Solution structure of a beta-neurotoxin from the New World scorpion Centruroides sculpturatus Ewing

We report the detailed solution structure of the 7.2 kDa protein CsE-I, a beta-neurotoxin from the New World scorpion Centruroides sculpturatus Ewing. This toxin binds to sodium channels, but unlike the alpha-neurotoxins, shifts the voltage of activation toward more negative potentials causing the membrane to fire spontaneously. Sequence-specific proton NMR assignments were made using 600 MHz 2D-NMR data. Distance geometry and dynamical simulated annealing refinements were performed using experimental distance and torsion angle constraints from NOESY and pH-COSY data. A family of 40 structures without constraint violations was generated, and an energy-minimized average structure was computed. The backbone conformation of the CsE-I toxin shows similar secondary structural features as the prototypical alpha-neurotoxin, CsE-v3, and is characterized by a short 2(1/2)-turn alpha-helix and a 3-strand antiparallel beta-sheet, both held together by disulfide bridges. The RMSD for the backbone atoms between CsE-I and CsE-v3 is 1.48 A. Despite this similarity in the overall backbone folding, the these two proteins show some important differences in the primary structure (sequence) and electrostatic potential surfaces. Our studies provide a basis for unravelling the role of these differences in relation to the known differences in the receptor sites on the voltage sensitive sodium channel for the alpha- and beta-neurotoxins.     

Three-dimensional solution structure of alpha-conotoxin MII by NMR spectroscopy: effects of solution environment on helicity

alpha-Conotoxin MII, a 16-residue polypeptide from the venom of the piscivorous cone snail Conus magus, is a potent and highly specific blocker of mammalian neuronal nicotinic acetylcholine receptors composed of alpha3 beta2 subunits. The role of this receptor type in the modulation of neurotransmitter release and its relevance to the problems of addiction and psychosis emphasize the importance of a structural understanding of the mode of interaction of MII with the alpha3 beta2 interface. Here we describe the three-dimensional solution structure of MII determined using 2D 1H NMR spectroscopy. Structural restraints consisting of 376 interproton distances inferred from NOEs and 12 dihedral restraints derived from spin-spin coupling constants were used as input for simulated annealing calculations and energy minimization in the program X-PLOR. The final set of 20 structures is exceptionally well-defined with mean pairwise rms differences over the whole molecule of 0.07 A for the backbone atoms and 0.34 A for all heavy atoms. MII adopts a compact structure incorporating a central segment of alpha-helix and beta-turns at the N- and C-termini. The molecule is stabilized by two disulfide bonds, which provide cross-links between the N-terminus and both the middle and C-terminus of the structure. The susceptibility of the structure to conformational change was examined using several different solvent conditions. While the global fold of MII remains the same, the structure is stabilized in a more hydrophobic environment provided by the addition of acetonitrile or trifluoroethanol to the aqueous solution. The distribution of amino acid side chains in MII creates distinct hydrophobic and polar patches on its surface that may be important for the specific interaction with the alpha3beta2 neuronal nAChR. A comparison of the structure of MII with other neuronal-specific alpha-conotoxins provides insights into their mode of interaction with these receptors.     

Proton nuclear magnetic resonance and distance geometry/simulated annealing studies on the variant-1 neurotoxin from the New World scorpion Centruroides sculpturatus Ewing

