The role of glycine residues at the C-terminal peptide segment in antinociceptive activity: a molecular dynamics simulation

Elucidating structural determinants in the functional regions of toxins can provide useful knowledge for designing novel analgesic peptides. Glycine residues at the C-terminal region of the neurotoxin BmK AGP-SYPU2 from the scorpion Buthus martensii Karsch (BmK) have been shown to be crucial to its analgesic activity. However, there has been no research on the structure–function relationship between the C-terminal segment of this toxin and its analgesic activity. To address this issue, we performed three MD simulations: one on the native structure and the other two on mutants of that structure. Results of these calculations suggest that the existence of glycine residues at the C-terminal segment stabilizes the protruding topology of the NC domain, which is considered an important determinant of the analgesic activity of BmK AGP-SYPU2.     

Soluble expression, purification and the role of C-terminal glycine residues in scorpion toxin BmK AGP-SYPU2

The existence of glycine residues in long-chain scorpion toxins has been well documented. However, their role as analgesics has not been evaluated. To address this issue, we investigated the functional role of glycines in the C-terminal end of Chinese-scorpion toxin from Buthus martensii Karsch (BmK AGP-SYPU2) using site-directed mutagenesis and analgesic activity assays. Recombinant BmK AGP-SYPU2 and its mutants were efficiently expressed in E. coli and purified to homogeneity using immobilized metal ion affinity chromatography (IMAC) and cation exchange chromatography. The mouse-twisting test was used to detect the analgesic activity of BmK AGP-SYPU2 and its mutants. As a result, we identified glycines at the C-terminal end that, when altered, significantly affected analgesic activity. Also, Mut6566 was significantly decreased compared to BmK AGP-SYPU2. These data indicate that the glycines at the C-terminal end are important for the analgesic activity of BmK AGP-SYPU2.     

Purification,characterization and cDNA cloning of an analgesic peptide from the chinese scorpion <Emphasis Type="Italic">Buthus martensii</Emphasis> Karsch (BmK AGP-SYPU2)

In this study an analgesic peptide was purified through five continuous chromatographic steps. The mouse twisting model test was used to identify the target peptides in every separation step. The purified BmK AGP-SYPU2 was further qualified by Reverse Phase-High Performance Liquid Chromatography and High Performance Capillary Electrophoresis. The molecular weight, isoelectric point, and N-terminal sequence of the purified peptide were determined. Based on the N-terminal sequence, the cDNA was cloned by rapid amplification of the cDNA ends from the cDNA pool of scorpion glands. Sequence determination showed that the mature BmK AGP-SYPU2 peptide is composed of 66 amino acid residues, and BmK AGP-SYPU2 is identical to BmK α2 (GenBank Acc. No. AF288608) and BmK αTX11 (GenBank Acc. No. AF155364). We report herein a purification procedure that yields substantial amounts of natural BmK AGP-SYPU2 with high analgesic activity.     

Purification, characterization and cDNA cloning of an analgesic peptide from the Chinese scorpion Buthus martensii Karsch (BmK AGP-SYPU2)

In this study an analgesic peptide was purified through five continuous chromatographic steps. The mouse twisting model test was used to identify the target peptides in every separation step. The purified BmK AGP-SYPU2 was further qualified by Reverse Phase-High Performance Liquid Chromatography and High Performance Capillary Electrophoresis. The molecular weight, isoelectric point, and N-terminal sequence of the purified peptide were determined. Based on the N-terminal sequence, the cDNA was cloned by rapid amplification of the cDNA ends from the cDNA pool of scorpion glands. Sequence determination showed that the mature BmK AGP-SYPU2 peptide is composed of 66 amino acid residues, and BmK AGP-SYPU2 is identical to BmK alpha2 (GenBank Acc. No. AF288608) and BmK alphaTX11 (GenBank Acc. No. AF155364). We report herein a purification procedure that yields substantial amounts of natural BmK AGP-SYPU2 with high analgesic activity.     

