Solution structure of PAFP-S: a new knottin-type antifungal peptide from the seeds of Phytolacca americana

The three-dimensional solution structure of PAFP-S, an antifungal peptide extracted from the seeds of Phytolacca americana, was determined using 1H NMR spectroscopy. This cationic peptide contains 38 amino acid residues. Its structure was determined from 302 distance restraints and 36 dihedral restraints derived from NOEs and coupling constants. The peptide has six cysteines involved in three disulfide bonds. The previously unassigned parings have now been determined from NMR data. The solution structure of PAFP-S is presented as a set of 20 structures using ab initio dynamic simulated annealing, with an average RMS deviation of 1.68 A for the backbone heavy atoms and 2.19 A for all heavy atoms, respectively. For the well-defined triple-stranded beta-sheet involving residues 8-10, 23-27, and 32-36, the corresponding values were 0.39 and 1.25 A. The global fold involves a cystine-knotted three-stranded antiparallel beta-sheet (residues 8-10, 23-27, 32-36), a flexible loop (residues 14-19), and four beta-reverse turns (residues 4-8, 11-14, 19-22, 28-32). This structure features all the characteristics of the knottin fold. It is the first structural model of an antifungal peptide that adopts a knottin-type structure. PAFP-S has an extended hydrophobic surface comprised of residues Tyr23, Phe25, Ile27, Tyr32, and Val34. The side chains of these residues are well-defined in the NMR structure. Several hydrophilic and positively charged residues (Arg9, Arg38, and Lys36) surround the hydrophobic surface, giving PAFP-S an amphiphilic character which would be the main structural basis of its biological function.     

New binding site on common molecular scaffold provides HERG channel specificity of scorpion toxin BeKm-1

The scorpion toxin BeKm-1 is unique among a variety of known short scorpion toxins affecting potassium channels in its selective action on ether-a-go-go-related gene (ERG)-type channels. BeKm-1 shares the common molecular scaffold with other short scorpion toxins. The toxin spatial structure resolved by NMR consists of a short alpha-helix and a triple-stranded antiparallel beta-sheet. By toxin mutagenesis study we identified the residues that are important for the binding of BeKm-1 to the human ERG K+ (HERG) channel. The most critical residues (Tyr-11, Lys-18, Arg-20, Lys-23) are located in the alpha-helix and following loop whereas the "traditional" functional site of other short scorpion toxins is formed by residues from the beta-sheet. Thus the unique location of the binding site of BeKm-1 provides its specificity toward the HERG channel.     

The three-dimensional structure of the first EGF-like module of human factor IX: comparison with EGF and TGF-alpha.

The three-dimensional structure of the first epidermal growth factor (EGF)-like module from human factor IX has been determined in solution using two-dimensional nuclear magnetic resonance (in the absence of calcium and at pH 4.5). The structure was found to resemble closely that of EGF and the homologous transforming growth factor-alpha (TGF-alpha). Residues 60-65 form an antiparallel beta-sheet with residues 68-73. In the C-terminal subdomain a type II beta-turn is found between residues 74 and 77 and a five-residue turn is found between residues 79 and 83. Glu 78 and Leu 84 pair in an antiparallel beta-sheet conformation. In the N-terminal region a loop is found between residues 50 and 55 such that the side chains of both are positioned above the face of the beta-sheet. Residues 56-60 form a turn that leads into the first strand of the beta-sheet. Whereas the global fold closely resembles that of EGF, the N-terminal residues of the module (46-49) do not form a beta-strand but are ill-defined in the structure, probably due to the local flexibility of this region. The structure is discussed with reference to recent site-directed mutagenesis data, which have identified certain conserved residues as ligands for calcium.     

