Refined three-dimensional solution structure of a snake cardiotoxin: Analysis of the side-chain organization suggests the existence of a possible phospholipid binding site

The solution structure of toxin γ (60 residues, 4 disulfides) from Naja nigricollis was determined by proton nmr and molecular modeling with DIANA and X-PLOR. The structures were calculated using 489 distance and 81 dihedral angle constraints. The average atomic rms deviation between the nine refined structures and the average structure is 0.118 nm for the backbone atoms. Toxin γ has an overall folding consisting of three loops stabilized by the four disulfides and forming a two- and a three-stranded β-sheet (loop I and loops II, III, respectively). The same type of folding has been observed for two homologous cardiotoxins. The very close similarity of the solution structure of toxin γ and the crystal structure of toxin V includes details of the topological arrangement of numerous side chains. Among these are the conserved residues K12, K18, K35, and Y22, known to be critical for the cytolytic activity of toxin γ. A cluster of hydrophobic side chains organized around Y22 is found on one side of the three-stranded β-sheet and is spatially close to a group of three lysines (K12, K18, K35). The side chains of these lysines form a cationic site that can accommodate the binding of a phosphate ion as found in the crystal structure of toxin V. The hydrophobic cluster constitutes a possible binding site for the hydrophobic moiety of phospholipids. Together with the complementary cationic site, this hydrophobic surface can form a conserved site by which cardiotoxins bind to membrane phospholipids. © 1993 John Wiley & Sons, Inc.     

Motions and structural variability within toxins: implication for their use as scaffolds for protein engineering

Animal toxins are small proteins built on the basis of a few disulfide bonded frameworks. Because of their high variability in sequence and biologic function, these proteins are now used as templates for protein engineering. Here we report the extensive characterization of the structure and dynamics of two toxin folds, the "three-finger" fold and the short alpha/beta scorpion fold found in snake and scorpion venoms, respectively. These two folds have a very different architecture; the short alpha/beta scorpion fold is highly compact, whereas the "three-finger" fold is a beta structure presenting large flexible loops. First, the crystal structure of the snake toxin alpha was solved at 1.8-A resolution. Then, long molecular dynamics simulations (10 ns) in water boxes of the snake toxin alpha and the scorpion charybdotoxin were performed, starting either from the crystal or the solution structure. For both proteins, the crystal structure is stabilized by more hydrogen bonds than the solution structure, and the trajectory starting from the X-ray structure is more stable than the trajectory started from the NMR structure. The trajectories started from the X-ray structure are in agreement with the experimental NMR and X-ray data about the protein dynamics. Both proteins exhibit fast motions with an amplitude correlated to their secondary structure. In contrast, slower motions are essentially only observed in toxin alpha. The regions submitted to rare motions during the simulations are those that exhibit millisecond time-scale motions. Lastly, the structural variations within each fold family are described. The localization and the amplitude of these variations suggest that the regions presenting large-scale motions should be those tolerant to large insertions or deletions.     

Comparison of three classes of snake neurotoxins by homology modeling and computer simulation graphics

We present a systematic structure comparison of three major classes of postsynaptic snake toxins, which include short and long chain alpha-type neurotoxins plus one angusticeps-type toxin of black mamba snake family. Two novel alpha-type neurotoxins isolated from Taiwan cobra (Naja naja atra) possessing distinct primary sequences and different postsynaptic neurotoxicities were taken as exemplars for short and long chain neurotoxins and compared with the major lethal short-chain neurotoxin in the same venom, i.e., cobrotoxin, based on the derived three-dimensional structure of this toxin in solution by NMR spectroscopy. A structure comparison among these two alpha-neurotoxins and angusticeps-type toxin (denoted as FS2) was carried out by the secondary-structure prediction together with computer homology-modeling based on multiple sequence alignment of their primary sequences and established NMR structures of cobrotoxin and FS2. It is of interest to find that upon pairwise superpositions of these modeled three-dimensional polypeptide chains, distinct differences in the overall peptide flexibility and interior microenvironment between these toxins can be detected along the three constituting polypeptide loops, which may reflect some intrinsic differences in the surface hydrophobicity of several hydrophobic peptide segments present on the surface loops of these toxin molecules as revealed by hydropathy profiles. Construction of a phylogenetic tree for these structurally related and functionally distinct toxins corroborates that all long and short toxins present in diverse snake families are evolutionarily related to each other, supposedly derived from an ancestral polypeptide by gene duplication and subsequent mutational substitutions leading to divergence of multiple three-loop toxin peptides.     