The sequence-specific proton resonance assignments for the variant-1 (CsE-v1) neurotoxin from the venom of the New World scorpion Centruroides sculpturatus Ewing (range Southwestern United States) have been performed by 2D 1H NMR spectroscopy at 600 MHz. The stereospecific assignments for the beta-methylene protons of 19 non-proline residues have been determined. A number of short-, medium-, and long-range NOESY contacts as well as the backbone and the side-chain vicinal coupling constants for several residues have been determined. Slowly exchanging amide hydrogens from a number of residues have been identified. On the basis of the NMR data, the solution structure of this protein has been determined by a hybrid procedure consisting of distance geometry and dynamical simulated annealing refinement calculations. Distance constraints from the NOESY data and torsion angle constraints from proton vicinal coupling constant data were used in the simulated annealing calculations. The three-dimensional structure of CsE-v1 is characterized by a three-stranded antiparallel beta-sheet, a short alpha-helix, a cis-proline, and intervening loops. A comparison with the solution NMR data of a homologous protein (CsE-v3) from the Centruroides venom, shows that the structures are essentially similar, except for some minor differences. Some of the NMR spectral perturbations are felt in regions far removed from sites of amino acid substitutions. The hydrophobic surface in CsE-v1 is slightly more extended than in CsE-v3.     

Determination of the three-dimensional solution structure of Raphanus sativus antifungal protein 1 by 1H NMR

Raphanus sativus Antifungal Protein 1 (Rs-AFP1) is a 51 amino acid residue plant defensin isolated from radish (Raphanus sativus L.) seeds. The three-dimensional structure in aqueous solution has been determined from two-dimensional 1H NMR data recorded at 500 MHz using the DIANA/REDAC calculation protocols. Experimental constraints consisted of 787 interproton distances extracted from NOE cross-peaks, 89 torsional constraints from 106 vicinal interproton coupling constants and 32 stereospecific assignments of prochiral protons. Further refinement by simulated annealing resulted in a set of 20 structures having pairwise root-mean-square differences of 1.35(+/- 0.35) A over the backbone heavy atoms and 2.11(+/- 0.46) A over all heavy atoms. The molecule adopts a compact globular fold comprising an alpha-helix from Asn18 till Leu28 and a triple-stranded beta-sheet (beta 1 = Lys2-Arg6, beta 2 = His33-Tyr38 and beta 3 = His43-Pro50). The central strand of this beta-sheet is connected by two disulfide bridges (Cys21-Cys45 and Cys25-Cys47) to the alpha-helix. The connection between beta-strand 2 and 3 is formed by a type VIa beta-turn. Even the loop (Pro7 to Asn17) between beta-strand 1 and the alpha-helix is relatively well defined. The structure of Raphanus sativus Antifungal Protein 1 features all the characteristics of the "cysteine stabilized alpha beta motif". A comparison of the complete structure and of the regions important for interaction with the fungal receptor according to a mutational study, is made with the structure of gamma-thionin, a plant defensin that has no antifungal activity. It is concluded that this interaction is both electrostatic and specific, and some possible scenarios for the mode of action are given.     

Solution structure of drosomycin, the first inducible antifungal protein from insects

Drosomycin is the first antifungal protein characterized recently among the broad family of inducible peptides and proteins produced by insects to respond to bacterial or septic injuries. It is a small protein of 44 amino acid residues extracted from Drosophila melanogaster that exhibits a potent activity against filamentous fungi. Its three-dimensional structure in aqueous solution was determined using 1H 2D NMR. This structure, involving an alpha-helix and a twisted three-stranded beta-sheet, is stabilized by three disulfide bridges. The corresponding Cysteine Stabilized alpha beta (CS alpha beta) motif, which was found in other defense proteins such as the antibacterial insect defensin A, short- and long-chain scorpion toxins, as well as in plant thionins and potent antifungal plant defensins, appears as remarkably persistent along evolution.     

Central nervous system infection due to Herpes simplex virus in AIDS

The secondary structure and global fold of the AVR9 elicitor protein of Cladosporium fulvum has been determined by 2D NMR and distance-geometry protocols. The protein consists of three anti-parallel strands forming a rigid region of beta-sheet. On the basis of the NMR-derived parameters and distance geometry calculations, it is evident that the AVR9 protein is structurally very homologuous to carboxy peptidase inhibitor (CPI) of which the X-ray structure is known. The AVR9 protein reveals the presence of a cystine knot, which consists of a ring formed by two disulfide bridges and the interconnecting backbone through which the third disulfide bridge penetrates. This structural motif is found in several small proteins such as proteinase inhibitors, ion channel blockers and growth factors. The implications of the structural relationship between AVR9 and other biologically active proteins are discussed.     