Investigation of the role of disulphide bond in modulating internal motions of BmK AGAP protein by molecular dynamics simulation

Elucidating structural determinants in the functional regions of toxins can provide useful knowledge for designing novel analgesic peptides. A series of 100 ns MD simulations were performed on the scorpion toxin BmK AGAP in native and disulphide bond broken states. The comparison of disulphide bond broken states with the native state showed the α-helix was found to be the key to the analgesic activity. Furthermore, our results revealed disulphide bonds have considerable influence on the functionally important essential modes of motions and the correlations between the motions of the Core domain and the C-terminal region which are involved in the analgesic activity. Therefore, we can conclude that disulphide bonds have a crucial role in modulating the function via adjusting the dynamics of scorpion toxin BmK AGAP molecule.     

Genetic engineering of Escherichia coli inorganic pyrophosphatase. Tyr55 and Tyr141 are important for the structural integrity.

Two tyrosines are supposed to be essential for the activity and to participate in the stabilization of Escherichia coli inorganic pyrophosphatase (PPiase) against heat denaturation [Samejima, T., Tamagawa, Y., Kondo, Y., Hachimori, A., Kaji, H., Takeda, A. and Shiroya, Y. (1988) J. Biochem. (Tokyo) 103, 766-772]. To locate these two tyrosines in the amino acid sequence, we substituted all the eight tyrosines of E. coli PPiase with phenylalanine and studied the properties of these YF mutant PPiases. Interestingly, substitution of the tyrosines (Tyr51, Tyr55 and Tyr141) conserved with the amino acid sequence of yeast PPiase [Lahti, R., Kolakowski, L. F., Heinonen, J., Vihinen, M., Pohjanoksa, K. and Cooperman, B. (1990) Biochim. Biophys. Acta 1038, 338-345] exerted the most drastic effects on the structure and activity of E. coli PPiase. PPiase variants YF51, YF55 and YF141 had 64%, 7% and 22% of the wild-type PPiase activity, respectively. Furthermore, PPiase variant YF141 had an increased sensitivity to heat denaturation, whereas mutant PPiase YF55 displayed a profound conformational change, as demonstrated by the binding of the fluorescent dye 9-(diethylamino)-5H-benzo(alpha) phenoxazine-5-one (Nile red) that monitors the hydrophobicity of protein surfaces. None of the tyrosines of E. coli PPiase seem to be essential for catalysis, but Tyr55 and Tyr141 are important for the structural integrity of E. coli PPiase.     

Phosphorylation of Tyr342 in the linker region of Syk is critical for Fc epsilon RI signaling in mast cells

The linker region of Syk and ZAP70 tyrosine kinases plays an important role in regulating their function. There are three conserved tyrosines in this linker region; Tyr317 of Syk and its equivalent residue in ZAP70 were previously shown to negatively regulate the function of Syk and ZAP70. Here we studied the roles of the other two tyrosines, Tyr342 and Tyr346 of Syk, in Fc epsilon RI-mediated signaling. Antigen stimulation resulted in Tyr342 phosphorylation in mast cells. Syk with Y342F mutation failed to reconstitute Fc epsilon RI-initiated histamine release. In the Syk Y342F-expressing cells there was dramatically impaired receptor-induced phosphorylation of multiple signaling molecules, including LAT, SLP-76, phospholipase C-gamma2, but not Vav. Compared to wild-type Syk, Y342F Syk had decreased binding to phosphorylated immunoreceptor tyrosine-based activation motifs and reduced kinase activity. Surprisingly, mutation of Tyr346 had much less effect on Fc epsilon RI-dependent mast cell degranulation. An anti-Syk-phospho-346 tyrosine antibody indicated that antigen stimulation induced only a very minor increase in the phosphorylation of this tyrosine. Therefore, Tyr342, but not Tyr346, is critical for regulating Syk in mast cells and the function of these tyrosines in immune receptor signaling appears to be different from what has been previously reported for the equivalent residues of ZAP70.     