Stability of a structural scaffold upon activity transfer: X-ray structure of a three fingers chimeric protein

Fasciculin 2 and toxin alpha proteins belong to the same structural family of three-fingered snake toxins. They act on different targets, but in each case the binding region involves residues from loops I and II. The superimposition of the two structures suggests that these functional regions correspond to structurally distinct zones. Loop I, half of loop II and the C-terminal residue of fasciculin 2 were therefore transferred into the toxin alpha. The inhibition constant of the resulting chimera is only 15-fold lower than that of fasciculin 2, and as expected the potency of binding to the toxin alpha target has been lost. In order to understand the structure-function relationship between the chimera and its "parent" molecules, we solved its structure by X-ray crystallography. The protein crystallized in space group P3(1)21 with a=b=58.5 A, and c=62.3 A. The crystal structure was solved by molecular replacement and refined to 2.1 A resolution. The structure belongs to the three-fingered snake toxin family with a core of four disulphide bridges from which emerge the three loops I, II and III. Superimposition of the chimera on fasciculin 2 or toxin alpha revealed an overall fold intermediate between those of the two parent molecules. The regions corresponding to toxin alpha and to fasciculin 2 retained their respective geometries. In addition, the chimera protein displayed a structural behaviour similar to that of fasciculin 2, i.e. dimerization in the crystal structure of fasciculin 2, and the geometry of the region that binds to acetylcholinesterase. In conclusion, this structure shows that the chimera retains the general structural characteristics of three-fingered toxins, and the structural specificity of the transferred function.     

Three-dimensional structure of ectatomin from Ectatomma tuberculatum ant venom

Summary Two-dimensional ¹H NMR techniques were used to determine the spatial structure of ectatomin, a toxin from the venom of the ant Ectatomma tuberculatum. Nearly complete proton resonance assignments for two chains of ectatomin (37 and 34 amino acid residues, respectively) were obtained using 2D TOCSY, DQF-COSY and NOESY experiments. The cross-peak volumes in NOESY spectra were used to define the local structure of the protein and generate accurate proton-proton distance constraints employing the MARDIGRAS program. Disulfide bonds were located by analyzing the global fold of ectatomin, calculated with the distance geometry program DIANA. These data, combined with data on the rate of exchange of amide protons with deuterium, were used to obtain a final set of 20 structures by DIANA. These structures were refined by unrestrained energy minimization using the CHARMm program. The resulting rms deviations over 20 structures (excluding the mobile N- and C-termini of each chain) are 0.75 ? for backbone heavy atoms, and 1.25 ? for all heavy atoms. The conformations of the two chains are similar. Each chain consists of two α-helices and a hinge region of four residues; this forms a hairpin structure which is stabilized by disulfide bridges. The hinge regions of the two chains are connected together by a third disulfide bridge. Thus, ectatomin forms a four-α-helical bundle structure.     

Tertiary structure of conotoxin GIIIA in aqueous solution

The three-dimensional structure of conotoxin GIIIA, an important constituent of the venom from the marine hunting snail Conus geographus L., was determined in aqueous solution by two-dimensional proton nuclear magnetic resonance and simulated annealing based methods. On the basis of 162 assigned nuclear Overhauser effect (NOE) connectivities obtained at the medium field strength frequency of 400 MHz, 74 final distance constraints of sequential and tertiary ones were derived and used together with 18 torsion angle (phi, chi 1) constraints and 9 distance constraints derived from disulfide bridges. A total of 32 converged structures were obtained from 200 runs of calculations. The atomic root-mean-square (RMS) difference about the mean coordinate positions (excluding the terminal residues 1 and 22) is 0.8 A for backbone atoms (N, C alpha, C). Conotoxin GIIIA is characterized by a particular folding of the 22 amino acid peptidic chain, which is stabilized by three disulfide bridges arranged in cage at the center of a discoidal structure of approximately 20-A diameter. The seven cationic side chains of lysine and arginine residues project radially into the solvent and form potential sites of interaction with the skeletal muscle sodium channel for which the toxin is a strong inhibitor. The present results provide a molecular basis to elucidate the remarkable physiological properties of this neurotoxin.     

