X-ray crystallographic analysis of time-dependent binding of guanidine hydrochloride to HEWL: First steps during protein unfolding.

Time-dependent binding of guanidine hydrochloride (GuHCl) to hen egg-white lysozyme (HEWL), and effects of this binding on the protein structure have been investigated by solving X-ray structures of crystalline complexes. The complexes have been prepared by soaking, for different periods of time, native lysozyme crystals in solutions containing 2.5M GuHCl. In the refined structures, the number of water molecules in the protein's first solvent shell has progressively decreased from 152 to 115, showing protein's preference for guanidinium over water. Guanidinium ions preferentially hydrogen bond with the backbone carbonyl oxygen atoms. In their van der Waals interactions, they do not show any preference for apolar residues. Guanidinium ions have replaced water molecules that form cages around exposed hydrophobic residues. Guanidinium binding has decreased the average length of water-water hydrogen bond by 0.1Å. The hydrogen bonds between main chain atoms have been weakened by GuHCl, and this may be the reason for increased potency of GuHCl compared to urea. Guanidinium binding destabilizes the ß-domain by causing loss of hydrogen bonds involving Asn 59 side chain. Interestingly, this loss is almost identical to that observed in structures of amyloidogenic variants of human lysozyme. Compounds preventing this loss could be anti-amyloidogenic.

Fasxiator, a novel factor XIa inhibitor from snake venom, and its site-specific mutagenesis to improve potency and selectivity.

BACKGROUND: Bleeding remains a major limitation of standard anticoagulant drugs that target the extrinsic and common coagulation pathways. Recently, intrinsic coagulation factors are increasingly being investigated as alternative targets for developing anticoagulant drugs with lower bleeding risk. OBJECTIVES: Goals were to (i) identify novel anticoagulants selectively targeting intrinsic coagulation pathway and (ii) characterize and further improve the properties of the identified anticoagulants. METHODS AND RESULTS: We have isolated and sequenced a specific factor XIa (FXIa) inhibitor, henceforth named Fasxiator, from the venom of the banded krait snake, Bungarus fasciatus. It is a Kunitz-type protease inhibitor that prolonged activated partial thromboplastin time without significant effects on prothrombin time. Fasxiator was recombinantly expressed (rFasxiator), purified, and characterized to be a slow-type inhibitor of FXIa that exerts its anticoagulant activities (doubled activated partial thromboplastin time at ~ 3 µmol L(-1) ) by selectively inhibiting human FXIa in in vitro assays. A series of mutants were subsequently generated to improve the potency and selectivity of recombinant rFasxiator. rFasxiatorN17R,L19E showed the best balance between potency (IC50 ~ 1 nmol L(-1) ) and selectivity (> 100 times). rFasxiatorN17R,L19E is a competitive slow-type inhibitor of FXIa (Ki  = 0.86 nmol L(-1) ), possesses anticoagulant activity that is ~ 10 times stronger in human plasma than in murine plasma, and prolonged the occlusion time of mice carotid artery in FeCl3 -induced thrombosis models. CONCLUSION: We have isolated an exogenous FXIa specific inhibitor, engineered it to improve its potency by ~ 1000 times and demonstrated its in vitro and in vivo efficacy. These proof-of-principle data supported the further development of Fasxiator as a novel anticoagulant candidate.

Toxins in thrombosis and haemostasis: potential beyond imagination.