Importance of the conserved aromatic residues in the scorpion alpha-like toxin BmK M1: the hydrophobic surface region revisited

About one-third of the amino acid residues conserved in all scorpion long chain Na+ channel toxins are aromatic residues, some of which constitute the so-called "conserved hydrophobic surface." At present, in-depth structure-function studies of these aromatic residues using site-directed mutagenesis are still rare. In this study, an effective yeast expression system was used to study the role of seven conserved aromatic residues (Tyr5, Tyr14, Tyr21, Tyr35, Trp38, Tyr42, and Trp47) from the scorpion toxin BmK M1. Using site-directed mutagenesis, all of these aromatic residues were individually substituted with Gly in association with a more conservative substitution of Phe for Tyr5, Tyr14, Tyr35, or Trp47. The mutants, which were expressed in Saccharomyces cerevisiae S-78 cells, were then subjected to a bioassay in mice, electrophysiological characterization on cloned Na+ channels (Nav1.5), and CD analysis. Our results show an eye-catching correlation between the LD50 values in mice and the EC50 values on Nav1.5 channels in oocytes, indicating large mutant-dependent differences that emphasize important specific roles for the conserved aromatic residues in BmK M1. The aromatic side chains of the Tyr5, Tyr35, and Trp47 cluster protruding from the three-stranded beta-sheet seem to be essential for the structure and function of the toxin. Trp38 and Tyr42 (located in the beta2-sheet and in the loop between the beta2- and beta3-sheets, respectively) are most likely involved in the pharmacological function of the toxin.     

Location of the analgesic domain in Scorpion toxin BmK AGAP by mutagenesis of disulfide bridges

An increasing number of analgesic peptides have been found in the tail toxicyst, but there has been little research into their analgesic domains. Where are the analgesic domains in a conservative βαββ topology conformation of the analgesic peptides? We have carried out research to address this question. On account of the importance of disulfide bonds in the study of protein structure, the conformational stability, catalytic activity and folding, and site-directed mutagenesis in disulfide bridges have been used to look for the analgesic domain in a mature antitumor-analgesic peptide from the venom of the Chinese scorpion Buthus martensii Karsch (BmK AGAP). The mouse-twisting assay was used to examine the analgesic activity of 12 mutants, in which two mutants (C22S, C46S) and (C16S, C36S), exhibited lower relative activity. Following the conformational analysis, one domain, called the “core domain”, was found to be the key to the analgesic activity.     

Exploration of the functional site of a scorpion alpha-like toxin by site-directed mutagenesis

Scorpion alpha-neurotoxins can be classified into distinct subgroups according to their sequence and pharmacological properties. Using toxicity tests, binding studies, and electrophysiological recordings, BmK M1, a toxin from the Asian scorpion Buthus martensi Karsch, was experimentally identified as an alpha-like toxin. Being the first alpha-like toxin available in a recombinant form, BmK M1 was then modified by site-directed mutagenesis for investigation of the molecular basis of its activity. The results suggested a functional site which protrudes from the molecular scaffold as a unique tertiary arrangement, constituted by the five-residue reverse turn 8-12 and the C-terminal segment. The C-terminal basic residues Lys62 and His64 together with Lys8 in the turn, which are critical for the bioactivities, may directly interact with the receptor site on the sodium channel. Residues Asn11 and Arg58, indispensable for the activities, are mainly responsible for stabilizing the distinct conformation of the putative bioactive site. Among others, His10 and His64 seem to be involved in the preference of BmK M1 for phylogenetically distinct target sites. The comparison of BmK M1 with Aah2 (classical alpha-toxin) and Lqh(alpha)IT (alpha-insect toxin) showed that the specific orientation of the C-terminus mediated by the reverse turn might be relevant to the preference of alpha-toxin subgroups for phylogenetically distinct yet closely related receptor sites. The Y5G mutation indicated the "conserved hydrophobic surface" might be structurally important for stabilizing the beta-sheet in the alpha/beta-scaffold. The observations in this work shed light on the nature and roles of the residues possibly involved in the biological activity of a scorpion alpha-like toxin.     