Solution structure of reduced plastocyanin from the blue-green alga Anabaena variabilis

The three-dimensional solution structure of plastocyanin from Anabaena variabilis (A.v.PCu) has been determined by nuclear magnetic resonance spectroscopy. Sixty structures were calculated by distance geometry from 1141 distance restraints and 46 dihedral angle restraints. The distance geometry structures were optimized by simulated annealing and restrained energy minimization. The average rms deviation from the mean structure for the 20 structures with the lowest total energy is 1.25 A for the backbone atoms and 1.75 A for all heavy atoms. Overall, the global tertiary fold of A.v.PCu resembles those of other plastocyanins which have been structurally characterized by X-ray diffraction and NMR methods. This holds even though A.v.PCu is longer than any other known plastocyanins, contains far less invariant amino acid residues, and has an overall charge that differs considerably from those of other plastocyanins (+1 vs -9 +/- 1 at pH > or = 7). The most striking feature of the A.v. PCu structure is the absence of the beta-turn, formed at the remote site by residues (58)-(61) in most higher plant plastocyanins. The displacement caused by the absence of this turn is compensated for by an extension of the small helix [from Ala53(51) to Ser60(58) in A.v.PCu] found in other plastocyanins. Moreover, the extra residues of A.v.PCu from Pro77 to Asp79 form an appended loop. These two features allow A.v.PCu to retain almost the same global fold as observed in other plastocyanins. From a comparison with the structures of other plastocyanins it is concluded that the lack of negatively charged residues at the remote site, rather than the specific structure of A.v.PCu, is the main reason for the failure of the remote site of this plastocyanin to function as a significant electron transfer site.     

Solution structure of human neuropeptide Y

The three-dimensional structure of synthetic human neuropeptide Y in aqueous solution at pH 3.2 and 37 degrees C was determined from two-dimensional 1H NMR data recorded at 600 MHz. A restraint set consisting of 440 interproton distance restraints inferred from NOEs and 11 backbone and 4 side-chain dihedral angle restraints derived from spin-spin coupling constants was used as input for distance geometry calculations on DIANA and simulated annealing and restrained energy minimization in X-PLOR. The final set of 26 structures is well defined in the region of residues 11-36, with a mean pairwise rmsd of 0.51 A for the backbone heavy atoms (N, C alpha and C) and 1.34 A for all heavy atoms. Residues 13-36 form an amphipathic alpha-helix. The N-terminal 10 residues are poorly defined relative to the helical region, although some elements of local structure are apparent. At least one of the three prolines in the N-terminal region co-exists in both cis and trans conformations. An additional set of 24 distances was interpreted as intermolecular distances within a dimer. A combination of distance geometry and restrained simulated annealing yielded a model of the dimer having antiparallel packing of two helical units, whose hydrophobic faces form a well-defined core. Sedimentation equilibrium experiments confirm the observation that neuropeptide Y associates to form dimers and higher aggregates under the conditions of the NMR experiments. Our results therefore support the structural features reported for porcine neuropeptide Y [Cowley, D.J. et al. (1992) Eur. J. Biochem., 205, 1099-1106] rather than the 'aPP' fold described previously for human neuropeptide Y [Darbon, H. et al. (1992) Eur. J. Biochem., 209, 765-771].     

