Structure-function relationships and site of action of apamin, a neurotoxic polypeptide of bee venom with an action on the central nervous system.

Specific chemical modifications of apamin have been used to study the residues involved in its toxic action. Transformation of Lys4 into homoarginine did not affect toxicity. Modification of the alpha-amino group of Cys1 and of the epsilon-amino group of Lys4 by acetic anhydride or fluorescamine decreased toxicity only by a factor of 2.5-2.8. Modification of the gamma-carboxylate of Glu7 with glycine ethyl ester in the presence of a soluble carbodiimide decreased toxicity by a factor of 2. Diethyl pyrocarbonate treated of the imidazole side chain of His18 decreased toxicity by a factor of 2.6. Thus none of these residues is essential for toxicity. However, combined modification of amino groups and of the imidazole side chain of His-18 completely abolished biological activity. Complete loss of toxicity also resulted from reduction and alkylation of both disulfide bridges, from chemical modification with cyclohexanedione of Arg-13 and Arg-14, and from removal of Arg-14 of acetylated apamin by digestion with trypsin. Incorporation of radioactive acetyl groups on both amino groups of apamin gave an active labeled toxin which has been used to localize the site of action of apamin in the spinal cord, principally in the lumbar part of the neuraxis.     

Structural requirements for conserved arginine of parathyroid hormone

Arg-20 is one of two residues conserved in all peptides known to activate the parathyroid hormone (PTH) receptor. Previous studies have failed to find any naturally encoded analogues of residue 20 that had any adenylyl cyclase (AC) stimulating activity. In this work we have studied substitutions of Arg-20 with nonencoded amino acids and conformationally constrained analogues with side chains mimicking that of Arg. No analogue had more than 20% of the AC-stimulating ability of the natural Arg-20-bearing peptide. In descending order of activity, the most active analogues had (S)-4-piperidyl-(N-amidino)glycine (PipGly), norleucine (Nle), citrulline (Cit), or ornithine (Orn) at residue 20. Analogues with Arg-20 substituted with L-4-piperidyl-(N-amidino)alanine, Lys, Glu, Ala, Gln, (S)-2-amino-4-[(2-amino)pyrimidinyl]butanoic acid, or L-(4-guanidino)phenylalanine had very low or negligible activity. Low or negligible activities of Lys or Orn analogues suggested ionic interactions play a minor role in the Arg interaction with the receptor. The conformational constraints imposed by the PipGly ring had a negative effect on its ability to substitute for Arg. The side-chain H-bonding potential of the Cit ureimido group was likely an important factor in its mimicry of Arg. The increase in amphiphilicity, as demonstrated by its greater high-performance liquid chromatographic retention, and increased alpha-helix, as shown by circular dichroic spectroscopy, likely contributed to the activity of the Nle-20 analogue. The data demonstrated that specific H-bonding, hydrophobicity of the side chain, stabilization of alpha-helix, and possibly specific cation positioning were all important in the interaction of Arg-20 with receptor groups.     

Chemical modification of arginine residues in alpha-bungarotoxin.

The reaction of alpha-bungarotoxin (alpha-BuTX) with 1,2-cyclohexanedione resulted in the modification of only Arg-72 but arginine at position 36 or 72, as well as both were modified by reaction of the toxin with p-hydroxyphenylglyoxal. No derivative modified at Arg-25 was obtained, indicating that this residue may be located in the interior region of alpha-BuTX molecule. Monoderivative at Arg-72 showed about 50% of the lethal toxicity and binding activity of alpha-BuTX to nicotinic acetylcholine receptor (AChR), while the activity was decreased to one-third when the invariant Arg-36 was modified, indicating that the latter residue is more closely related to the interaction of the toxin with AChR. Approx. 13% of the residual activity was observed when both arginine residues at 36 and 72 were modified. The antigenicity of alpha-BuTX was still retained essentially intact after Arg-36 or -72 was modified, whereas it decreased to 50% when both these arginine residues were modified. The present study indicates that Arg-36 and -72 in alpha-BuTX may be involved in the multipoint contact between the toxin and AChR, but neither is absolutely essential for the binding.     