Structurally conserved aromaticity of Tyr249 and Phe264 in helix 7 is important for toxicity of the Bacillus thuringiensis Cry4Ba toxin

Functional elements of the conserved helix 7 in the poreforming domain of the Bacillus thuringiensis Cry delta- endotoxins have not yet been clearly identified. Here, we initially performed alanine substitutions of four highly conserved aromatic residues, Trp(243), Phe(246), Tyr(249) and Phe(264), in helix 7 of the Cry4Ba mosquito-larvicidal protein. All mutant toxins were overexpressed in Escherichia coli as 130-kDa protoxins at levels comparable to the wild-type. Bioassays against Stegomyia aegypti mosquito larvae revealed that only W243A, Y249A or F264A mutant toxins displayed a dramatic decrease in toxicity. Further mutagenic analysis showed that replacements with an aromatic residue particularly at Tyr(249) and Phe(264) still retained the high-level toxin activity. In addition, a nearly complete loss in larvicidal activity was found for Y249L/F264L or F264A/ Y249A double mutants, confirming the involvement in toxicity of both aromatic residues which face towards the same direction. Furthermore, the Y249L/F264L mutant was found to be structurally stable upon toxin solubilisation and trypsin digestion, albeit a small change in the circular dichroism spectrum. Altogether, the present study provides for the first time an insight into the highly conserved aromaticity of Tyr(249) and Phe(264) within helix 7 playing an important role in larvicidal activity of the Cry4Ba toxin.     

High-level expression and purification of an analgesic peptide from Buthus martensii Karch

BmK AngM1, a scorpion peptide isolated from Buthus martensii Karch was reported to exhibit potential analgesic effect. But the relative low content of this toxin in crude venom limits its further characterization. In this study, we constructed an expression vector and transformed into E.coli. The BmK AngM1 was expressed as a fusion protein in the soluble fraction and was purified by Nickel affinity chromatography. Subsequently, the purified fusion protein was cleaved by enterokinase and was further purified by reverse-phase HPLC. We purified 25 mg recombinant BmK AngM1 (rBmK AngM1) from 1 L bacterial culture. The molecular weight of rBmK AngM1 determined by ESI-MS was 7240.4 Da which was the expected size for correctly processed. Analgesic bioassay studies of rBmK AngM1 exhibited its potential analgesic effect comparable to that of the natural BmK AngM1 peptide.     

Fluorescence and folding properties of Tyr mutant tryptophan synthase alpha-subunits from Escherichia coli

The fluorescence of tyrosine has been used to monitor a folding process of tryptophan synthase alpha-subunit from Escherichia coli, because this protein has 7 tyrosines, but not tryptophan. Here to assess the contribution of each Tyr to fluorescence properties of this protein during folding, mutant proteins in which Tyr was replaced with Phe were analyzed. The result shows that a change of Tyr fluorescence occurring during folding of this protein is contributed to approximately 40% each by Tyr(4) and Tyr(115), and to the remaining approximately 20% by Tyr(173) and Tyr(175). Y173F and Y175F mutant proteins showed an increase in their fluorescence intensity by approximately 40% and approximately 10%, respectively. These increases appear to be due to multiple effects of increased hydrophobicity, quenching effect of nearby residue Glu(49), and/or energy transfer between Tyrs. Two data for Y173F alpha-subunit of urea-induced unfolding equilibrium monitored by UV and fluorescence were different. This result, together with ANS binding and far UV CD, shows that folding intermediate(s) of Y173F alpha-subunit, contrary to that of wild-type, may contain self-inconsistent properties such as more buried hydrophobicity, highly quenched fluorescence, and different dependencies on urea of UV absorbance, suggesting an ensemble of heterogeneous structures.     

Electrophysiological characterization of BmK M1, an alpha-like toxin from Buthus martensi Karsch venom

The present study investigates the electrophysiological actions of BmK M1, an alpha-like toxin purified from the venom of the scorpion Buthus martensi Karsch, on voltage-gated Na+ channels. Using the voltage clamp technique, we assessed the BmK M1 activity on the cardiac Na+ channel (hH1) functionally expressed in Xenopus oocytes. The main actions of the toxin are a concentration-dependent slowing of the inactivation process and a hyperpolarizing shift of the steady-state inactivation. This work is the first electrophysiological characterization of BmK M1 on a cloned Na+ channel, demonstrating that this toxin belongs to the class of scorpion alpha-toxins. Our results also show that BmK M1 can be considered as a cardiotoxin.     