Solution structure of P01, a natural scorpion peptide structurally analogous to scorpion toxins specific for apamin-sensitive potassium channel

The venom of the North African scorpion Androctonus mauretanicus mauretanicus possesses numerous highly active neurotoxins that specifically bind to various ion channels. One of these, P05, has been found to bind specifically to calcium-activated potassium channels and also to compete with apamin, a toxin extracted from bee venom. Besides the highly potent ones, several of these peptides (including that of P01) have been purified and been found to possess only a very weak, although significant, activity in competition with apamin. The amino acid sequence of P01 shows that it is shorter than P05 by two residues. This deletion occurs within an alpha-helix stretch (residues 5-12). This alpha-helix has been shown to be involved in the interaction of P05 with its receptor via two arginine residues. These two arginines are absent in the P01 sequence. Furthermore, a proline residue in position 7 of the P01 sequence may act as an alpha-helix breaker. We have determined the solution structure of P01 by conventional two-dimensional 1H nuclear magnetic resonance and show that 1) the proline residue does not disturb the alpha-helix running from residues 5 to 12; 2) the two arginines are topologically replaced by two acidic residues, which explains the drop in activity; 3) the residual binding activity may be due to the histidine residue in position 9; and 4) the overall secondary structure is conserved, i.e., an alpha-helix running from residues 5 to 12, two antiparallel stretches of beta-sheet (residues 15-20 and 23-27) connected by a type I' beta-turn, and three disulfide bridges connecting the alpha-helix to the beta-sheet.     

Solution structure of eotaxin, a chemokine that selectively recruits eosinophils in allergic inflammation

The solution structure of the CCR3-specific chemokine, eotaxin, has been determined by NMR spectroscopy. The quaternary structure of eotaxin was investigated by ultracentrifugation and NMR, and it was found to be in equilibrium between monomer and dimer under a wide range of conditions. At pH 相似文献   

Solution structure of the superactive monomeric des-[Phe(B25)] human insulin mutant: elucidation of the structural basis for the monomerization of des-[Phe(B25)] insulin and the dimerization of native insulin

The three-dimensional solution structure of des-[Phe(B25)] human insulin has been determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Thirty-five structures were calculated by distance geometry from 581 nuclear Overhauser enhancement-derived distance constraints, ten phi torsional angle restraints, the restraints from 16 helical hydrogen bonds, and three disulfide bridges. The distance geometry structures were optimized using simulated annealing and restrained energy minimization. The average root-mean-square (r.m.s.) deviation for the best 20 refined structures is 1.07 angstroms for the backbone and 1.92 angstroms for all atoms if the less well-defined N and C-terminal residues are excluded. The helical regions are more well defined, with r.m.s. deviations of 0.64 angstroms for the backbone and 1.51 angstroms for all atoms. It is found that the des-[Phe(B25)] insulin is a monomer under the applied conditions (4.6 to 4.7 mM, pH 3.0, 310 K), that the overall secondary and tertiary structures of the monomers in the 2Zn crystal hexamer of native insulin are preserved, and that the conformation-averaged NMR solution structure is close to the structure of molecule 1 in the hexamer. The structure reveals that the lost ability of des-[Phe(B25)] insulin to self-associate is caused by a conformational change of the C-terminal region of the B-chain, which results in an intra-molecular hydrophobic interaction between Pro(B28) and the hydrophobic region Leu(B11)-Leu(B15) of the B-chain alpha-helix. This interaction interferes with the inter-molecular hydrophobic interactions responsible for the dimerization of native insulin, depriving the mutant of the ability to dimerize. Further, the structure displays a series of features that may explain the high potency of the mutant on the basis of the current model for the insulin-receptor interaction. These features are: a change in conformation of the C-terminal region of the B-chain, the absence of strong hydrogen bonds between this region and the rest of the molecule, and a relatively easy accessibility to the Val(A3) residue.     

Three-dimensional molecular modeling of bovine caseins: a refined, energy-minimized kappa-casein structure

A refined three-dimensional molecular model of kappa-casein has been produced using energy minimization techniques and a Kollman force field on a previously reported predicted three-dimensional structure. This initial model was constructed via molecular modeling techniques from sequence-based secondary structural prediction algorithms. Both the initial and refined structures agreed with global secondary structure analysis from vibration spectroscopy. The refined structure contained many of the features of the initial model, including two sets of antiparallel beta-sheet structures containing predominantly hydrophobic side chains, which could form interaction sites with alpha s1-casein. Two types of energy-minimized dimer and tetramer models are presented: 1) using Cys as potential intermolecular disulfide binding sites and 2) using the two sheets as possible hydrophobic self-association sites, without Cys interactions. All structures yielded good stabilization energies and are in agreement with chemical, biochemical, and physical chemical results obtained for kappa-casein.     