Exogenous factors isolated from venoms of snakes and saliva of haematophagous animals that affect thrombosis and haemostasis have contributed significantly to the development of diagnostic agents, research tools and life-saving drugs. Here, I discuss recent advances in the discovery, structural and functional characterisation, and mechanism of action of new procoagulant and anti-haemostatic proteins. In nature, these factors have evolved to target crucial 'bottlenecks' in the coagulation cascade and platelet aggregation. Several simple protein scaffolds are used to target a wide variety of target proteins and receptors exhibiting functional divergence. Different protein scaffolds have also evolved to target identical, physiologically relevant key enzymes or receptors exhibiting functional convergence. At times, exogenous factors bind to the same target protein, but at distinct sites, to differentially attenuate their functions exhibiting mechanistic divergence within the same family of proteins. The structure-function relationships of these factors are subtle and complicated but represent an exciting challenge. These studies provide ample opportunities to design highly specific and precise ligands to achieve desired biological target function. Although only a small number of them have been characterised to date, the molecular and mechanical diversities of these exogenous factors and their contributions to understanding molecular and cellular events in thrombosis and haemostasis as well as developing diagnostic and research tools and therapeutic agents, is outstanding. Based on the current status, I have attempted to identify future potential and prospects in this area of research.

Isthmin exerts pro-survival and death-promoting effect on endothelial cells through alphavbeta5 integrin depending on its physical state.

Isthmin (ISM) is a 60 kDa secreted-angiogenesis inhibitor that suppresses tumor growth in mouse and disrupts vessel patterning in zebrafish embryos. It selectively binds to alphavbeta5 (αvß5) integrin on the surface of endothelial cells (ECs), but the mechanism of its antiangiogenic action remains unknown. In this work, we establish that soluble ISM suppresses in vitro angiogenesis and induces EC apoptosis by interacting with its cell surface receptor αvß5 integrin through a novel 'RKD' motif localized within its adhesion-associated domain in MUC4 and other proteins domain. ISM induces EC apoptosis through integrin-mediated death (IMD) by direct recruitment and activation of caspase-8 without causing anoikis. On the other hand, immobilized ISM loses its antiangiogenic function and instead promotes EC adhesion, survival and migration through αvß5 integrin by activating focal adhesion kinase (FAK). ISM unexpectedly has both a pro-survival and death-promoting effect on ECs depending on its physical state. This dual function of a single antiangiogenic protein may impact its antiangiogenic efficacy in vivo.

Crystal structure of bucain, a three-fingered toxin from the venom of the Malayan krait (Bungarus candidus).

Bucain, a potent neurotoxin isolated from the venom of the Malayan krait (Bungarus candidus), induces paralysis and death. Its crystal structure has been determined at 2.10 A resolution and based on the molecular topology and hydrophobicity profile is structurally classified as a three-fingered alpha-neurotoxin possessing a positively charged AChR-binding site.

Protein complexes in snake venom.

Snake venom contains mixture of bioactive proteins and polypeptides. Most of these proteins and polypeptides exist as monomers, but some of them form complexes in the venom. These complexes exhibit much higher levels of pharmacological activity compared to individual components and play an important role in pathophysiological effects during envenomation. They are formed through covalent and/or non-covalent interactions. The subunits of the complexes are either identical (homodimers) or dissimilar (heterodimers; in some cases subunits belong to different families of proteins). The formation of complexes, at times, eliminates the non-specific binding and enhances the binding to the target molecule. On several occasions, it also leads to recognition of new targets as protein-protein interaction in complexes exposes the critical amino acid residues buried in the monomers. Here, we describe the structure and function of various protein complexes of snake venoms and their role in snake venom toxicity.

Structure of two genes encoding parallel prothrombin activators in Tropidechis carinatus snake: gene duplication and recruitment of factor X gene to the venom gland.

BACKGROUND: Proteins with new function originate through gene duplication followed by divergence. In nature, occurrence of structurally and functionally similar proteins performing highly diverse physiological roles within an organism is rare. Several Australian elapid snakes have two parallel prothrombin activating systems with distinct physiological roles. For example, in Tropidechis carinatus, trocarin D exists in the venom and acts as toxin, whereas coagulation factor (F) X exists in plasma and plays a role in hemostasis. RESULTS: Here, we show that FX and the trocarin D genes are expressed in a highly tissue-specific manner in T. carinatus. To understand their origin, recruitment and evolution, we determined the complete structure of their genes. Both genes have eight exons with identical exon-intron boundaries. All the introns are 92-99% identical with the exception of intron 1, indicating a recent gene duplication event. The first intron of the trocarin D gene is also nearly identical to that of the FX gene, except for two deletions (255 and 1406 bp) and three insertions (214, 1975, and 2174 bp). The third insertion has a potential scaffold/matrix attached region. The putative promoter of the snake FX gene shares similar cis-elements compared with those of human and murine FX genes. Interestingly, the trocarin D promoter has a 264-bp insertion with core promoter sequences and cis-elements that are known to induce high-level expression. This insertion might be responsible for switching from constitutive expression of the FX gene to inducible expression of trocarin D. Thus, we named this segment as VERSE (Venom Recruitment/Switch Element). CONCLUSION: To our knowledge, this is the first molecular evidence for the recruitment of a duplicated gene for expression in venom glands by a simple insertion.

