Crystals of fasciculin 2 from green mamba snake venom. Preparation and preliminary x-ray analysis

Fasciculin 2 from the venom of the green mamba, Dendroaspis angusticeps, has been crystallized. The crystals are tetragonal, with unit cell dimensions a = 48.9 A and c = 82.0 A, space group P 41 21 2 or P 43212. Density measurements and pseudocentering of the hko zone indicate that there are 16 molecules in the unit cell.     

1.9-A resolution structure of fasciculin 1, an anti-acetylcholinesterase toxin from green mamba snake venom.

The crystal structure of fasciculin 1, a potent acetylcholinesterase inhibitor from green mamba snake venom, has been solved by the multiple isomorphous replacement method complemented with anomalous scattering and subsequently refined at 1.9-A resolution. The overall structure of fasciculin is similar to those of the short alpha-neurotoxins and cardiotoxins, with a dense core rich in disulfide bridges and three long loops disposed as the central fingers of a hand. A comparison of these three prototypic toxin types shows that fasciculin 1 has structural features that are intermediate between those of the other two molecules. Its core region, which can be defined as a continuous stretch of conserved residues, is very similar to that of erabutoxin b, whereas the orientation of its long loops resembles that of cardiotoxin VII4. This result introduces a new element in the study of phylogenetic relationships of snake toxins and suggests that, after divergency from an ancestral gene, convergent evolution may have played an important factor in the evolution of these proteins. In fasciculin 1, several arginine and lysine residues are well ordered and relatively exposed to the solvent medium and may play a role in the binding to the peripheral site of acetylcholinesterases.     

Fasciculins, anticholinesterase toxins from the venom of the green mamba Dendroaspis angusticeps

Two toxins that are potent inhibitors of acetylcholinesterase have been isolated from the venom of the green mamba, Dendroaspis angusticeps. The toxins have been called fasciculins since after injection into mice (i.p. 0.5-3 micrograms/g body weight) they cause severe, generalized and long-lasting (5-7 h) fasciculations. Homogenates of diaphragm, tibialis anterior and gastrocnemius muscles from mice injected with fasciculins showed a decrease in acetylcholinesterase activity by 45-60% compared to muscles from control animals. Histochemical staining revealed a greatly reduced acetylcholinesterase activity at neuromuscular junctions. Fasciculins have 61 amino acid residues and four disulfides. The molecular weights are 6765 (fasciculin 1) and 6735 (fasciculin 2). The sequences of the two toxins differ probably only at one position by a replacement of Tyr with Asp/Asn. 1 g of venom contained about 40 mg of fasciculins, 2/3 of which was fasciculin 2. A similar inhibitor has also been isolated from D. polylepis (black mamba) venom. The sequence of fasciculin 2 is known. Most of the positive charges are concentrated in a small section of the central part of the molecule, and most of the negative charges are in the C-terminal region. Fasciculins appear to have a pronounced dipole character. Fasciculin binds to the peripheral anionic site, since it can displace propidium, a probe for that site, from acetylcholinesterase. In vitro, in Krebs-Henseleit solution containing 2 mM NaH2PO4 (pH 7.4), fasciculin 2 inhibits acetylcholinesterase from human erythrocytes (Ki = 1.1 X 10(-10) M, 37 degrees C), rat muscle (Ki = 1.2 X 10(-10) M, 37 degrees C) and Electrophorus electricus (Ki = 3.0 X 10(-10) M, 22 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of alpha-dendrotoxin from the green mamba venom and its comparison with the structure of bovine pancreatic trypsin inhibitor.

The three-dimensional structure of alpha-dendrotoxin (alpha-DTX) from the green mamba (Dendroaspis angusticeps) venom has been determined crystallographically using the method of isomorphous replacement and refined at 2.2 A resolution using a restrained least-squares method. The crystallographic R-factor is 0.169 for all 3451 measured reflections between 7.0 and 2.2 A. Although the main-chain fold of alpha-DTX is similar to that of homologous bovine pancreatic trypsin inhibitor (BPTI), there are significant differences involving segments of the polypeptide chain close to the "antiprotease site" of BPTI. Comparison of the structure of alpha-DTX with the existing models of BPTI and its complexes with trypsin and kallikrein reveals structural differences that explain the inability of alpha-DTX to inhibit trypsin and chymotrypsin.     