1H NMR assignment and secondary structure of the Ca2(+)-free form of the amino-terminal epidermal growth factor like domain in coagulation factor X

Blood coagulation factor X is composed of discrete domains, two of which are homologous to the epidermal growth factor (EGF). The N-terminal EGF like domain in factor X (fX-EGFN), residues 45-86 of the intact protein, contains a beta-hydroxylated aspartic acid and has one Ca2(+)-binding site. Using 2D NMR techniques, we have made a full assignment of the 500-MHz 1H NMR spectrum of Ca2(+)-free fX-EGFN. On the basis of this assignment and complementary NOESY experiments, we have also determined the secondary structure of Ca2(+)-free fX-EGFN in water solution. Residues 45-49 are comparatively mobile, whereas residues 50-56 are constrained by two disulfide bonds to one side of an antiparallel beta-sheet involving residues 59-64 and 67-72. Another antiparallel beta-sheet involves residues 76-77 and 83-84. A small, parallel beta-sheet connects residues 80-81 and 55-56 and thereby orients the two antiparallel beta-sheets relative to each other. Four beta-turns are identified, involving residues 50-53, 56-59, 64-67, and 73-76. Residues 78-82 adopt an extended bend structure. On the basis of secondary structure and the location of the three disulfide bonds, we find that Asp 46, Asp 48, and Hya 63 are sufficiently close to each other to form a Ca2(+)-binding site. However, the amino terminus of the Ca2(+)-free form of fX-EGFN is not part of a triple-stranded beta-sheet as in other EGF like peptides. Differences and similarities between fX-EFGN and murine EGF with respect to secondary structure and conformational shifts are discussed.     

1H NMR assignment and secondary structural elements of human transforming growth factor alpha

The 1H NMR spectrum of human transforming growth factor alpha (hTGF-alpha) has been completely assigned, and secondary structural elements have been identified as a preliminary step in determining the structure of this protein by distance geometry methods. Many of these structural elements closely correspond to those previously found in a truncated human EGF [Cooke et al. (1987) Nature (London) 327, 339-341] and murine EGF [Montelione et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5226-5230]. These include the presence of an antiparallel beta-sheet between residues G19 and C34 with a type I beta-turn at V25-D28, a type II beta-turn at H35-Y38, and another short beta-sheet between residues Y38-V39 and H45-A46.     

Structure and activity of the insect cytokine growth-blocking peptide. Essential regions for mitogenic and hemocyte-stimulating activities are separate.

Growth-blocking peptide (GBP) is a 25-amino acid insect cytokine found in Lepidopteran insects that possesses diverse biological activities such as larval growth regulation, cell proliferation, and stimulation of immune cells (plasmatocytes). The tertiary structure of GBP consists of a structured core that contains a disulfide bridge and a short antiparallel beta-sheet (Tyr(11)-Arg(13) and Cys(19)-Pro(21)) and flexible N and C termini (Glu(1)-Gly(6) and Phe(23)-Gln(25)). In this study, deletion and point mutation analogs of GBP were synthesized to investigate the relationship between the structure of GBP and its mitogenic and plasmatocyte spreading activity. The results indicated that deletion of the N-terminal residue, Glu(1), eliminated all plasmatocyte spreading activity but did not reduce mitogenic activity. In contrast, deletion of Phe(23) along with the remainder of the C terminus destroyed all mitogenic activity but only slightly reduced plasmatocyte spreading activity. Therefore, the minimal structure of GBP containing mitogenic activity is 2-23 GBP, whereas that with plasmatocyte spreading activity is 1-22 GBP. NMR analysis indicated that these N- and C-terminal deletion mutants retained a similar core structure to wild-type GBP. Replacement of Asp(16) with either a Glu, Leu, or Asn residue similarly did not alter the core structure of GBP. However, these mutants had no mitogenic activity, although they retained about 50% of their plasmatocyte spreading activity. We conclude that specific residues in the unstructured and structured domains of GBP differentially affect the biological activities of GBP, which suggests the possibility that multifunctional properties of this peptide may be mediated by different forms of a GBP receptor.     