Direct expression in E. coli of a functionally active protein A--snake toxin fusion protein

We constructed a recombinant expression plasmid encoding a protein A--neurotoxin fusion protein. The fused toxin is directly expressed in the periplasmic space of Escherichia coli and can be purified in the milligram range by a single immuno-affinity step. The LD50 values of the fused toxin and native toxin are 130 and 20 nmol/kg mouse respectively. The Kd values characterizing their binding to the nicotinic acetylcholine receptor (AcChoR) are respectively 4.8 +/- 0.8 and 0.07 +/- 0.03 nM. In contrast, the fused and native toxins are equally well recognized by a toxin-specific monoclonal antibody which recognizes the AcChoR binding site. The lower toxicity of the fused toxin might result, therefore, from a steric hindrance, due to the presence of the bulky protein A moiety (mol. wt = 31 kd) rather than to a direct alteration of the 'toxic' site. The fused toxin is more immunogenic than native toxin, since 1 nmol of hybrid toxin and 14 nmol of native toxin give rise to comparable titers of antitoxin antibodies which, furthermore, are equally potent at neutralizing neurotoxicity. The work described in this paper shows that the use of fused toxins may be of paramount importance for future development of serotherapy against envenomation by snake bites.     

Chemical engineering of a three-fingered toxin with anti-alpha7 neuronal acetylcholine receptor activity

Though it possesses four disulfide bonds the three-fingered fold is amenable to chemical synthesis, using a Fmoc-based method. Thus, we synthesized a three-fingered curaremimetic toxin from snake with high yield and showed that the synthetic and native toxins have the same structural and biological properties. Both were characterized by the same 2D NMR spectra, identical high binding affinity (K(d) = 22 +/- 5 pM) for the muscular acetylcholine receptor (AChR) and identical low affinity (K(d) = 2.0 +/- 0.4 microM) for alpha7 neuronal AchR. Then, we engineered an additional loop cyclized by a fifth disulfide bond at the tip of the central finger. This loop is normally present in longer snake toxins that bind with high affinity (K(d) = 1-5 nM) to alpha7 neuronal AchR. Not only did the chimera toxin still bind with the same high affinity to the muscular AchR but also it displayed a 20-fold higher affinity (K(d) = 100 nM) for the neuronal alpha7 AchR, as compared with the parental short-chain toxin. This result demonstrates that the engineered loop contributes, at least in part, to the high affinity of long-chain toxins for alpha7 neuronal receptors. That three-fingered proteins with four or five disulfide bonds are amenable to chemical synthesis opens new perspectives for engineering new activities on this fold.     

Role of disulfide bonds in folding and activity of leiurotoxin I: just two disulfides suffice

The aim of this study is to investigate the contribution of each disulfide bond in the folding and function of leiurotoxin I, a short scorpion toxin that blocks small conductance K(+) channels. The structure of leiurotoxin I contains a motif conserved in all scorpion toxins, formed by a helix and a double-stranded beta-sheet and stabilized by three disulfide bridges. We synthesized three analogues, each presenting two alpha-aminobutyric acid (Abu) moieties replacing two bridged cysteine residues: LeTx1 ([Abu 3,21] Leiurotoxin I), LeTx2 ([Abu 8,26] Leiurotoxin I), and LeTx3 ([Abu 12,28] Leiurotoxin I). All three analogues fold into a major product containing two native disulfide bonds, while LeTx3 forms an additional isomer, containing non-native disulfides. In denaturing conditions, analogues LeTx2 and LeTx3 yield non-native isomers, while LeTx1 only forms the isomer with native disulfides. All isomers with native disulfides contain nativelike alpha-helical conformations and bind to synaptosomal membranes with affinities within a log of that shown by the native toxin. By contrast, the non-native LeTx3A analogue exhibits a disordered conformation and a decreased biological potency. Our results indicate that the "CxxxC, CxC" cysteine spacing, conserved in all scorpion toxins and preserved in LeTx1, may play an active role in folding, and that only two native disulfide bonds in leiurotoxin I are sufficient to preserve a nativelike and active conformation. Thus, in the scorpion toxin scaffold, modifications of conserved and interior cysteine residues may permit modulation of function, without significantly affecting folding efficiency and structure.     