Arginine residues 47 and 70 of human flap endonuclease-1 are involved in DNA substrate interactions and cleavage site determination

Flap endonuclease-1 (FEN-1) is a critical enzyme for DNA replication and repair. Intensive studies have been carried out on its structure-specific nuclease activities and biological functions in yeast cells. However, its specific interactions with DNA substrates as an initial step of catalysis are not defined. An understanding of the ability of FEN-1 to recognize and bind a flap DNA substrate is critical for the elucidation of its molecular mechanism and for the explanation of possible pathological consequences resulting from its failure to bind DNA. Using human FEN-1 in this study, we identified two positively charged amino acid residues, Arg-47 and Arg-70 in human FEN-1, as candidates responsible for substrate binding. Mutation of the Arg-70 significantly reduced flap endonuclease activity and eliminated exonuclease activity. Mutation or protonation of Arg-47 shifted cleavage sites with flap substrate and significantly reduced the exonuclease activity. We revealed that these alterations are due to the defects in DNA-protein interactions. Although the effect of the single Arg-47 mutation on binding activities is not as severe as R70A, its double mutation with Asp-181 had a synergistic effect. Furthermore the possible interaction sites of these positively charged residues with DNA substrates were discussed based on FEN-1 cleavage patterns using different substrates. Finally data were provided to indicate that the observed negative effects of a high concentration of Mg(2+) on enzymatic activity are probably due to the competition between the arginine residues and metal ions with DNA substrate since mutants were found to be less tolerant.     

Effect of substitution of glycine for arginine at position 146 of the A1 subunit on biological activity of Escherichia coli heat-labile enterotoxin.

The ADP-ribosyltransferase activity of polypeptide A1 of cholera toxin and that of Escherichia coli heat-labile enterotoxin (LT) are primarily responsible for the toxic activities of these toxins. Since the amino acid sequences of the two A1 polypeptides are very similar, their functional mechanisms are considered to be the same. Arg-146 of polypeptide A1 is thought to be involved in the active site, because this amino acid of cholera toxin has been identified as the site of self-ADP-ribosylation. However, the exact role of Arg-146 and the significance of self-ADP-ribosylation in toxicity remain unclear. We substituted Arg-146 of polypeptide A1 of LT with Gly by oligonucleotide-directed mutagenesis and examined the biological property of the resultant mutant LT. The substitution changed the mobility of subunit A on sodium dodecyl sulfate-polyacrylamide gel but did not reduce the vascular permeability activity of LT. This result indicates that Arg-146 is not absolutely required for toxic activity and that LT can express its toxic activity without self-ADP-ribosylation at Arg-146.     

Preparation of a pure monoiodo derivative of the bee venom neurotoxin apamin and its binding properties to rat brain synaptosomes

The preparation and purification of an active monoiodo derivative of apamin is described. Radiolabeled monoiodoapamin (2000 Ci/mmol) binds specifically to rat brain synaptosomes at 0 degrees C and pH 7.5 with a second order rate constant of association (ka = 2.6 x 10(7) M-1 s-1) and a first order rate constant of dissociation (kd = 3.8 x 10(-4) s-1). The maximal binding capacity is 12.5 fmol/mg of protein and the dissociation constant is 15-25 pM for the monoiodo derivative and 10 pM for the native toxin. The apamin receptor is destroyed by proteases suggesting that it is of a proteic nature. Neurotensin and its COOH-terminal partial sequences are the only molecules unrelated to apamin that are able to displace monoiodoapamin from its receptor at low concentrations. Half-displacement occurs at 170 nM neurotensin. This property is due to the presence in the COOH-terminal sequence of neurotensin of two contiguous arginine residues, a structure analogous to that of the apamin active site. The binding of monoiodoapamin to its receptor is sensitive to cations. Increasing K+ or Rb+ concentrations from 10 microM to 5 mM selectively enhances the binding by a factor of 1.8. Increasing the concentration of any cation from 1 to 100 mM completely inhibits iodoapamin binding. Both effects are due to a cation-induced modulation of the affinity of monoidoapamin for its receptor without any change of the maximal toxin binding capacity of synaptosomes. Guanidinium and molecules containing a guanidinium group are better inhibitors of iodoapamin binding than other inorganic cations or positively charged organic molecules.     