The in vitro and in vivo pharmacological activity of Boiga dendrophila (mangrove catsnake) venom.

The great taxonomic and prey base diversity of colubrids (non-front-fanged snakes) suggests that their venoms may represent a 'literal gold mine' for scientists eager to find novel pharmacological probes. While pharmacological characterization is lacking for most of these venoms, this is even more so with regard to activity of colubrid venoms on the mammalian autonomic nervous system. This study characterizes the activity of venom from the colubrid, Boiga dendrophila using in vitro smooth muscle preparations and the anaesthetized rat. In the prostatic segment of the rat vas deferens, cumulative additions of venom (1-150 microg ml(-1)) induced concentration-dependent inhibition of electrically evoked (0.2 Hz, 0.3 ms, 70-100 V) twitches. The inhibitory effect of venom (100 microg ml(-1)) was attenuated by 8-phenyltheophylline (8-PT) (20 microM) and 8-cyclopentyl-1, 3-dipropylxanthine (20 microM) but not idazoxan (1 microM), or a combination of ranitidine (0.2 microM) and thioperamide (10 microM). The inhibitory effect of venom (100 microg ml(-1)) was augmented by dipyridamole (10 microM) but abolished by pretreatment with adenosine deaminase (7.5 units/100 microl) suggesting that it contains components with adenosine A(1) receptor activity, most likely adenosine. In isolated segments of guinea-pig ileum, venom (10-100 microg ml(-1)) caused concentration-dependent contractions which were inhibited by the muscarinic receptor antagonist atropine (0.1 microM) but not by the histamine receptor antagonist mepyramine (0.5 microM). In the anaesthetized rat, venom (5-7.5 mg kg(-1), i.v.) caused a hypotensive effect. Our data suggest that the venom contains components with purinergic and muscarinic receptor activity.

Molecular evolution and phylogeny of elapid snake venom three-finger toxins.

Animal venom components are of considerable interest to researchers across a wide variety of disciplines, including molecular biology, biochemistry, medicine, and evolutionary genetics. The three-finger family of snake venom peptides is a particularly interesting and biochemically complex group of venom peptides, because they are encoded by a large multigene family and display a diverse array of functional activities. In addition, understanding how this complex and highly varied multigene family evolved is an interesting question to researchers investigating the biochemical diversity of these peptides and their impact on human health. Therefore, the purpose of our study was to investigate the long-term evolutionary patterns exhibited by these snake venom toxins to understand the mechanisms by which they diversified into a large, biochemically diverse, multigene family. Our results show a much greater diversity of family members than was previously known, including a number of subfamilies that did not fall within any previously identified groups with characterized activities. In addition, we found that the long-term evolutionary processes that gave rise to the diversity of three-finger toxins are consistent with the birth-and-death model of multigene family evolution. It is anticipated that this "three-finger toxin toolkit" will prove to be useful in providing a clearer picture of the diversity of investigational ligands or potential therapeutics available within this important family.

Occurrence of O-linked Xyl-GlcNAc and Xyl-Glc disaccharides in trocarin, a factor Xa homolog from snake venom.