Determination of the three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata: a study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing

The three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata has been determined on the basis of 489 interproton and 24 hydrogen-bonding distance restraints supplemented by 23 phi backbone and 21 chi 1 side-chain torsion angle restraints derived from nuclear magnetic resonance (NMR) measurements. A total of 42 structures is calculated by a hybrid metric matrix distance geometry-dynamical simulated annealing approach. Both the backbone and side-chain atom positions are well defined. The average atomic rms difference between the 42 individual SA structures and the mean structure obtained by averaging their coordinates is 0.67 +/- 0.12 A for the backbone atoms and 0.90 +/- 0.17 A for all atoms. The core of the protein is formed by a triple-stranded antiparallel beta-sheet composed of residues 14-16 (strand 1), 30-34 (strand 2), and 37-41 (strand 3) with an additional mini-antiparallel beta-sheet at the N-terminus (residues 6-9). The first and second strands of the triple-stranded antiparallel beta-sheet are connected by a long exposed loop (residues 17-30). A number of side-chain interactions are discussed in light of the structure.     

Sequence-specific 1H-n.m.r. assignments and peptide backbone conformation in rat epidermal growth factor.

The solution conformation of rat epidermal growth factor (EGF) has been investigated by proton n.m.r. techniques. Two-dimensional proton n.m.r. experiments have allowed sequential resonance assignments to be made for most protons. On the basis of these assignments, two regions of anti-parallel beta-sheet structure have been derived from the n.m.r. data. A beta-sheet segment running from about V19 to V23 (capital letters refer to amino acids in the single-letter notation) is folded onto a beta-sheet segment running from R28 to N32 and joined by a chain reversal from E24 to D27. A second region involves a beta-turn from V34 to Y37, which starts a short beta-sheet up to G39, followed by a chain reversal up to Q43, which leads to folding of the C-terminal beta-sheet segment, i.e. H44-R45, running antiparallel to the short Y37 beta-sheet segment. The N-terminal segment up to G18 exists in a multiple bend conformation and is folded on to the V29-V23/R28-N32 beta-sheet such that Y10, Y13, Y22 and Y29 are proximal to each other. Structural comparison of rat, murine and human EGFs indicates a number of highly conserved structural features common to at least these species of EGF.     

Solution structure of BmP01 from the venom of scorpion Buthus martensii Karsch

From the venom of scorpion Buthus martensii Karsch,a short peptide (BmP01, 29 amino acid residues) was isolated and characterized as previously reported (Lebren, R. R., et al. (1997) Eur. J. Biochem. 245, 457-464). It was shown to reduce 33% outward K(+) channel (hippocampal neurons) currents at 10 microM. The solution structure of BmP01 was determined by 2D (1)H NMR spectroscopy. The NOEs, coupling constants, and H-D exchange obtained from NMR spectroscopy were used in structural calculations. The conformation of BmP01 is composed of a short alpha-helix (Cys 3-Thr 12) and a two-stranded antiparallel beta-sheet (Ala 15-Asp 20 and Lys 23-Pro 28). There are three disulfide bridges (Cys 3-Cys 19, Cys 6-Cys 24 and Cys 10-Cys 26) connecting the alpha-helix and beta-sheet. Asp 20 to Lys 23 form a type II turn linking the two strands. Structural and electrostatic potential comparison between BmP01 and its analogues are also presented.     

Properties of an aldose reductase from pig lens. Comparative studies of an aldehyde reductase from pig lens

THe characteristic feature of the crystal structure of erabutoxin b, a short neurotoxin from Laticauda semifasciata, and alpha-cobratoxin, a long neurotoxin from Naja naja siamensis, is the presence of a triple-stranded antiparallel pleated beta-sheet structure formed by the central and the third peptide loops. In the present study, we have studied the assignment of slowly exchangeable amide protons of Laticauda semifasciata III from L. semifasciata, using nuclear Overhauser effects (NOE) and spin-decoupling methods. The results show that nearly all of the slowly exchangeable amide protons are to be assigned to the back-bone amide protons, involved in the triple-stranded antiparallel pleated beta-sheet structure, indicating that this sheet is stable in 2H2O solution. In contrast, the amide protons in short neurotoxins are readily exchangeable under the same experimental condition, suggesting that long neurotoxins have a more rigid sheet structure than short ones. This rigidity may come from the hydrophobic and hydrogen bond interaction between the central loop and the tail, which is not present in short neurotoxins. Since the functionally important residues are located on this beta-sheet, the different kinetic properties of the neurotoxins are well correlated with the difference in the rigidity of the beta-sheet.     