Specific binding and pharmacological interactions of apamin,the neurotoxin from bee venom,with guinea pig colon

This paper describes the interaction of apamin, the bee venom neurotoxin, with its receptor in the guinea pig colon. The pharmacological activity of the toxin was assayed by measuring its contracting effect on guinea pig colon preparations that had been previously relaxed by neurotensin. The IC₅₀ value of apamin in this $\text{in vitro}$ bioassay is 7 nM. These pharmacological data are compared to the binding properties of apamin to smooth muscle membranes prepared from guinea pig colon. The highly radiolabeled monoiododerivative of apamin binds to its colon receptor with a dissociation constant $\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}^1\text{= 36}\text{pM}$. The maximal binding capacity of colonic membranes is 30^dfmol/mg of protein. The dissociation constant of the unmodified toxin is 23 pM. The difference between the toxin concentrations that produce half-maximal effects in the binding and pharmacological studies arises from the different experimental conditions used for the two assays.     

The cleavage site specificity of human prostate specific antigen for insulin-like growth factor binding protein-3

The cleavage site of human insulin-like growth factor binding protein-3 by urinary prostate specific antigen was examined. Human insulin-like growth factor binding protein-3 was incubated with urinary prostate specific antigen at 37 degrees C and its proteolyzed fragments were separated by a reversed phase HPLC followed by N-terminal amino acid sequence analysis, demonstrating that the cleavage mainly occurred at Tyr-159. The synthetic peptide including Tyr-159 was also cleaved at the same site, although its reaction rate was relatively low. These results indicate that human insulin-like growth factor binding protein-3 is specifically cleaved at Tyr-159 by prostate specific antigen. Human insulin-like growth factor binding protein-3 was previously reported to be cleaved at five sites including Arg-97, Arg-132, Tyr-159, Phe-173 and Arg-179 by another group, however, prostate specific antigen preparation is possibly contaminated by trypsin-like protease. In contrast, our purified urinary prostate specific antigen had only a chymotrypsin-like activity, demonstrating that prostate specific antigen has the high substrate specificity for human insulin-like growth factor binding protein-3.     

Structure-function relationships in human epidermal growth factor studied by site-directed mutagenesis and 1H NMR

In order to elucidate the mechanism of interaction between human epidermal growth factor (EGF) and its receptor, selected variants of EGF, differing by single amino acid substitutions, have been made by site-directed mutagenesis. The receptor affinity of these mutants was determined by a receptor binding competition assay, and the effects of the substitution on the structure of the protein were assessed by 1H nuclear magnetic resonance techniques. Various substitutions of Arg-41 resulted in substantial reduction in receptor affinity of EGF whereas change of Tyr-13 did not affect binding to the receptor. The 1H resonances of all nonexchangeable protons of the Tyr-13----Leu, Arg-41----His, and Leu-47----Glu variants were assigned and compared in order to assess the structural integrity of these mutants, which possess very different spectral and biological properties. In the case of the Leu-47----Glu mutant, only minor localized spectral changes were observed, confirming that the tertiary structure of the protein is preserved upon mutation. In contrast, for both the Arg-41----His and Tyr-13----Leu variants, significant and strikingly similar spectra changes were observed for many residues located far away from the mutated residues. This implies that similar structural alterations have taken place in both proteins, an idea further supported by hydrogen-exchange experiments where the exchange rates of hydrogen-bonded amide protons for both the Tyr-13----Leu and the Arg-41----His mutants were found to be about 4 times faster than in the wild-type protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Certification of the critical importance of L-3-(2-naphthyl)alanine at position 3 of a specific CXCR4 inhibitor, T140, leads to an exploratory performance of its downsizing study