Trocarin is a 46515-Da group D prothrombin-activating glycoprotein from the venom of the Australian elapid, Tropidechis carinatus. Amino acid sequencing and functional characterization of trocarin demonstrated that it is a structural and functional homolog of mammalian blood coagulation factor (F)Xa. In this study we show that, in contrast to mammalian Xa, which is not glycosylated, trocarin contains an O-linked carbohydrate moiety in its light chain and an N-linked carbohydrate oligosaccharide in its heavy chain. Mass spectrometry and sugar compositional analysis indicate that the O-linked carbohydrate moiety is a mixture of Xyl-GlcNAc-, GlcNAc-, Xyl-Glc- and Glc- structures linked to Ser 52. The N-linked carbohydrate on Asn 45 of the heavy chain is a sialylated, diantennary oligosaccharide that is located at the lip of the active site of the prothrombin activator.

Neuromuscular effects of candoxin, a novel toxin from the venom of the Malayan krait (Bungarus candidus).

1 Candoxin (MW 7334.6), a novel toxin isolated from the venom of the Malayan krait Bungarus candidus, belongs to the poorly characterized subfamily of nonconventional three-finger toxins present in Elapid venoms. The current study details the pharmacological effects of candoxin at the neuromuscular junction. 2 Candoxin produces a novel pattern of neuromuscular blockade in isolated nerve-muscle preparations and the tibialis anterior muscle of anaesthetized rats. In contrast to the virtually irreversible postsynaptic neuromuscular blockade produced by curaremimetic alpha-neurotoxins, the neuromuscular blockade produced by candoxin was rapidly and completely reversed by washing or by the addition of the anticholinesterase neostigmine. 3 Candoxin also produced significant train-of-four fade during the onset of and recovery from neuromuscular blockade, both, in vitro and in vivo. The fade phenomenon has been attributed to a blockade of putative presynaptic nicotinic acetylcholine receptors (nAChRs) that mediate a positive feedback mechanism and maintain adequate transmitter release during rapid repetitive stimulation. In this respect, candoxin closely resembles the neuromuscular blocking effects of d-tubocurarine, and differs markedly from curaremimetic alpha-neurotoxins that produce little or no fade. 4 Electrophysiological experiments confirmed that candoxin produced a readily reversible blockade (IC(50) approximately 10 nM) of oocyte-expressed muscle (alphabetagammadelta) nAChRs. Like alpha-conotoxin MI, well known for its preferential binding to the alpha/delta interface of the muscle (alphabetagammadelta) nAChR, candoxin also demonstrated a biphasic concentration-response inhibition curve with a high- (IC(50) approximately 2.2 nM) and a low- (IC(50) approximately 98 nM) affinity component, suggesting that it may exhibit differential affinities for the two binding sites on the muscle (alphabetagammadelta) receptor. In contrast, curaremimetic alpha-neurotoxins have been reported to antagonize both binding sites with equal affinity.

Non-conventional toxins from Elapid venoms.

Non-conventional toxins constitute a poorly characterized class of three-finger toxins isolated exclusively from Elapidae venoms. These toxins are monomers of 62-68 amino acid residues and contain five disulfide bridges. However, unlike alpha/kappa-neurotoxins and kappa-neurotoxins which have the fifth disulfide bridge in their middle loop (loop II), the fifth disulfide bridge in non-conventional toxins is located in loop I (N-terminus loop). Overall, non-conventional toxins share approximately 28-42% identity with other three-finger toxins including alpha-neurotoxins, alpha/kappa-neurotoxins and kappa-neurotoxins. Recent structural studies have revealed that non-conventional toxins also display the typical three-finger motif. Non-conventional toxins are typically characterized by a lower order of toxicity (LD(50) approximately 5-80 mg/kg) in contrast to prototype alpha-neurotoxins (LD(50) approximately 0.04-0.3 mg/kg) and hence they are also referred to as 'weak toxins'. Further, it is generally assumed that non-conventional toxins target muscle (alpha(2)beta gamma delta) receptors with low affinities several orders of magnitude lower than alpha-neurotoxins and alpha/kappa-neurotoxins. However, it is now known that some non-conventional toxins also antagonize neuronal alpha 7 nicotinic acetylcholine receptors. Hence, non-conventional toxins are not a functionally homogeneous group and other, yet unknown, molecular targets for this class of snake venom toxins may exist. Non-conventional toxins may therefore be a useful source of ligands with novel biological activity targeting the plethora of neuronal nicotinic receptors as well as other physiological processes.