Sequential resonance assignment and secondary structure determination of the Ascaris trypsin inhibitor, a member of a novel class of proteinase inhibitors

The solution conformation of the Ascaris trypsin inhibitor, a member of a novel class of proteinase inhibitors, has been investigated by nuclear magnetic resonance spectroscopy. Complete sequence-specific assignments of the 1H NMR spectrum have been obtained by using a number of two-dimensional techniques for identifying through-bond and through-space (less than 5-A) connectivities. Elements of regular secondary structure have been identified on the basis of a qualitative interpretation of the nuclear Overhauser enhancement, coupling constant, and amide exchange data. These are two beta-sheet regions. One double-stranded antiparallel beta-sheet comprises residues 11-14 (strand 1) and 37-39 (strand 2). The other triple-stranded sheet is formed by two antiparallel strands comprising residues 45-49 (strand 4) and 53-57 (strand 5) connected by a turn (residues 50-52), and a small strand consisting of residues 20-22 (strand 3) that is parallel to strand 4.     

The NMR structure of the activation domain isolated from porcine procarboxypeptidase B.

The three-dimensional structure of the activation domain isolated from porcine pancreatic procarboxypeptidase B was determined using 1H NMR spectroscopy. A group of 20 conformers is used to describe the solution structure of this 81 residue polypeptide chain, which has a well-defined backbone fold from residues 11-76 with an average root mean square distance for the backbone atoms of 1.0 +/- 0.1 A relative to the mean of the 20 conformers. The molecular architecture contains a four-stranded beta-sheet with the polypeptide segments 11-17, 36-39, 50-56 and 75-76, two well defined alpha-helices from residues 20-30 and 60-70, and a 3(10) helix from residues 43-46. The three helices are oriented almost exactly antiparallel to each other, are all on the same side of the beta-sheet, and the helix axes from an angle of approximately 45 degrees relative to the direction of the beta-strands. Three segments linking beta-strands and helical secondary structures, with residues 32-35, 39-43 and 56-61, are significantly less well ordered than the rest of the molecule. In the three-dimensional structure two of these loops (residues 32-35 and 56-61) are located close to each other near the protein surface, forming a continuous region of increased mobility, and the third disordered loop is separated from this region only by the peripheral beta-strand 36-39 and precedes the short 3(10) helix.     

Solution structure of BmKK4, the first member of subfamily alpha-KTx 17 of scorpion toxins

BmKK4 is a 30 amino acid peptide purified from the venom of the Chinese scorpion Buthus martensi Karsch. It has been classified as the first member of scorpion toxin subfamily alpha-KTx 17. The 3D structure of BmKK4 in solution has been determined by 2D NMR spectroscopy. This toxin adopts a common alpha/beta-motif, but shows a distinctive local conformation. The most novel feature is that the regular arrangements of the side chains of the residues involved in the beta-sheet of BmKK4 are distorted by a classic beta-bulge structure, which involves two residues (Asp18 and Arg19) in the first strand opposite a single residue (Tyr26) in the second strand. The bulge produces two main changes in the structure of the antiparallel beta-sheet: (1) It disrupts the normal alteration of the side chain direction; the side chain of Asp18 turns over to form a salt bridge with that of Arg19. (2) It accentuates the twist of the sheet, and alters the direction of the antiparallel beta-sheet. The unusual structural feature of the toxin is attributed to the shorter peptide segment (Leu15-Arg19) between the third and fourth Cys residues and two unique residues (Asp18 and Arg19) at the position preceding the fourth Cys. In addition, the lower affinity of the peptide for the Kv channel is correlated to the structural features: residue Arg19 instead of a Lys residue at the critical position for binding and the salt bridge formed between residues Arg19 and Asp18.     