Structure of the insect cytokine peptide plasmatocyte-spreading peptide 1 from Pseudoplusia includens

The structure of the recently identified plasmatocyte spreading peptide from the moth Pseudoplusia includens (PSP1) has been determined by NMR spectroscopy. This novel insect cytokine consists of 23 amino acid residues and a single disulfide bond. Torsion angle dynamics calculations utilizing a total of 337 distance constraints yielded an ensemble of 30 structures with an average backbone root mean square deviation for residues 7-22 of 0.18 A from the mean structure. The structure consists of a disordered N-terminal region and a well defined core that is stabilized by numerous hydrophobic interactions and a short beta-hairpin. Structural comparisons confirm that PSP1 adopts an epidermal growth factor (EGF)-like fold with close similarity to the C-terminal subdomain of EGF-like module 5 of human thrombomodulin. The combination of the three-dimensional structure of PSP1 and the extensive literature on EGF-receptor interactions should accelerate the process of identifying the specific residues responsible for receptor binding activity of this family of immunoregulatory peptides.     

Solution structure by NMR of circulin A: a macrocyclic knotted peptide having anti-HIV activity

The three-dimensional solution structure of circulin A, a 30 residue polypeptide from the African plant Chassalia parvifolia, has been determined using two-dimensional 1H-NMR spectroscopy. Circulin A was originally identified based upon its inhibition of the cytopathic effects and replication of the human immunodeficiency virus. Structural restraints consisting of 369 interproton distances inferred from nuclear Overhauser effects, and 21 backbone dihedral and nine chi1 angle restraints from spin-spin coupling constants were used as input for simulated annealing calculations and energy minimisation in the program X-PLOR. The final set of 12 structures had mean pairwise rms differences over the whole molecule of 0.91 A for the backbone atom, and 1.68 A for all heavy atoms. For the well-defined region encompassing residues 2-12 and 18-27, the corresponding values were 0.71 and 1.66 A, respectively. Circulin A adopts a compact structure consisting of beta-turns and a distorted segment of triple-stranded beta-sheet. Fluorescence spectroscopy provided additional evidence for a solvent-exposed Trp residue. The molecule is stabilised by three disulfide bonds, two of which form an embedded loop completed by the backbone fragments connecting the cysteine residues. A third disulfide bond threads through the centre of this loop to form a "cystine-knot" motif. This motif is present in a range of other biologically active proteins, including omega-contoxin GVIA and Cucurbita maxima trypsin inhibitor. Circulin A belongs to a novel class of macrocyclic peptides which have been isolated from plants in the Rubiaceae family. The global fold of circulin A is similar to kalata B1, the only member of this class for which a structure has previously been determined.     

Three-dimensional structure of leucocin A in trifluoroethanol and dodecylphosphocholine micelles: spatial location of residues critical for biological activity in type IIa bacteriocins from lactic acid bacteria

The first three-dimensional structure of a type IIa bacteriocin from lactic acid bacteria is reported. Complete 1H resonance assignments of leucocin A, a 37 amino acid antimicrobial peptide isolated from the lactic acid bacterium Leuconostoc gelidum UAL187, were determined in 90% trifluoroethanol (TFE)-water and in aqueous dodecylphosphocholine (DPC) micelles (1:40 ratio of leucocin A:DPC) using two-dimensional NMR techniques (e.g., DQF-COSY, TOCSY, NOESY). Circular dichroism spectra, NMR chemical shift indices, amide hydrogen exchange rates, and long-range nuclear Overhauser effects indicate that leucocin A adopts a reasonably well defined structure in both TFE and DPC micelle environments but exists as a random coil in water or aqueous DMSO. Distance geometry and simulated annealing calculations were employed to generate structures for leucocin A in both lipophilic media. While some differences were noted between the structures calculated for the two different solvent systems, in both, the region encompassing residues 17-31 assumes an essentially identical amphiphilic alpha-helix conformation. A three-strand antiparallel beta-sheet domain (residues 2-16), anchored by the disulfide bridge, is also observed in both media. In TFE, these two regions have a more defined relationship relative to each other, while, in DPC micelles, the C-terminus is folded back onto the alpha-helix. The implications of these structural features with regard to the antimicrobial mechanism of action and target recognition are discussed.     