Solution structures of the cis- and trans-Pro30 isomers of a novel 38-residue toxin from the venom of Hadronyche Infensa sp. that contains a cystine-knot motif within its four disulfide bonds

The primary sequence and three-dimensional structure of a novel peptide toxin isolated from the Australian funnel-web spider Hadronyche infensa sp. is reported. ACTX-Hi:OB4219 contains 38 amino acids, including eight-cysteine residues that form four disulfide bonds. The connectivities of these disulfide bonds were previously unknown but have been unambiguously determined in this study. Three of these disulfide bonds are arranged in an inhibitor cystine-knot (ICK) motif, which is observed in a range of other disulfide-rich peptide toxins. The motif incorporates an embedded ring in the structure formed by two of the disulfides and their connecting backbone segments penetrated by a third disulfide bond. Using NMR spectroscopy, we determined that despite the isolation of a single native homologous product by RP-HPLC, ACTX-Hi:OB4219 possesses two equally populated conformers in solution. These two conformers were determined to arise from cis/trans isomerization of the bond preceding Pro30. Full assignment of the NMR spectra for both conformers allowed for the calculation of their structures, revealing the presence of a triple-stranded antiparallel beta sheet consistent with the inhibitor cystine-knot (ICK) motif.     

Evaluation of comparative protein modeling by MODELLER

We evaluate 3D models of human nucleoside diphosphate kinase, mouse cellular retinoic acid binding protein I, and human eosinophil neurotoxin that were calculated by MODELLER , a program for comparative protein modeling by satisfaction of spatial restraints. The models have good stereochemistry and are at least as similar to the crystallographic structures as the closest template structures. The largest errors occur in the regions that were not aligned correctly or where the template structures are not similar to the correct structure. These regions correspond predominantly to exposed loops, insertions of any length, and non-conserved side chains. When a template structure with more than 40% sequence identity to the target protein is available, the model is likely to have about 90% of the mainchain atoms modeled with an rms deviation from the X-ray structure of ≈ 1 Å, in large part because the templates are likely to be that similar to the X-ray structure of the target. This rms deviation is comparable to the overall differences between refined NMR and X-ray crystallography structures of the same protein. © 1995 Wiley-Liss, Inc.     

Refined solution structure of the anti-mammal and anti-insect LqqIII scorpion toxin: Comparison with other scorpion toxins

The solution structure of the anti-mammal and anti-insect LqqIII toxin from the scorpion Leiurus quinquestriatus quinquestriatuswas refined and compared with other long-chain scorpion toxins. This structure, determined by ¹H-NMR and molecular modeling, involves an α-helix (18–29) linked to a three-stranded β-sheet (2–6, 33–39, and 43–51) by two disulfide bridges. The average RMSD between the 15 best structures and the mean structure is 0.71 Å for Cα atoms. Comparison between LqqIII, the potent anti-mammal AaHII, and the weakly active variant-3 toxins revealed that the LqqIII three-dimensional structure is closer to that of AaHII than to the variant-3 structure. Moreover, striking analogies were observed between the electrostatic and hydrophobic potentials of LqqIII and AaHII. Several residues are well conserved in long-chain scorpion toxin sequences and seem to be important in protein structure stability and function. Some of them are involved in the CSαβ (Cysteine Stabilized α-helix β-sheet) motif. A comparison between the sequences of the RII rat brain and the Drosophila extracellular loops forming scorpion toxin binding-sites of Na⁺ channels displays differences in the subsites interacting with anti-mammal or anti-insect toxins. This suggests that hydrophobic as well as electrostatic interactions are essential for the binding and specificity of long-chain scorpion toxins. Proteins 28:360–374, 1997 © 1997 Wiley-Liss, Inc.     