Crystallization and preliminary X-ray analysis of bucain, a novel toxin from the Malayan krait Bungarus candidus.

Bucain is a three-finger toxin, structurally homologous to snake-venom muscarinic toxins, from the venom of the Malayan krait Bungarus candidus. These proteins have molecular masses of approximately 6000-8000 Da and encompass the potent curaremimetic neurotoxins which confer lethality to Elapidae and Hydrophidae venoms. Bucain was crystallized in two crystal forms by the hanging-drop vapour-diffusion technique in 0.1 M sodium citrate pH 5.6, 15% PEG 4000 and 0.15 M ammonium acetate. Form I crystals belong to the monoclinic system space group C2, with unit-cell parameters a = 93.73, b = 49.02, c = 74.09 A, beta = 111.32 degrees, and diffract to a nominal resolution of 1.61 A. Form II crystals also belong to the space group C2, with unit-cell parameters a = 165.04, b = 49.44, c = 127.60 A, beta = 125.55 degrees, and diffract to a nominal resolution of 2.78 A. The self-rotation function indicates the presence of four and eight molecules in the crystallographic asymmetric unit of the form I and form II crystals, respectively. Attempts to solve these structures by molecular-replacement methods have not been successful and a heavy-atom derivative search has been initiated.

Electrospray liquid chromatography/mass spectrometry fingerprinting of Acanthophis (death adder) venoms: taxonomic and toxinological implications.

Death adders (genus Acanthophis) are unique among elapid snakes in both morphology and venom composition. Despite this genus being among the most divergent of all elapids, the venom has been historically regarded as relatively quite simple. In this study, liquid chromatography/mass spectrometry (LC/MS) analysis has revealed a much greater diversity in venom composition, including the presence of molecules of novel molecular weights that may represent a new class of venom component. Furthermore, significant variation exists between species and populations, which allow for the LC/MS fingerprinting of each species. Mass profiling of Acanthophis venoms clearly demonstrates the effectiveness of this technique which underpins fundamental studies ranging from chemotaxonomy to drug design.

SCORPION, a molecular database of scorpion toxins.

Increasing interest in the studies of toxins and the requirements for better structural and functional annotations have created a need for improved data management in the field of toxins. The molecular database, SCORPION, contains more than 200 entries of fully referenced scorpion toxin data including primary sequences, three-dimensional structures, structural and functional annotations of scorpion toxins along with relevant literature references. SCORPION has a set of search tools that allow users to extract data and perform specific queries. These entries have been compiled from public databases and literature, cleaned of errors and enriched with additional structural and functional information. The grouping of scorpion toxins provides a basis for extending and clarifying the existing structural and functional classifications. The bioinformatics modules in SCORPION facilitate analyses aimed at classification of scorpion toxins and identification of sequence patterns associated with specific structural or functional properties of scorpion toxins. The SCORPION database is accessible via the Internet at sdmc.krdl.org.sg:8080/scorpion.

NMR structure of bucandin, a neurotoxin from the venom of the Malayan krait (Bungarus candidus).