Graded compression ultrasonography in the assessment of the "tough decision" acute abdomen in childhood

The three-dimensional structure of omega-conotoxin MVIID has been determined in aqueous solution by two-dimensional 1H NMR techniques. A total of 267 relevant upper-bound distance restraints were used to obtain a family of convergent structures using molecular dynamics methods. A standard simulated annealing protocol using the XPLOR program included in ARIA provided a total of 18 final structures. The averaged RMSD between these structures and the mean atomic coordinates was 0.8 +/- 0.3 A for the backbone atoms. The highest mobility was observed in the segments between residues 10 to 13, comprising Tyr 13, one of the residues shown to be important for binding of omega-conotoxin GVIA and MVIIA to N-type calcium channels. The three-dimensional structure is stabilised by the three disulfide bonds and includes a short antiparallel beta-strand between residues 5-8, 23-25 and 19-21. The folding for this non-N-type calcium channel blocker is similar to that previously calculated for omega-conotoxins GVIA, MVIIA and MVIIC. This suggests the disulfide bond pattern fixes the structure. The reported three-dimensional information can be used to advantage in order to highlight the structural parameters involved in discrimination among calcium channel subtypes.     

Nuclear magnetic resonance solution structure of the plasminogen-activator protein staphylokinase

Staphylokinase, a 15.5 kDa protein from Staphylococcus aureus, is a plasminogen activator which is currently undergoing clinical trials for the therapy of myocardial infarction and peripheral thrombosis. The three-dimensional (3D) NMR solution structure has been determined by multidimensional heteronuclear NMR spectroscopy on uniformly 15N- and 15N,13C-labeled samples of staphylokinase. Structural constraints were obtained from 82 3JHNH alpha as well as 22 3JNH beta scalar coupling constants and 2345 NOE cross-peaks, derived from 15N-edited and 13C-edited 3D NOE spectra. NOE cross-peak assignments were confirmed by analysis of ?15N,13C?-edited and ?13C,13C?-edited 4D NOE spectra. The structure is presented as a family of 20 conformers which show an average rmsd of 1.02 +/- 0.15 A from the mean structure for the backbone atoms. The tertiary structure of staphylokinase shows a well-defined global structure consisting of a central 13-residue alpha-helix flanked by a two-stranded beta-sheet, both of which are located above a five-stranded beta-sheet. Two of the connecting loops exhibit a higher conformational heterogeneity. Overall, staphylokinase shows a strong asymmetry of hydrophilic and hydrophobic surfaces. The N-terminal sequence, including Lys10 which is the site of the initial proteolytic cleavage during activation of plasminogen, folds back onto the protein core, thereby shielding amino acids with functional importance in the plasminogen activation process. From a comparison of the structure with mutational studies, a binding region for plasminogen is proposed.     