Model of Alzheimer's disease amyloid-beta peptide based on a RNA binding protein

Although Alzheimer's Abeta peptide has been shown to form beta-sheet structure, a high-resolution molecular structure is still unavailable to date. A search for a sequence neighbor using Abeta(10-42) as the query in the Protein Data-Bank (PDB) revealed that an RNA binding protein, AF-Sm1 from Archaeoglobus fulgidus (PDB entry: 1i4k chain Z), shared 36% identical residues. Using AF-Sm1 as a template, we built a molecular model of Abeta(10-42) by applying comparative modeling methods. The model of Abeta(10-42) contains an antiparallel beta-sheet formed by residues 16-23 and 32-41. Hydrophobic surface constituted by residues 17-20 (LVFF) separates distinctly charged regions. Residues that interact with RNA in the AF-Sm1 crystal structure were found to be conserved in Abeta. Using a native gel we demonstrate for the first time that RNA can interact with Abeta and selectively retard the formation of fibrils or higher-order oligomers. We hypothesize that in a similar fashion to AF-Sm1, RNA interacts with Abeta in the beta-hairpin (beta-turn-beta) structure and prevents fibril formation.     

Circular dichroism studies of acetylcholinesterase conformation. Comparison of the 11 S and 5.6 S species and the differences induced by inhibitory ligands

Circular dichroism studies were carried out in the vacuum ultraviolet region for 11 S and 5.6 S species of acetylcholinesterase from Torpedo. As the 5.6 S acetylcholinesterase forms larger oligomers in the absence of detergent, the CD spectrum was measured both with and without detergent. Secondary structure analysis of the CD spectrum for 11 S acetylcholinesterase shows 33% alpha-helix, 23% beta-sheet (14% antiparallel and 9% parallel), 17% turns and 26% other structure. Binding of edrophonium to the active site of 11 S acetylcholinesterase increases alpha-helix, while binding of propidium to the peripheral site increases beta-sheet. The beta-sheet content is slightly higher for 5.6 S than 11 S acetylcholinesterase in water. When the detergent is added to 5.6 S acetylcholinesterase, the 190 nm and 220 nm bands become less intense, although the analyses of the two spectra are similar. No significant change is observed for the 5.6 S form in either solvent on binding ligands. The prediction of both parallel and antiparallel beta-sheet suggests that at least one domain in these multidomain proteins belongs to the alpha/beta tertiary structural type.     

Solution structure of Eucommia antifungal peptide: a novel structural model distinct with a five-disulfide motif

The three-dimensional structure in aqueous solution of Eucommia antifungal peptide 2 (EAFP2) from Eucommia ulmoides Oliv was determined using (1)H NMR spectroscopy. EAFP2 is a newly discovered 41-residue peptide distinct with a five-disulfide cross-linked motif. This peptide exhibits chitin-binding activity and inhibitory effects on the growth of cell wall chitin-containing fungi and chitin-free fungi. The structure was calculated by using torsion angle dynamic simulated annealing with a total of 614 distance restraints and 16 dihedral restraints derived from NOESY and DQF-COSY spectra, respectively. The five disulfide bonds were assigned from preliminary structures using a statistical analysis of intercystinyl distances. The solution structure of EAFP2 is presented as an ensemble of 20 conformers with a backbone RMS deviation of 0.65 (+/-0.13) A for the well-defined Cys3-Cys39 segment. The tertiary structure of EAFP2 represents the first five-disulfide cross-linked structural model of the plant antifungal peptide. EAFP2 adopts a compact global fold composed of a 3(10) helix (Cys3-Arg6), an alpha-helix (Gly26-Cys30), and a three-strand antiparallel beta-sheet (Cys16-Ser18, Tyr22-Gly24, and Arg36-Cys37). The tertiary structure of EAFP2 shows a chitin-binding domain (residues 11-30) with a hydrophobic face and a characteristic sector formed by the N-terminal 10 residues and the C-terminal segment cross-linked through the unique disulfide bond Cys7-Cys37, which brings all four positively charged residues (Arg6, Arg9, Arg36, and Arg40) onto a cationic face. On the basis of such a structural feature, the possible structural basis for the functional properties of EAFP2 is discussed.     