Microglial activation of p38 contributes to scorpion envenomation-induced hyperalgesia

Intraplantar (i.pl.) injection of BmK I, a receptor site 3-specific modulator of voltage-gated sodium channels (VGSCs) from the venom of scorpion Buthus martensi Karsch (BmK), was shown to induce long-lasting and spontaneous nociceptive responses as demonstrated through experiments utilizing primary thermal and mirror-imaged mechanical hypersensitivity with different time course of development in rats. In this study, microglia was activated on both sides of L4–L5 spinal cord by i.pl. injection of BmK I. Meanwhile, the activation of p38/MAPK in L4–L5 spinal cord was found to be co-expressed with OX-42, the cell marker of microglia. The unilateral thermal and bilateral mechanical pain hypersensitivity of rat induced by BmK I was suppressed in a dose-dependent manner following pretreatment with SB203580 (a specific inhibitor of p-p38). Interestingly, microglia activity was also reduced in the presence of SB203580, which suggests that BmK I-induced microglial activation is mediated by p38/MAPK pathway. Combined with previously published literature, the results of this study demonstrate that p38-dependent microglial activation plays a role in scorpion envenomation-induced pain-related behaviors.     

Identification of Tyr504 as an alternative tyrosyl radical site in human prostaglandin H synthase-2

Hydroperoxides induce formation of a tyrosyl radical on Tyr385 in prostaglandin H synthase (PGHS). The Tyr385 radical initiates hydrogen abstraction from arachidonic acid, thereby mechanistically connecting the peroxidase and cyclooxygenase activities. In both PGHS isoforms the tyrosyl radical undergoes a time-dependent transition from a wide doublet to a wide singlet species; pretreatment with cyclooxygenase inhibitors results in a third type of signal, a narrow singlet [Tsai, A.-L.; Kulmacz, R. J. (2000) Prost. Lipid Med. 62, 231-254]. These transitions have been interpreted as resulting from Tyr385 ring rotation, but could also be due to radical migration from Tyr385 to another tyrosine residue. PATHWAYS analysis of PGHS crystal structures identified four tyrosine residues with favorable predicted electronic coupling: residues 148, 348, 404, and 504 (ovine PGHS-1 numbering). We expressed recombinant PGHS-2 proteins containing single Tyr --> Phe mutations at the target residues, a quadruple mutant with all four tyrosines mutated, and a quintuple mutant, which also contains a Y385F mutation. All mutants bind heme and display appreciable peroxidase activity, and with the exception of the quintuple mutant, all retain cyclooxygenase activity, indicating that neither of the active sites is significantly perturbed. Reaction of the Y148F, Y348F, and Y404F mutants with EtOOH generates a wide singlet EPR signal similar to that of native PGHS-2. However, reaction of the Y504F and the quadruple mutants with peroxide yields persistent wide doublets, and the quintuple mutant is EPR silent. Nimesulide pretreatment of Y504F and the quadruple mutant results in an abnormally small amount of wide doublet signal, with no narrow singlet being formed. Therefore, the formation of an alternative tyrosine radical on Tyr504 probably accounts for the transition from a wide doublet to a wide singlet in native PGHS-2 and for formation of a narrow singlet in complexes of PGHS-2 with cyclooxygenase inhibitors.     

Cloning,expression, and pharmacological activity of BmK AS,an active peptide from scorpion <Emphasis Type="Italic">Buthus martensii</Emphasis> Karsch

BmK AS is a β long-chain scorpion peptide from the venom of Buthus martensii Karsch (BmK). It was efficiently expressed as a soluble and functional peptide in Escherichia coli, and purified by metal chelating chromatography. About 4.2 mg/l purified recombinant BmK AS could be obtained. The recombinant BmK AS maintained a similar analgesic activity to the natural one in both the mouse-twisting test and hot-plate procedure. It also exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria. BmK AS is the first long-chain scorpion peptide reported to have antimicrobial activity, and is a valuable molecular scaffold for pharmacological research.     