We have previously found that a 14-amino acid residue-peptide, T140, inhibits infection of target cells by T cell line-tropic HIV-1 (X4-HIV-1) through its specific binding to a chemokine receptor, CXCR4. Here, the importance of an L-3-(2-naphthyl)alanine (Nal) residue at position 3 in T140 for high anti-HIV activity and inhibitory activity against Ca(2+) mobilization induced by stromal cell-derived factor (SDF)-1alpha-stimulation through CXCR4 has initially been shown by the synthesis and biological evaluation of several analogues, where Nal(3) is substituted by diverse aromatic amino acids. Next, the order of the N-terminal 3 residues (Arg(1)-Arg(2)-Nal(3)) has been proved to be important from the structure--activity relationship (SAR) study shuffling these residues. Based on these results, we have found 10-residue peptides possessing modest anti-HIV activity by systematic antiviral evaluation of a series of synthetic, shortened analogues of T140.     

Scyllatoxin, a blocker of Ca(2+)-activated K+ channels: structure-function relationships and brain localization of the binding sites.

Chemical modifications of scyllatoxin (leiurustoxin I) have shown that two arginines in the sequence, Arg6 and Arg13, are essential both for binding to the Ca(2+)-activated K+ channel protein and for the functional effect of the toxin. His31 is important both for the binding activity of the toxin and for the induction of contractions on taenia coli. However, although its iodination drastically decreases the toxin activity, it does not abolish it. Chemical modification of lysine residues or of Glu27 does not significantly alter toxin binding, but it drastically decreases potency with respect to contraction of taenia coli. The same observation has been made after chemical modification of the lysine residues. The brain distribution of scyllatoxin binding sites has been analyzed by quantitative autoradiographic analysis. It indicates that apamin (a bee venom toxin) binding sites are colocalized with scyllatoxin binding sites. The results are consonant with the presence of apamin/scyllatoxin binding sites associated with Ca(2+)-activated K+ channels. High-affinity binding sites for apamin can be associated with very-high-affinity (less than 70 pM), high-affinity (approximately 100-500 pM), or moderate-affinity (greater than 800 pM) binding sites for scyllatoxin.     

Identification of functional arginines in human angiogenin by site-directed mutagenesis.

Chemical modifications of human angiogenin had suggested that arginines are essential for its ribonucleolytic activity [Shapiro, R., Weremowicz, S., Riordan, J. F., & Vallee, B. L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8783-8787]. Each of the six arginines within or near angiogenin's catalytic or cell-binding sites--i.e., those at positions 5, 31, 32, 33, 66, and 70--was therefore mutated to alanine. Two of these residues, Arg-5 and Arg-33, indeed play a role, albeit noncrucial, in enzymatic activity, although neither one is implicated in the abolition of activity by arginine reagents. R5A-angiogenin, while nearly fully active toward dinucleotides, is one-fourth as active as angiogenin toward tRNA, suggesting that Arg-5 may participate in the binding of peripheral components of the substrate. In contrast, the activity of R33A-angiogenin toward both polynucleotide and dinucleotide substrates is reduced similarly, reflecting a decrease in kcat. These results, together with its position in the calculated three-dimensional structure of angiogenin, imply an indirect role for Arg-33 in catalysis. Three arginines are important for angiogenesis: mutation of Arg-5, Arg-33, or Arg-66 dramatically reduces the angiogenic potency of angiogenin on the chicken embryo chorioallantoic membrane. Arg-66 lies within a segment previously proposed to be part of a cell-surface receptor binding site. Arg-5 and Arg-33 are outside of this site as defined at present, and the decreased angiogenicity of R5A- and R33A-angiogenin may be a consequence of their reduced ribonucleolytic activities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Locating the carboxylate group of GABA in the homomeric rho GABA(A) receptor ligand-binding pocket