Two-dimensional NMR studies of the antimicrobial peptide NP-5

Nearly complete proton resonance assignment of the rabbit antimicrobial peptide NP-5 has been made from two-dimensional NMR data taken at a single temperature. The assignment procedure involved acquisition of phase-sensitive double-quantum-filtered correlation spectra, relayed coherence-transfer spectra, total correlation (homonuclear Hartmann-Hahn) spectra, double- and triple-quantum spectra, and nuclear Overhauser effect spectra. The combination of these complementary experiments simplified and accelerated resonance assignment of the peptide. Individual assignments were made at 20 degrees C for all amide and C alpha protons in the peptide, and for all nonlabile side-chain protons on 26 of the 33 amino acid residues in NP-5. Analysis of the proton-proton nuclear Overhauser effect connectivities, the slowly exchanging amide protons, and the proton chemical shifts in NP-5 indicates that the peptide has a stable, ordered structure in solution. These data also indicate that residues 19-29 in NP-5 are involved in an antiparallel beta-sheet that has a hairpin conformation.     

1H assignments and secondary structure determination of the soybean trypsin/chymotrypsin Bowman-Birk inhibitor

The 1H resonance assignments and secondary structure of the trypsin/chymotrypsin Bowman-Birk inhibitor from soybeans were determined by nuclear magnetic resonance spectroscopy (NMR) at 600 MHz in an 18% acetonitrile-d3/aqueous cosolvent. Resonances from 69 of 71 amino acids were assigned sequence specifically. Residues Q11-T15 form an antiparallel beta-sheet with residues Q21-S25 in the tryptic inhibitory domain and an analogous region of antiparallel sheet forms between residues S38-A42 and Q48-V52 in the chymotryptic inhibitory domain. The inhibitory sites of each fragment (K16-S17 for trypsin, L43-S44 for chymotrypsin) are each part of a type VI like turn at one end of their respective region of the antiparallel beta-sheet. These structural elements are compared to those found in other Bowman-Birk inhibitors.     

Sequence-specific 1H-NMR assignments and determination of the secondary structure in aqueous solution of the cardiotoxins CTXIIa and CTXIIb from Naja mossambica mossambica

Sequence-specific assignments of the 1H-nuclear magnetic resonance (NMR) spectra of the cardiotoxins CTXIIa and CTXIIb from Naja mossambica mossambica were obtained using two-dimensional NMR experiments at 500 MHz and the independently determined amino acid sequences. Assignments were obtained from data at 25 degrees C and 45 degrees C for all but one backbone proton of the 60 residues in each protein. Complete or partial assignments are also reported for the side-chain protons. These assignments supercede those published previously for the toxin preparation VII2 [Hosur, R. V., Wider, G. & Wüthrich K. (1983) Eur. J. Biochem. 130, 497-508]. The 1H/2H-exchange kinetics were measured in 2H2O at 20 degrees C for the amide protons and the N-terminal amino group. These and additional NMR data enabled the determination of the secondary structure in aqueous solution, which is virtually identical in CTXIIa and CTXIIb. Both proteins contain a short double-stranded antiparallel beta-sheet comprising the residues 2-4 and 11-13, and a triple-stranded antiparallel beta-sheet consisting of the residues 20-26, 35-39, and 49-55. The two peripheral strands of the triple-stranded beta-structure were found to be connected by a right-handed cross-over, and the locations of several tight turns were also identified.