Preliminary X-ray analysis of crystals of plasminogen activator inhibitor-1

Crystals of bacterially expressed plasminogen activator inhibitor (PAI-1) suitable for X-ray diffraction analysis have been obtained from 8% (w/v) PEG 1500, pH 8.25. The space group is P1, and the lattice constants are a = 82.17 A, b = 47.82 A, c = 62.89 A, alpha = 90.00 degrees, beta = 106.90 degrees, gamma = 106.84 degrees. The diffraction limit is 2.3 A, and the unit cell contains two molecules of PAI-1. The crystals contain latent PAI-1 which can be partly reactivated by exposure to denaturants.     

Characterization of a potent platelet aggregation inhibitor from Agkistrodon rhodostoma snake venom

By means of CM-Sephadex C-50 column chromatography and gel filtration on Sephadex G-75 and G-50 columns, a potent platelet aggregation inhibitor was purified and characterized. It was a glycoprotein with a molecular weight of 31,000. It was devoid of phospholipase A, ADPase, esterase and fibrino(geno)lytic activities. It inhibited dose-dependently the aggregation of washed platelets induced by collagen, thrombin, sodium arachidonate, platelet activating factor and ionophore A23187 with a similar IC50 (5-10 micrograms/ml). It was also active in platelet-rich plasma, with an IC50 of 10-15 micrograms/ml. The venom inhibitor reduced the elasticity of whole blood clot and inhibited the thrombin-induced clot retraction of platelet-rich plasma. These activities were related to its inhibitory activity on platelet aggregation rather than blood coagulation. The venom inhibitor had various effects on [14C]serotonin release stimulated by aggregation agonists. It had no effect on thromboxane B2 formation of platelets stimulated by sodium arachidonate, collagen and ionophore A23187. The presence of this venom inhibitor prior to the initiation of aggregation was a prerequisite for the maintenance of its maximal activity. It showed a similar inhibitory effect on collagen or thrombin-induced aggregation even when it was added after the platelets had undergone the shape change. High fibrinogen levels partially antagonized its activity. The venom inhibitor completely inhibited the fibrinogen-induced aggregation of alpha-chymotrypsin-treated platelets. It is concluded that this venom inhibitor interferes with the interaction of fibrinogen with fibrinogen receptors, leading to inhibition of aggregation.     

Synthetic peptides mimicking the binding site of human acetylcholinesterase for its inhibitor fasciculin 2

Molecules capable of mimicking protein binding and/or functional sites present useful tools for a range of biomedical applications, including the inhibition of protein–ligand interactions. Such mimics of protein binding sites can currently be generated through structure‐based design and chemical synthesis. Computational protein design could be further used to optimize protein binding site mimetics through rationally designed mutations that improve intermolecular interactions or peptide stability. Here, as a model for the study, we chose an interaction between human acetylcholinesterase (hAChE) and its inhibitor fasciculin‐2 (Fas) because the structure and function of this complex is well understood. Structure‐based design of mimics of the hAChE binding site for Fas yielded a peptide that binds to Fas at micromolar concentrations. Replacement of hAChE residues known to be essential for its interaction with Fas with alanine, in this peptide, resulted in almost complete loss of binding to Fas. Computational optimization of the hAChE mimetic peptide yielded a variant with slightly improved affinity to Fas, indicating that more rounds of computational optimization will be required to obtain peptide variants with greatly improved affinity for Fas. CD spectra in the absence and presence of Fas point to conformational changes in the peptide upon binding to Fas. Furthermore, binding of the optimized hAChE mimetic peptide to Fas could be inhibited by hAChE, providing evidence for a hAChE‐specific peptide–Fas interaction. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Preliminary X-ray crystallographic analysis of canine parvovirus crystals

The first diffraction pattern of a crystalline single-stranded DNA virus has been obtained. Canine parvovirus was crystallized in a monoclinic P21 unit cell with a = 264.4 A, b = 350.3 A, c = 267.8 A and beta = 90.86 degrees (1 A = 0.1 nm). The diffraction pattern extends to at least 2.8 A resolution. Packing of the particles suggests that they have a diameter around 257 A, in excellent agreement with the reported molecular weight of 5.5 x 10(6).     