Conserved structural determinants in three-fingered protein domains

The three-dimensional structures of some components of snake venoms forming so-called 'three-fingered protein' domains (TFPDs) are similar to those of the ectodomains of activin, bone morphogenetic protein and transforming growth factor-beta receptors, and to a variety of proteins encoded by the Ly6 and Plaur genes. The analysis of sequences of diverse snake toxins, various ectodomains of the receptors that bind activin and other cytokines, and numerous gene products encoded by the Ly6 and Plaur families of genes has revealed that they differ considerably from each other. The sequences of TFPDs may consist of up to six disulfide bonds, three of which have the same highly conserved topology. These three disulfide bridges and an asparagine residue in the C-terminal part of TFPDs are essential for the TFPD-like fold. Analyses of the three-dimensional structures of diverse TFPDs have revealed that the three highly conserved disulfides impose a major stabilizing contribution to the TFPD-like fold, in both TFPDs contained in some snake venoms and ectodomains of several cellular receptors, whereas the three remaining disulfide bonds impose specific geometrical constraints in the three fingers of some TFPDs.     

Solution structure of crotamine, a Na+ channel affecting toxin from Crotalus durissus terrificus venom.

Crotamine is a component of the venom of the snake Crotalus durissus terrificus and it belongs to the myotoxin protein family. It is a 42 amino acid toxin cross-linked by three disulfide bridges and characterized by a mild toxicity (LD50 = 820 micro g per 25 g body weight, i.p. injection) when compared to other members of the same family. Nonetheless, it possesses a wide spectrum of biological functions. In fact, besides being able to specifically modify voltage-sensitive Na+ channel, it has been suggested to exhibit analgesic activity and to be myonecrotic. Here we report its solution structure determined by proton NMR spectroscopy. The secondary structure comprises a short N-terminal alpha-helix and a small antiparallel triple-stranded beta-sheet arranged in an alphabeta1beta2beta3 topology never found among toxins active on ion channels. Interestingly, some scorpion toxins characterized by a biological activity on Na+ channels similar to the one reported for crotamine, exhibit an alpha/beta fold, though with a beta1alphabeta2beta3 topology. In addition, as the antibacterial beta-defensins, crotamine interacts with lipid membranes. A comparison of crotamine with human beta-defensins shows a similar fold and a comparable net positive potential surface. To the best of our knowledge, this is the first report on the structure of a toxin from snake venom active on Na+ channel.     

Effects of eglin-c binding to thermitase: three-dimensional structure comparison of native thermitase and thermitase eglin-c complexes.

Thermitase is a thermostable member of the subtilisin family of serine proteases. Four independently determined crystal structures of the enzyme are compared in this study: a high resolution native one and three medium resolution complexes of thermitase with eglin-c, grown from three different calcium concentrations. It appeared that the B-factors of the thermitase eglin complex obtained at 100 mM CaCl2 and elucidated at 2.0 A resolution are remarkably similar to those of the 1.4 A native structure: the main chain atoms have an rms difference of only 2.3 A2; for all atoms this difference is 4.6 A2. The rms positional differences between these two structures of thermitase are 0.31 A for the main chain atoms and 0.58 A for all atoms. There results show that not only atomic positions but also temperature factors can agree well in X-ray structures determined entirely independently by procedures which differ in virtually every possible technical aspect. A detailed comparison focussed on the effects of eglin binding on the structure of thermitase. Thermitase can be considered as consisting of (1) a central core of 94 residues, plus (2) four segments of 72 residues in total which shift as rigid bodies with respect to the core, plus (3) the remaining 113 residues which show small changes but, however, cannot be described as rigid bodies. The central cores of native thermitase and the 100 mM CaCl2 thermitase:eglin complex have an rms deviation of 0.13 A for 376 main chain atoms. One of the segments, formed by loops of the strong calcium binding site, shows differences up to 1.0 A in C alpha positions. These are probably due to crystal packing effects. The three other segments, comprising 51 residues, are affected conformational changes upon eglin binding so that the P1 to P3 binding pockets of thermitase broaden by 0.4 to 0.7 A. The residues involved in these changes correspond with residues which change position upon inhibitor binding in other subtilisins. This suggests that an induced fit mechanism is operational during substrate recognition by subtilisins.     