Three-dimensional solution structure of beta cryptogein, a beta elicitin secreted by a phytopathogenic fungus Phytophthora cryptogea

Cryptogein belongs to a new family of 10-kDa proteins called elicitins. Elicitins are necrotic and signaling proteins secreted by Phytophthora spp. responsible for the incompatible reaction and systemic hypersensitive-like necroses of diverse plant species leading to resistance against fungal or bacterial plant pathogens. The solution structure of beta cryptogein from Phytophthora cryptogea fungus was determined by using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. A set of 18 structures was calculated using 1360 NOE-derived distance restraints and 40 dihedral angle restraints obtained from 3JHNH alpha couplings. The RMS deviation from the mean structure is 0.87 +/- 0.14 A for backbone atoms and 1.34 +/- 0.14 A for all the non-hydrogen atoms of residues 2 to 98. The structure of beta cryptogein reveals a novel protein fold, with five helices and a double-stranded beta-sheet facing an omega-loop. One edge of the beta-sheet and the adjacent face of the omega-loop form a hydrophobic cavity. This cavity made of highly conserved residues represents a plausible binding site. Residue 13, which has been identified from directed mutagenesis and natural sequence comparison studies as a key amino acid involved in the differential control of necrosis, is surface exposed and could contribute to the binding to a ligand or a receptor. The solution structure is close to the X-ray structure, with slight differences lightly due to the crystal packing.     

Solution structure of the transforming growth factor beta-binding protein-like module, a domain associated with matrix fibrils

Here we describe the high resolution nuclear magnetic resonance (NMR) structure of a transforming growth factor beta (TGF-beta)-binding protein-like (TB) domain, which comes from human fibrillin-1, the protein defective in the Marfan syndrome (MFS). This domain is found in fibrillins and latent TGF-beta-binding proteins (LTBPs) which are localized to fibrillar structures in the extracellular matrix. The TB domain manifests a novel fold which is globular and comprises six antiparallel beta-strands and two alpha-helices. An unusual cysteine triplet conserved in the sequences of TB domains is localized to the hydrophobic core, at the C-terminus of an alpha-helix. The structure is stabilized by four disulfide bonds which pair in a 1-3, 2-6, 4-7, 5-8 pattern, two of which are solvent exposed. Analyses of MFS-causing mutations and the fibrillin-1 cell-binding RGD site provide the first clues to the surface specificity of TB domain interactions. Modelling of a homologous TB domain from LTBP-1 (residues 1018-1080) suggests that hydrophobic contacts may play a role in its interaction with the TGF-beta1 latency-associated peptide.     

The crystal structure of zinc-containing ferredoxin from the thermoacidophilic archaeon Sulfolobus sp. strain 7

The crystal structure of ferredoxin from the thermoacidophilic archaeon Sulfolobus sp. strain 7 was determined by multiple isomorphous replacement supplemented with anomalous scattering effects of iron atoms in the Fe-S clusters, and refined at 2.0 A resolution to a crystallographic R value of 0.173. The structural model contains a polypeptide chain of 103 amino acid residues, 2 [3Fe-4S] clusters, and 31 water molecules; in this model, the cluster corresponding to cluster II in bacterial dicluster ferredoxins loses the fourth iron atom although it may originally be a [4Fe-4S] cluster. The structure of the archaeal ferredoxin consists of two parts: the core fold part (residues 37-103) and the N-terminal extension part (residues 1-36). The "core fold" part has an overall main-chain folding common to bacterial dicluster ferredoxins, containing two clusters as the active center, two alpha-helices near the clusters, and two sheets of two-stranded antiparallel beta-sheet (the terminal and central beta-sheets). The "N-terminal extension" part is mainly formed by a one-turn alpha-helix and a three-stranded antiparallel beta-sheet. The beta-sheet in the N-terminal extension is hydrogen-bonded with the terminal beta-sheet in the core fold to form a larger beta-sheet. The distinct structural feature of this archaeal ferredoxin lies in the zinc-binding center where the zinc ion is tetrahedrally ligated by four amino acid residues (His 16, His 19, and His 34 from the N-terminal extension, and Asp 76 from the core fold). The zinc ion in the zinc-binding center is located at the interface between the core fold and the N-terminal extension, and connects the beta-sheet in the N-terminal extension and the central beta-sheet in the core fold through the zinc ligation. Thus, the zinc ion plays an important role in stabilizing the structure of the present archaeal ferredoxin by connecting the N-terminal extension and the core fold, which may be common to thermoacidophilic archaeal ferredoxins.