Synthesis and characterization of a chimeric peptide derived from fasciculin that inhibits acetylcholinesterase.

Fasciculins are peptides isolated from mamba (Dendroaspis) venoms which exert their toxic action by inhibiting acetylcholinesterase (AChE). They contain a characteristic triple stranded antiparallel beta-sheet formed by residues 22-27, 34-39 and 48-53. A chimeric peptide named Fas-C, encompassing most of these sequences was synthesized using SPPS/Boc-chemistry and characterized chemically, structurally and functionally. Fas-C has two disulfide bridges, formed sequentially using dual cysteine protection. SDS-PAGE patterns, HPLC profiles and MS proved the peptide identity. Circular dichroism indicated the presence of 13.6% and 41.6% of beta-sheet and beta-turn, respectively, comparable to values observed in the native toxin. An inhibitory effect on eel AChE was displayed by the peptide (Ki71.6 +/- 18.3 microM), although not reaching the affinity level of the parent native toxin (Ki 0.3 nM). It is confirmed that the principal binding region of fasciculin to AChE resides within loop II.     

Preliminary X-ray analysis of crystals of murine adenosine deaminase

We have obtained single crystals of a cloned mammalian adenosine deaminase (Mr = 41,000), a key enzyme in purine degradation and in normal development of the immune system, that are suitable for high-resolution structural analysis. The crystals belong to the space group C2 with unit cell parameters a = 101.68 A (1 A = 0.1 nm), b = 94.38 A, c = 85.51 A, and beta = 96.54 degrees. The asymmetric unit contains two enzyme molecules.     

Purification and characterization of a unique, potent inhibitor of apamin binding from Leiurus quinquestriatus hebraeus venom

An inhibitor of apamin binding has been purified to homogeneity in three chromatographic steps from the venom of the scorpion, Leiurus quinquestriatus hebraeus. The inhibitor, which we have named leiurotoxin I, represents less than 0.02% of the venom protein. It is a 3.4-kDa peptide with little structural homology to apamin although it has some homology to other scorpion toxins such as charybdotoxin, noxiustoxin, and neurotoxin P2. Leiurotoxin I completely inhibits 125I-apamin binding to rat brain synaptosomal membranes (Ki = 75 pM). Thus, it is 10-20-fold less potent than apamin. Leiurotoxin I is not a strictly competitive inhibitor of this binding reaction. Like apamin, leiurotoxin I blocks the epinephrine-induced relaxation of guinea pig teniae coli (ED50 = 6.5 nM), while having no effect on the rate or force of contraction in guinea pig atria or rabbit portal vein preparations. Thus, leiurotoxin I of scorpion venom and apamin of honeybee venom demonstrate similar activities in a variety of tissues, yet are structurally unrelated peptides. These two peptides should be useful in elucidating the role of the small conductance, Ca2+-activated K+ channels in different tissues.     

X-ray crystal structure of sangivamycin, a potent inhibitor of protein kinases

The X-ray crystal structure of sangivamycin, a potent nucleoside inhibitor of protein kinases, has been determined. Sangivamycin crystallizes from water with its purine ring in a conformation anti to its ribose sugar. Such an anti conformation has been detected in solution for sangivamycin and other potent protein kinase inhibitors and appears to correlate with inhibitor potency [(1990) Biochemistry (in press)]. An intramolecular hydrogen bond between purine ring substituents is detected in the X-ray structure and may be an important structural feature of sangivamycin related to its degree of inhibition of rhodopsin kinase and of protein kinases C and A.     