Three-dimensional solution structure of the E3-binding domain of the dihydrolipoamide succinyltransferase core from the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli.

The three-dimensional solution structure of a 51-residue synthetic peptide comprising the dihydrolipoamide dehydrogenase (E3)-binding domain of the dihydrolipoamide succinyltransferase (E2) core of the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli has been determined by nuclear magnetic resonance spectroscopy and hybrid distance geometry-dynamical simulated annealing calculations. The structure is based on 630 approximate interproton distance and 101 torsion angle (phi, psi, chi 1) restraints. A total of 56 simulated annealing structures were calculated, and the atomic rms distribution about the mean coordinate positions for residues 12-48 of the synthetic peptide is 1.24 A for the backbone atoms, 1.68 A for all atoms, and 1.33 A for all atoms excluding the six side chains which are disordered at chi 1 and the seven which are disordered at chi 2; when the irregular partially disordered loop from residues 31 to 39 is excluded, the rms distribution drops to 0.77 A for the backbone atoms, 1.55 A for all atoms, and 0.89 A for ordered side chains. Although proton resonance assignments for the N-terminal 11 residues and the C-terminal 3 residues were obtained, these two segments of the polypeptide are disordered in solution as evidenced by the absence of nonsequential nuclear Overhauser effects. The solution structure of the E3-binding domain consists of two parallel helices (residues 14-23 and 40-48), a short extended strand (24-26), a five-residue helical-like turn, and an irregular (and more disordered) loop (residues 31-39). This report presents the first structure of an E3-binding domain from a 2-oxo acid dehydrogenase complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

The impact of the fourth disulfide bridge in scorpion toxins of the alpha-KTx6 subfamily

Animal toxins are highly reticulated and structured polypeptides that adopt a limited number of folds. In scorpion species, the most represented fold is the alpha/beta scaffold in which an helical structure is connected to an antiparallel beta-sheet by two disulfide bridges. The intimate relationship existing between peptide reticulation and folding remains poorly understood. Here, we investigated the role of disulfide bridging on the 3D structure of HsTx1, a scorpion toxin potently active on Kv1.1 and Kv1.3 channels. This toxin folds along the classical alpha/beta scaffold but belongs to a unique family of short-chain, four disulfide-bridged toxins. Removal of the fourth disulfide bridge of HsTx1 does not affect its helical structure, whereas its two-stranded beta-sheet is altered from a twisted to a nontwisted configuration. This structural change in HsTx1 is accompanied by a marked decrease in Kv1.1 and Kv1.3 current blockage, and by alterations in the toxin to channel molecular contacts. In contrast, a similar removal of the fourth disulfide bridge of Pi1, another scorpion toxin from the same structural family, has no impact on its 3D structure, pharmacology, or channel interaction. These data highlight the importance of disulfide bridging in reaching the correct bioactive conformation of some toxins.     