gamma-Aminobutyric acid, type A (GABA(A)) receptors, of which the GABA(C) receptor family is a subgroup, are members of the Cys loop family of neurotransmitter receptors. Homology modeling of the extracellular domain of these proteins has revealed many molecular details, but it is not yet clear how GABA is orientated in the binding pocket. Here we have examined the role of arginine residues that the homology model locates in or close to the binding site of the GABA(C) receptor (Arg-104, Arg-170, Arg-158, and Arg-249) using mutagenesis and functional studies. The data suggest that Arg-158 is critical for GABA binding and/or function; substitution with Lys, Ala, or Glu resulted in nonfunctional receptors, and modeling placed the carboxylate of GABA within 3A of this residue. Substitution of Arg-104 with Ala or Glu resulted in >10,000-fold increases in EC(50) values compared with wild type receptors, and modeling indicated a role of this residue both in binding GABA and in the structure of the binding pocket. Substitution of Arg-170 with Asp or Ala yielded nonfunctional receptors, whereas Lys caused an approximately 10-fold increase in EC(50). Arg-249 was substituted with Ala, Glu, or Asp with relatively small ( approximately 4-30-fold) changes in EC(50). These and data from other residues that the model suggested could interact with GABA (His-105, Ser-168, and Ser-243) support a location for GABA in the binding site with its carboxylate pincered between Arg-158 and Arg-104, with Arg-104, Arg-170, and Arg-249 contributing to the structure of the binding pocket through salt bridges and/or hydrogen bonds.     

Molecular mapping and functional characterization of the VEGF164 heparin-binding domain

The longer splice isoforms of vascular endothelial growth factor-A (VEGF-A), including mouse VEGF164, contain a highly basic heparin-binding domain (HBD), which imparts the ability of these isoforms to be deposited in the heparan sulfate-rich extracellular matrix and to interact with the prototype sulfated glycosaminoglycan, heparin. The shortest isoform, VEGF120, lacks this highly basic domain and is freely diffusible upon secretion. Although the HBD has been attributed significant relevance to VEGF-A biology, the molecular determinants of the heparin-binding site are unknown. We used site-directed mutagenesis to identify amino acid residues that are critical for heparin binding activity of the VEGF164 HBD. We focused on basic residues and found Arg-13, Arg-14, and Arg-49 to be critical for heparin binding and interaction with extracellular matrix in tissue samples. We also examined the cellular and biochemical consequences of abolishing heparin-binding function, measuring the ability of the mutants to interact with VEGF receptors, induce endothelial cell gene expression, and trigger microvessel outgrowth. Induction of tissue factor expression, vessel outgrowth, and binding to VEGFR2 were unaffected by the HBD mutations. In contrast, the HBD mutants showed slightly decreased binding to the NRP1 (neuropilin-1) receptor, and analyses suggested the heparin and NRP1 binding sites to be distinct but overlapping. Finally, mutations that affect the heparin binding activity also led to an unexpected reduction in the affinity of VEGF164 binding specifically to VEGFR1. This finding provides a potential basis for previous observations suggesting enhanced potency of VEGF164 versus VEGF120 in VEGFR1-mediated signaling in inflammatory cells.     