Role of Tyr348 in Tyr385 radical dynamics and cyclooxygenase inhibitor interactions in prostaglandin H synthase-2

Both prostaglandin H synthase (PGHS) isoforms utilize a radical at Tyr385 to abstract a hydrogen atom from arachidonic acid, initializing prostaglandin synthesis. A Tyr348-Tyr385 hydrogen bond appears to be conserved in both isoforms; this hydrogen bonding has the potential to modulate the positioning and reactivity of the Tyr385 side chain. The EPR signal from the Tyr385 radical undergoes a time-dependent transition from a wide doublet to a wide singlet species in both isoforms. In PGHS-2, this transition results from radical migration from Tyr385 to Tyr504. Localization of the radical to Tyr385 in the recombinant human PGHS-2 Y504F mutant was exploited in examining the effects of blocking Tyr385 hydrogen bonding by introduction of a further Y348F mutation. Cyclooxygenase and peroxidase activities were found to be maintained in the Y348F/Y504F mutant, but the Tyr385 radical was formed more slowly and had greater rotational freedom, as evidenced by observation of a transition from an initial wide doublet species to a narrow singlet species, a transition not seen in the parent Y504F mutant. The effect of disrupting Tyr385 hydrogen bonding on the cyclooxygenase active site structure was probed by examination of cyclooxygenase inhibitor kinetics. Aspirin treatment eliminated all oxygenase activity in the Y348F/Y504F double mutant, with no indication of the lipoxygenase activity observed in aspirin-treated wild-type PGHS-2. Introduction of the Y348F mutation also strengthened the time-dependent inhibitory action of nimesulide. These results suggest that removal of Tyr348-Tyr385 hydrogen bonding in PGHS-2 allows greater conformational flexibility in the cyclooxygenase active site, resulting in altered interactions with inhibitors and altered Tyr385 radical behavior.     

Role of the activation loop tyrosines in regulation of the insulin-like growth factor I receptor-tyrosine kinase

The tyrosine kinase activity of insulin-like growth factor I receptor (IGF1R) is under tight control. Ligand binding to the extracellular portion of IGF1R stimulates autophosphorylation at three sites (Tyr1131, Tyr1135, and Tyr1136) in the activation loop within the tyrosine kinase catalytic domain. Autophosphorylation at all three sites is required for maximum enzyme activity, and for IGF1-stimulated cellular activity of the receptor. Previous studies have not clarified the contributions of the individual tyrosines to enzymatic activation. Here, we produced single Tyr-to-Phe mutations at these positions, and compared activities of the purified kinase domains (unphosphorylated and phosphorylated) with wild-type IGF1R. Rates of autophosphorylation of the three mutants were more rapid than for wild-type IGF1R; this was most apparent for the Y1135F mutant. Substrate phosphorylation studies on the unphosphorylated forms of IGF1R confirmed that the value of Vmax for Y1135F was elevated relative to wild-type IGF1R, consistent with a disruption of an autoinhibitory interaction. In contrast, activity measurements on the fully phosphorylated enzymes indicated that kcat/Km values were lowered relative to wild-type IGF1R; this effect was most dramatic for Y1136F. We confirmed these findings using limited proteolysis and tryptophan fluorescence experiments. The results demonstrate that Tyr1135 plays a particularly important role in stabilizing the autoinhibited conformation of the activation loop, while Tyr1136 plays the key role in stabilizing the open, activated conformation of IGF1R.     

A new insect neurotoxin AngP1 with analgesic effect from the scorpion Buthus martensii Karsch: purification and characterization.

An insect toxin named BmK AngP1 was purified from the venom of the scorpion Buthus martensii Karsch (BmK). It also shows an evident analgesic effect on mice, but is interestingly devoid of mammalian toxicity. Bioassay showed that the CPU value of AngP1 was 0.01 microg/body ( approximately 30 mg) for the excitatory insect toxicity and 43.0% inhibition efficiency for analgesia at a dose of 5 mg/kg. However, even at the dosage of 10 mg/kg no detectable toxicity on mice could be found. The isoelectric point (pI) value for AngP1 was 4.0, and its molecular mass analyzed by MALDI-TOF MS was 8141.0. The first 15 N-terminal residues of AngP1 were determined by Edman degradation and showed high similarity to that of other excitatory scorpion insect toxins. The circular dichroism spectroscopy measured on a JASCO J-720 system showed that there were 10.4% alpha-helix, 46.2% beta-strand and 14.1% turn structure in this peptide. Under two conditions single crystals of AngP1 were obtained.