Solution structure of the major alpha-amylase inhibitor of the crop plant amaranth.

alpha-Amylase inhibitor (AAI), a 32-residue miniprotein from the Mexican crop plant amaranth (Amaranthus hypochondriacus), is the smallest known alpha-amylase inhibitor and is specific for insect alpha-amylases (Chagolla-Lopez, A., Blanco-Labra, A., Patthy, A., Sanchez, R., and Pongor, S. (1994) J. Biol. Chem. 269, 23675-23680). Its disulfide topology was confirmed by Edman degradation, and its three-dimensional solution structure was determined by two-dimensional 1H NMR spectroscopy at 500 MHz. Structural constraints (consisting of 348 nuclear Overhauser effect interproton distances, 8 backbone dihedral constraints, and 9 disulfide distance constraints) were used as an input to the X-PLOR program for simulated annealing and energy minimization calculations. The final set of 10 structures had a mean pairwise root mean square deviation of 0.32 A for the backbone atoms and 1.04 A for all heavy atoms. The structure of AAI consists of a short triple-stranded beta-sheet stabilized by three disulfide bonds, forming a typical knottin or inhibitor cystine knot fold found in miniproteins, which binds various macromolecular ligands. When the first intercystine segment of AAI (sequence IPKWNR) was inserted into a homologous position of the spider toxin Huwentoxin I, the resulting chimera showed a significant inhibitory activity, suggesting that this segment takes part in enzyme binding.     

NMR structure of free RGS4 reveals an induced conformational change upon binding Galpha

Heterotrimeric guanine nucleotide-binding proteins (G-proteins) are transducers in many cellular transmembrane signaling systems where regulators of G-protein signaling (RGS) act as attenuators of the G-protein signal cascade by binding to the Galpha subunit of G-proteins (G(i)(alpha)(1)) and increasing the rate of GTP hydrolysis. The high-resolution solution structure of free RGS4 has been determined using two-dimensional and three-dimensional heteronuclear NMR spectroscopy. A total of 30 structures were calculated by means of hybrid distance geometry-simulated annealing using a total of 2871 experimental NMR restraints. The atomic rms distribution about the mean coordinate positions for residues 5-134 for the 30 structures is 0.47 +/- 0.05 A for the backbone atoms, 0. 86 +/- 0.05 A for all atoms, and 0.56 +/- 0.04 A for all atoms excluding disordered side chains. The NMR solution structure of free RGS4 suggests a significant conformational change upon binding G(i)(alpha)(1) as evident by the backbone atomic rms difference of 1. 94 A between the free and bound forms of RGS4. The underlying cause of this structural change is a perturbation in the secondary structure elements in the vicinity of the G(i)(alpha)(1) binding site. A kink in the helix between residues K116-Y119 is more pronounced in the RGS4-G(i)(alpha)(1) X-ray structure relative to the free RGS4 NMR structure, resulting in a reorganization of the packing of the N-terminal and C-terminal helices. The presence of the helical disruption in the RGS4-G(i)(alpha)(1) X-ray structure allows for the formation of a hydrogen-bonding network within the binding pocket for G(i)(alpha)(1) on RGS4, where RGS4 residues D117, S118, and R121 interact with residue T182 from G(i)(alpha)(1). The binding pocket for G(i)(alpha)(1) on RGS4 is larger and more accessible in the free RGS4 NMR structure and does not present the preformed binding site observed in the RGS4-G(i)(alpha)(1) X-ray structure. This observation implies that the successful complex formation between RGS4 and G(i)(alpha)(1) is dependent on both the formation of the bound RGS4 conformation and the proper orientation of T182 from G(i)(alpha)(1). The observed changes for the free RGS4 NMR structure suggest a mechanism for its selectivity for the Galpha-GTP-Mg(2+) complex and a means to facilitate the GTPase cycle.     

Structural properties of mojave toxin of crotalus scutulatus (Mojave rattlesnake) determined by laser Raman spectroscopy.

Laser Raman Spectra were obtained on aqueous and solid samples of Mojave toxin isolated from the venom of the Mojave rattlesnake ($\text{Crotalus}\text{scutulatus}$). The Raman spectra reveal that the Mojave toxin, an acidic protein of molecular weight about 22,000, contains a predominantly α-helical secondary structure and that the tyrosyl residues, on the basis of the Raman frequencies and intensities, are exposed to the solvent. These features of the Mojave toxin distinguish it structurally from the neurotoxins of sea snake venoms. However, like the sea snake venom toxins, Mojave toxin contains four disulfide bridges and is not greatly altered in structure by removal of the aqueous solvent.