Analysis of structure and function of the B subunit of cholera toxin by the use of site-directed mutagenesis

Oligonucleotide-directed mutagenesis of ctxB was used to produce mutants of cholera toxin B subunit (CT-B) altered at residues Cys-9, Gly-33, Lys-34, Arg-35, Cys-86 and Trp-88. Mutants were identified phenotypically by radial passive immune haemolysis assays and genotypically by colony hybridization with specific oligonucleotide probes. Mutant CT-B polypeptides were characterized for immunoreactivity, binding to ganglioside GM1, ability to associate with the A subunit, ability to form holotoxin, and biological activity. Amino acid substitutions that caused decreased binding of mutant CT-B to ganglioside GM1 and abolished toxicity included negatively charged or large hydrophobic residues for Gly-33 and negatively or positively charged residues for Trp-88. Substitution of lysine or arginine for Gly-33 did not affect immunoreactivity or GM1-binding activity of CT-B but abolished or reduced toxicity of the mutant holotoxins, respectively. Substitutions of Glu or Asp for Arg-35 interfered with formation of holotoxin, but none of the observed substitutions for Lys-34 or Arg-35 affected binding of CT-B to GM1. The Cys-9, Cys-86 and Trp-88 residues were important for establishing or maintaining the native conformation of CT-B or protecting the CT-B polypeptide from rapid degradation in vivo.     

The Ca2+-dependent slow K+ conductance in cultured rat muscle cells: characterization with apamin.

The interaction of apamin, a bee venom neurotoxin, with rat skeletal muscle cell membranes has been followed using both an electrophysiological and a biochemical approach. Voltage-clamp analyses have shown that apamin, at low concentrations, specifically blocks the Ca2+-dependent slow K+ conductance in rat myotubes and myosacs . A specific binding site for apamin in rat muscle cell membranes has been characterized with the use of a highly radiolabelled apamin derivative [( 125I]apamin). The dissociation constant for the apamin-receptor complex is 36-60 pM and the maximal binding capacity is 3.5 fmol/mg of protein. [125I]Apamin binding to rat muscle membranes is displaced by quinine and quinidine with K0.5 values of 110 microM and 200 microM, respectively.     

Interpretation of biological activity data of bacterial endotoxins by simple molecular models of mechanism of action.

Lipid A moiety has been identified as the bioactive component of bacterial endotoxins (lipopolysaccharides). However, the molecular mechanism of biological activity of lipid A is still not fully understood. This paper contributes to understanding of the molecular mechanism of action of bacterial endotoxins by comparing molecular modelling results for two possible mechanisms with the underlying experimental data. Mechanisms of action involving specific binding of lipid A to a protein receptor as well as nonspecific intercalation into phospholipid membrane of a host cell were modelled and analysed. As the cellular receptor for endotoxin has not been identified, a model of a peptidic pseudoreceptor was proposed, based on molecular structure, symmetry of the lipid A moiety and the observed character of endotoxin-binding sites in proteins. We have studied the monomeric form of lipid A from Escherichia coli and its seven synthetic analogues with varying numbers of phosphate groups and correlated them with known biological activities determined by the Limulus assay. Gibbs free energies associated with the interaction of lipid A with the pseudoreceptor model and intercalation into phospholipid membrane calculated by molecular mechanics and molecular dynamics methods were used to compare the two possible mechanisms of action. The results suggest that specific binding of lipid A analogues to the peptidic pseudoreceptor carrying an amphipathic cationic binding pattern BHPHB (B, basic; H, hydrophobic; P, polar residue, respectively) is energetically more favourable than intercalation into the phospholipid membrane. In addition, binding affinities of lipid A analogues to the best minimum binding sequence KFSFK of the pseudoreceptor correlated with the experimental Limulus activity parameter. This correlation enabled us to rationalize the observed relationship between the number and position of the phosphate groups in the lipid A moiety and its biological activity in terms of specific ligand-receptor interactions. If lipid A-receptor interaction involves formation of phosphate-ammonium ion-pair(s) with cationic amino-acid residues, the specific mechanism of action was fully consistent with the underlying experimental data. As a consequence, recognition of lipid A variants by an amphipathic binding sequence BHPHB of a host-cell protein receptor might represent the initial and/or rate-determining molecular event of the mechanism of action of lipid A (or endotoxin). The insight into the molecular mechanism of action and the structure of the lipid A-binding pattern have potential implications for rational drug design strategies of endotoxin-neutralizing agents or binding factors.