A high-resolution solution structure of bucandin, a neurotoxin from Malayan krait (Bungarus candidus), was determined by (1)H-NMR spectroscopy and molecular dynamics. The average backbone root-mean-square deviation for the 20 calculated structures and the mean structure is 0.47 A (1 A=0.1 nm) for all residues and 0.24 A for the well-defined region that spans residues 23-58. Secondary-structural elements include two antiparallel beta-sheets characterized by two and four strands. According to recent X-ray analysis, bucandin adopts a typical three-finger loop motif and yet it has some peculiar characteristics that set it apart from other common alpha-neurotoxins. The presence of a fourth strand in the second antiparallel beta-sheet had not been observed before in three-finger toxins, and this feature was well represented in the NMR structure. Although the overall fold of the NMR structure is similar to that of the X-ray crystal structure, there are significant differences between the two structures that have implications for the pharmacological action of the toxin. These include the extent of the beta-sheets, the conformation of the region spanning residues 42-49 and the orientation of some side chains. In comparison with the X-ray structure, the NMR structure shows that the hydrophobic side chains of Trp(27) and Trp(36) are stacked together and are orientated towards the tip of the middle loop. The NMR study also showed that the two-stranded beta-sheet incorporated in the first loop, as defined by residues 1-22, and the C-terminus from Asn(59), is probably flexible relative to the rest of the molecule. On the basis of the dispositions of the hydrophobic and hydrophilic side chains, the structure of bucandin is clearly different from those of cytotoxins.

DNA binding mediated by the wheat HMGa protein: a novel instance of selectivity against alternating GC sequence.

The high-mobility-group (HMG) chromosomal protein wheat HMGa was purified to homogeneity and tested for its binding characteristics to double-stranded DNA. Wheat HMGa was able to bind to P268, an A/T-rich fragment derived from the pea plastocyanin gene promoter, producing a small mobility shift in gel retardation assays where the bound complex was sensitive to addition of proteinase K but resistant to heat treatment of the protein, consistent with the identity of wheat HMGa as a putative HMG-I/Y protein. Gel retardation assays and southwestern hybridization analysis revealed that wheat HMGa could selectively interact with the DNA polynucleotides poly(dA).poly(dT), poly(dAdT).poly(dAdT), and poly(dG).poly(dC), but not with poly(dGdC).poly(dGdC). Surface plasmon resonance analysis determined the kinetic and affinity constants of sensor chip-immobilized wheat HMGa for double-stranded DNA 10-mers, revealing a good affinity of the protein for various dinucleotide combinations, except that of alternating GC sequence. Thus contrary to prior reports of a selectivity of wheat HMGa for A/T-rich DNA, the protein appears to be able to interact with sequences containing guanine and cytosine residues as well, except where G/C residues alternate directly in the primary sequence.

Purification and properties of three new phospholipase A2 isoenzymes from Micropechis ikaheka venom.

Three new phospholipase A2 (PLA2) isoenzymes were purified from the Micropechis ikaheka venom by successive chromatographies. The homogeneity of them was accessed by capillary zone electrophoresis and mass spectrometry. Their N-terminal sequences showed high identity (94, 88 and 90, respectively) with MiPLA-1, a group IB PLA2 also from this venom. In addition, strong immuno-cross-reaction with anti-MiPLA-1 serum was observed. These results suggested that three newly purified PLA2 belonged to group IB. Beside enzymatic activity, they induced various pharmacological effects, including myotoxic, anticoagulant effects and insulin secretion stimulating effects. Our results indicated that enzymatic activity is essential for their myotoxic and anticoagulant effects. On the other hand, no direct correlation between their insulin secretion stimulating effect and enzymatic activity was observed, suggesting that they may stimulate insulin secretion through a non-enzymatic mechanism.

Tamulustoxin: a novel potassium channel blocker from the venom of the Indian red scorpion Mesobuthus tamulus.

We have characterized tamulustoxin, a novel 35-amino-acid peptide found in the venom of the Indian red scorpion (Mesobuthus tamulus). Tamulustoxin was identified through a [125I]toxin I screen, designed to identify toxins that block voltage-activated potassium channels. Tamulustoxin has also been cloned by RT-PCR, using RNA extracted from scorpion venom glands. Tamulustoxin shares no homology with other scorpion venom toxins, although the positions of its six cysteine residues would suggest that it shares the same structural scaffold. Tamulustoxin rapidly inhibited both peak and steady-state currents (18.9 +/- 1.0 and 37 +/- 1.1%, respectively) produced by injecting CHO cells with mRNA encoding the hKv1.6 channel.