxin to form a five-stranded intermolecular, antiparallel β-sheet. Peptide residues Y3 P , E5 P , and L8 P have the highest intermolecular contact area, indicating their importance in the binding of α-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 α-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 α-bungarotoxin, using a phage-display peptide library, ( ii ) the information about the three-dimensional structure of α-bungarotoxin/lead-peptide complex, and ( iii ) the amino acid sequence analysis of different AChRs.

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.

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.

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 (β2GPI)-dependent Abs or anti-β2GPI Abs by themselves. Using a hexapeptide phage display library, we identified three hexapeptides that react specifically with the anti-β2GPI 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 β2GPI 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 bothin vitroandin vivothe biological functions of the corresponding anti-β2GPI mAbs. Exposure of endothelial cells to anti-β2GPI 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 vivoinfusion of each of the anti-β2GPI 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-β2GPI Abs represents a possible new therapeutic approach to APS.

The solution structure of the complex between α-bungarotoxin (α-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 α-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 α-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 α-BTX. The structure of the complex may explain the fact that the library-derived peptide binds α-BTX with a 15-fold higher affinity than that shown by the acetylcholine receptor peptide (α185–196). Although both peptides bind to similar sites on α-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 α-BTX binding site.

The nicotinic acetylcholine receptor (AcChoR) is a ligand-gated ion channel that is activated upon binding of acetylcholine. α-Neurotoxins, in particular α-bungarotoxin (α-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 α-BTX. Phages bearing the motif YYXSSL as a consensus sequence were found to bind with high affinity to α-BTX. The library-derived peptide (MRYYESSLKSYPD) bears amino acid sequence similarities to a region of the α-subunit of theTorpedomuscle AcChoR, as well as of other muscle and neuronal AcChoRs that bind α-BTX. The library-derived peptide and the corresponding peptides containing residues 187–199 of theTorpedoAcChoR α-subunit (WVYYTCCPDTPYL), as well as peptides analogous to the above region in the neuronal AcChoR (e.g., human α7; ERFYECCKEPYPD) that binds α-BTX, inhibit the binding of α-BTX to the intactTorpedoAcChoR with IC50values of 10−6M. A synthetic peptide from a neuronal AcChoR that does not bind α-BTX (e.g., human α2; ERKYECCKEPYPD) which differs by just one amino acid from the homologous peptide from the α-BTX-binding protein (α7)—i.e., Lys in α2and Tyr in α7—does not inhibit the binding of α-BTX toTorpedoAcChoR. These results indicate the requirement for two adjacent aromatic amino acid residues for binding to α-BTX.

The three-dimensional (3D) structure of most protein complexes reveals a close geometric match between those parts of the respective surfaces of the protein and the ligand that are in contact. In many cases the 3D structure of the components in the complex closely resembles that of the molecules in their free, native state. Geometric matching thus appears to play an important role in determining the structure of a complex. A geometric recognition algorithm was developed to identify molecular surface complementarity. It is based on a purely geometric approach and takes advantage of techniques applied in the field of pattern recognition. The algorithm provides a list of correlation values indicating the extent of geometric match between the surfaces of the molecules. The procedure is equivalent to a six-dimensional search, but is much faster by design, and the computations are only moderately dependent on the molecular size. The procedure was tested and validated by using five known complexes for which the relative position of the molecules in the respective adducts was successfully predicted. The molecular pairs were the α, β subunits of deoxy-hemoglobin and methemoglobin, tRNA synthetase-tyrosinyl adenylate, aspartate proteinase-peptide inhibitor, and trypsin-trypsin inhibitor. The algorithm developed is being extended to include electrostatic match and hydrophobic interactions. In view of the above findings, the parameters determining biological specificity on the molecular level are discussed and evaluated.

Seven murine anti-hen egg-white lysozyme (HEL) monoclonal antibodies (MAbs), which recognize distinct epitopes of the native enzyme, were used as macromolecular probes to detect structural or conformational alterations occurring in HEL on heating at 95 degrees C, pH 5. As the interactions of the heat-treated HEL with its corresponding MAbs were carried out at room temperature, only irreversible structural and/or conformational alterations could be detected. The transformation of the native enzyme into its denatured form was followed electrophoretically and chromatographically. The denatured enzyme was more negatively charged at pH 8.4 and exhibited a longer retention time on reverse-phase HPLC than native HEL. Its specific catalytic activity was considerably lower than that of the native enzyme. Of the seven MAbs tested in competitive ELISA assays with native and heat-treated HEL only one, MAb D74.3, failed to recognize the heat-treated enzyme. This antibody, which is directed toward the active site region of the enzyme, was ineffective in inhibiting the catalytic activity of the heat-treated HEL using M. lysodeikticus as substrate. In contrast, the monoclonal antibody D1.3, which recognizes an epitope remote from the active site of HEL, inhibited the catalytic activity of the native as well as the heat-treated enzyme. The results indicate that the active site of HEL undergoes an irreversible structural alteration on heating for 2 hr at 95 degrees C, pH 5. No irreversible structural changes could be detected in the other regions of HEL recognized by the corresponding MAbs.

The preparation and properties of a glucose oxidase-Blue Dextran complex are described. At pH 7.4, in 0.1 M phosphate buffer, glucose oxidase and Blue Dextran interact strongly with each other. Under these conditions a soluble complex is formed, as shown by ultrafiltration experiments using a Diafio XM-300 membrane (nominal molecular weight cut-off 300,000). In this complex, the enzyme is about 40% more active than when free in solution, and is also more stable towards acid denaturation. Comparative studies were carried out using a high-molecular-weight Dextran fraction devoid of dye residues (Dextran T-2000). The results obtained suggest that the dye moiety of Blue Dextran is directly involved in the binding of the protein, and possibly also in the enhancement of its enzymatic activity.

The movement of enzymes along the surfaces of biopolymers containing enzyme-susceptible sites can be described as a lateral diffusion process characterized by an apparent diffusion coefficient [E. Katchalski-Katzir, J. Rishpon, E. Sahar, R. Lamed, and Y. I. Henis (1985) Biopolymers24, 257–277]. Studies on the diffusion of enzymes on biopolymer substrates can therefore provide important information on the mechanism of enzyme–biopolymer interaction. For this reason, the motion of fluorescently labeled β-amylase [α-D-(1 → 4)glucan maltohydrolase; E.C. 3.2.1.2] on the surface of starch gels was studied by fluorescence photobleaching recovery. The results indicate that the motion of β-amylase on the surface of the gel substrate occurs by both lateral diffusion along the surface (over micron distances) and exchange between bound and free enzyme molecules in the solution covering the gel, and that the two processes occur concomitantly and in a random manner. Surface diffusion also appears an important process with respect to the action of the enzyme on the substrate sites, since this component of the motion disappears upon inactivation of the enzyme, leaving only exchange to contribute to the measured motion.