Synthesis of more potent analogues of the acetylcholinesterase inhibitor,huperzine B

The synthesis and acetylcholinesterase inhibition activity of analogues of huperzine B are reported. These new racemic analogues show a better AChE inhibitory activity than the natural product huperzine B.     

Paecilomide,a new acetylcholinesterase inhibitor from Paecilomyces lilacinus

Fungi are some of the most important organisms in the production of bioactive secondary metabolites. This success is related to the advances in biotechnology and also to the possibility of working with techniques such as the “OSMAC” (one strain-many compounds) to achieve different fungal secondary metabolites profiles upon modifying the culturing conditions. Using this approach, the fungal species Paecilomyces lilacinus was cultivated in potato dextrose broth under 14 different fermentative conditions by adding the bacterium Salmonella typhimurium to the growing medium in order to provide biotic stress. S. typhimurium was added alive or after inactivation by autoclave or microwave irradiation in different stages of fungal growth. Extracts were prepared by liquid–liquid extraction using ethyl acetate, a medium polarity solvent in order to avoid extracting culturing media components. Production of fatty acids of relevance for the pharmaceutical and food industries was enhanced by the modified fermentative conditions and they were identified and quantified. The extracts were evaluated for acetylcholinesterase inhibition and the more active extract (91 ± 2.91% inhibition) was prepared in large scale. From this active P. lilacinus extract, a novel pyridone alkaloid, named Paecilomide, was isolated and its structure was elucidated by modern nuclear magnetic resonance techniques and mass spectrometric analyses. Paecilomide (1) was also evaluated for acetylcholinesterase inhibition, presenting 57.5 ± 5.50% of acetylcholinesterase inhibition.     

Preliminary X-ray diffraction analysis of crystals of Bacillus thuringiensis toxin, a cell membrane disrupting protein

Crystals suitable for high resolution X-ray diffraction analysis have been reproducibly grown of the 24,000 Mr protein insect toxin from Bacillus thuringiensis. This protein, which demonstrates substantial insecticidal activity by inserting into phospholipid membranes, crystallizes as long square needles from polyethylene glycol 4000 at neutral pH. The crystals are of space group P4(1) and have cell dimensions of a = b = 33 A and c = 235 A, which suggests to us a predominantly helical motif for the protein's structure.     

Soluble monomeric acetylcholinesterase from mouse: expression, purification, and crystallization in complex with fasciculin.

A soluble, monomeric form of acetylcholinesterase from mouse (mAChE), truncated at its carboxyl-terminal end, was generated from a cDNA encoding the glycophospholipid-linked form of the mouse enzyme by insertion of an early stop codon at position 549. Insertion of the cDNA behind a cytomegalovirus promoter and selection by aminoglycoside resistance in transfected HEK cells yielded clones secreting large quantities of mAChE into the medium. The enzyme sediments as a soluble monomer at 4.8 S. High levels of expression coupled with a one-step purification by affinity chromatography have allowed us to undertake a crystallographic study of the fasciculin-mAChE complex. Complexes of two distinct fasciculins, Fas1-mAChE and Fas2-mAChE, were formed prior to the crystallization and were characterized thoroughly. Single hexagonal crystals, up to 0.6 mm x 0.5 mm x 0.5 mm, grew spontaneously from ammonium sulfate solutions buffered in the pH 7.0 range. They were found by electrophoretic migration to consist entirely of the complex and diffracted to 2.8 A resolution. Analysis of initial X-ray data collected on Fas2-mAChE crystals identified the space group as P6(1)22 or P6(5)22 with unit cell dimensions a = b = 75.5 A, c = 556 A, giving a Vm value of 3.1 A3/Da (or 60% of solvent), consistent with a single molecule of Fas2-AChE complex (72 kDa) per asymmetric unit. The complex Fas1-mAChE crystallizes in the same space group with identical cell dimensions.     

Toxins from the venom of the green mamba Dendroaspis angusticeps that inhibit the binding of quinuclidinyl benzilate to muscarinic acetylcholine receptors

Two protein toxins that displace the muscarinic antagonist quinuclidinyl benzilate from rat cortex synaptosomal membranes have been isolated from the green mamba (Dendroaspis angusticeps) venom by gel filtration on sephadex G-50, chromatography on the ion-exchangers Bio-Rex 70 and Sulphopropyl-Sephadex C-25 and reversed-phase HPLC. Toxin 1 has 64 amino acids and four disulfides and a formula weight of 7200 and the corresponding values for toxin 2 are 63, 4 and 6840, respectively. Ultracentrifugation gave a molecular weight of 6900 for toxin 1 and 6700 for toxin 2, Quinuclidinyl benzilate that binds to all types of muscarinic cholinergic receptor was displaced to about 50% by both toxins. This partial displacement indicates that the toxins might be specific for one subtype of receptor.     

Theoretical analysis of the structure of the peptide fasciculin and its docking to acetylcholinesterase.

The fasciculins are a family of closely related peptides that are isolated from the venom of mambas and exert their toxic action by inhibiting acetylcholinesterase (AChE). Fasciculins belong to the structural family of three-fingered toxins from Elapidae snake venoms, which include the alpha-neurotoxins that block the nicotinic acetylcholine receptor and the cardiotoxins that interact with cell membranes. The features unique to the known primary and tertiary structures of the fasciculin molecule were analyzed. Loop I contains an arginine at position 11, which is found only in the fasciculins and could form a pivotal anchoring point to AChE. Loop II contains five cationic residues near its tip, which are partly charge-compensated by anionic side chains in loop III. By contrast, the other three-fingered toxins show full charge compensation within loop II. The interaction of fasciculin with the recognition site on acetylcholinesterase was investigated by estimating a precollision orientation followed by determination of the buried surface area of the most probable complexes formed, the electrostatic field contours, and the detailed topography of the interaction surface. This approach has led to testable models for the orientation and site of bound fasciculin.     

Preliminary X-ray study of papD crystals from uropathogenic Escherichia coli

The product of the Escherichia coli papD gene is a periplasmic transport protein that forms complexes with pilus subunits, thereby stabilizing them as they cross the periplasmic space to the site of pilus assembly. Purified PapD protein was crystallized by the hanging drop method of vapour diffusion with polyethylene glycol 8000 as the precipitating agent at pH 6.5. The space group is P2(1)2(1)2(1), with unit cell dimensions of a = 67.0 A, b = 58.1 A and c = 64.0 A. The crystal data, together with a subunit molecular weight of 24,500 suggest that there is one monomer in the asymmetric unit. The crystals are stable in X-rays and diffract to a resolution beyond 2.0 A.     

Lebecetin, a potent antiplatelet C-type lectin from Macrovipera lebetina venom

A novel C-type lectin protein (CLP), lebecetin, was purified to homogeneity from the venom of Macrovipera lebetina by gel filtration on a Sephadex G75 column and ion exchange chromatography on Mono S column. Lebecetin is a basic protein with a pHi=9.9 and migrates in SDS-PAGE as a single band or two distinct bands under nonreducing and reducing conditions, respectively. These results are further confirmed by MALDI-TOF mass spectrometry that indicates a molecular mass of 29779 Da for native lebecetin and molecular masses of 15015 and 16296 Da for alpha and beta subunits, respectively. The N-terminal amino acid sequences of lebecetin subunits show a high degree of similarity with those of C-type lectin-like proteins. In addition, functional studies showed that lebecetin has a potent inhibitory effect on platelet aggregation induced by thrombin in a concentration-dependent manner. In contrast, no inhibitory effect is observed when platelets are exposed to thromboxane A2 (TxA2) mimetic (U46619) or arachidonic acid. Moreover, there was no effect either on blood coagulation or A, B and O washed human erythrocytes agglutination. Furthermore, flow cytometric analysis revealed that fluoro-isothiocyanate (FITC)-labelled lebecetin bound to human formalin fixed platelets in a saturable and concentration manner and this binding was specifically prevented by anti-glycoprotein Ib (GPIb) mAb. These observations suggest that lebecetin is a C-type lectin-like protein that selectively binds to platelet GPIb.