Simultaneous determination of disulphide bridge topology and three-dimensional structure using ambiguous intersulphur distance restraints: Possibilities and limitations

Knowledge of the native disulphide bridge topology allows the introduction of conformational restraints between remote parts of the peptide chain. This information is therefore of great importance for the successful determination of the three-dimensional structure of cysteine-rich proteins by NMR spectroscopy. In this paper we investigate the limitations of using ambiguous intersulphur restraints [Nilges, M. (1995) J. Mol. Biol., 245, 645–660] associated with NMR experimental information to determine the native disulphide bridge pattern. Using these restraints in a simulated annealing protocol we have determined the correct topology of numerous examples, including a protein with seven disulphide bridges (phospholipase A₂) and a protein in which 25% of the total number of residues are cysteines (-conotoxin GIIIB). We have also characterised the behaviour of the method when only limited experimental data is available, and find that the proposed protocol permits disulphide bridge determination even with a small number of restraints (around 5 NOEs – including a long-range restraint – per residue). In addition, we have shown that under these conditions the use of a reduced penalty function allows the identification of misassigned NOE restraints. These results indicate that the use of ambiguous intersulphur distances with the proposed simulated annealing protocol is a general method for the determination of disulphide bridge topology, particularly interesting in the first steps of NMR study of cysteine-rich proteins. Comparison with previously proposed protocols indicates that the presented method is more reliable and the interpretation of results is straightforward.     

Disulphide bridges of bovine factor X.

Evidence is presented for the disulphide bridges in bovine Factor X. The protein was degraded by chemical and enzymic means, and all 12 disulphide bridges were isolated in separate peptides except for bridges nos. 6/7 in the light chain. All the disulphide bridges were found to be in positions corresponding to those found in other homologous domains. This report is the first verification of an epidermal-growth-factor-homologous domain having the same disulphide-bonding pattern as that found in mouse epidermal growth factor.     

Large-scale prediction of disulphide bridges using kernel methods, two-dimensional recursive neural networks, and weighted graph matching

The formation of disulphide bridges between cysteines plays an important role in protein folding, structure, function, and evolution. Here, we develop new methods for predicting disulphide bridges in proteins. We first build a large curated data set of proteins containing disulphide bridges to extract relevant statistics. We then use kernel methods to predict whether a given protein chain contains intrachain disulphide bridges or not, and recursive neural networks to predict the bonding probabilities of each pair of cysteines in the chain. These probabilities in turn lead to an accurate estimation of the total number of disulphide bridges and to a weighted graph matching problem that can be addressed efficiently to infer the global disulphide bridge connectivity pattern. This approach can be applied both in situations where the bonded state of each cysteine is known, or in ab initio mode where the state is unknown. Furthermore, it can easily cope with chains containing an arbitrary number of disulphide bridges, overcoming one of the major limitations of previous approaches. It can classify individual cysteine residues as bonded or nonbonded with 87% specificity and 89% sensitivity. The estimate for the total number of bridges in each chain is correct 71% of the times, and within one from the true value over 94% of the times. The prediction of the overall disulphide connectivity pattern is exact in about 51% of the chains. In addition to using profiles in the input to leverage evolutionary information, including true (but not predicted) secondary structure and solvent accessibility information yields small but noticeable improvements. Finally, once the system is trained, predictions can be computed rapidly on a proteomic or protein-engineering scale. The disulphide bridge prediction server (DIpro), software, and datasets are available through www.igb.uci.edu/servers/psss.html.     

Engineering new peptidic inhibitors from a natural chymotrypsin inhibitor.

Three model peptides of different sizes (17-24 amino acid residues) mimicking the chymotrypsin inhibitor SCGI (a peptide of 35 amino acid residues) isolated from Schistocerca gregaria were designed and prepared by convergent peptide synthesis. Selective formation of disulphide bridges in the closing step was achieved without selective protection of cysteine residues. The natural pattern of the two disulphide bridges was determined by 2D homonuclear 1H NMR techniques. All three model peptides were characterized by amino acid analysis. MS and CD spectra. Preliminary results revealed that the two smaller model peptides exhibit no Inhibitory activity, whereas the larger one shows limited inhibition of chymotrypsin.     

1H NMR structure of an antifungal γ-thionin protein SIα1: Similarity to scorpion toxins

The three-dimensional structure of the Sorghum bicolor seed protein γ-thionin SIα1 has been determined by 2D ¹H nuclear magnetic resonance (NMR) spectroscopy. The secondary structure of this 47-residue antifungal protein with four disulphide bridges consists of a three-stranded antiparallel sheet and one helix. The helix is tethered to the sheet by two disulphide bridges which link two successive turns of the helix to alternate residues i, i + 2 in one strand. Possible binding sites for antifungal activity are discussed. The same fold has been observed previously in several scorpion toxins. Proteins 32:334–349, 1998. © 1998 Wiley-Liss, Inc.     

Protein stability and plasticity of the hydrophobic cavity in wheat ns-LTP

Plant ns-LTPs display an original structure with four helices and a flexible C-terminus, maintained together by four disulphide bridges and delineating an elongated central hydrophobic cavity. In order to relate these structural features to the protein stability and plasticity, combined molecular mechanics and simulated annealing calculations were undertaken on a wheat ns-LTP "mutant" with Cys-Ala replacement and with the application of core inter-residue restraints up to 2 A, reducing the cross-section size of the hydrophobic cavity. Analysis of the energy-minimized structures shows that removal of the disulphide bridges results in structures with a lower total energy and a smaller cavity volume. A 1-ns MD simulation at 300K in water, underlines that, despite the absence of a well-packed hydrophobic core, the native structure is extremely stable at room temperature and the cavity is not hydrated. This confirms that the disulphide bridges are essential for the existence of the cavity, whereas its plasticity depends both on the hydrophobic chain lining the cavity and on the C-terminal flexibility. A high temperature (500K) MD simulation confirms the stability of the secondary structure elements and the flexibility of the loops and of the C-terminal segment. Two important structural transitions during this simulation are discussed and possible routes for the insertion and release of hydrophobic ligands are suggested.     

Assignment of the complete disulphide bridge pattern in the human recombinant follitropin beta-chain

The chemical assessment of the complete disulphide bridge pattern in the beta-chain of human recombinant follicotropin (betaFSH) was accomplished by integrating classical biochemical methodologies with mass spectrometric procedures. A proteolytic strategy consisting of a double digestion of native betaFSH using the broad-specificity protease subtilisin first, followed by trypsin, was employed. The resulting peptide mixture was directly analysed by FAB-MS, leading to the assignment of the first three disulphide bridges. The remaining S-S bridges were determined by HPLC fractionation of the proteolytic digest followed by ESMS analysis of the individual fractions. The pattern of cysteine couplings in betaFSH was determined as: Cys3-Cys5l, Cys17-Cys66, Cys20-Cys104, Cys28-Cys82, Cys32-Cys84 and Cys87-Cys94, confirming the arrangement inferred from the crystal structure of the homologous betaCG. A subset of the S-S bridge pattern comprising Cys3-Cys51, Cys28-Cys82 and Cys32-Cys84 constitutes a cysteine knot motif similar to that found in the growth factor superfamily.     

The primary structure of porcine pancreatic elastase: The N-terminus and disulphide bridges

1. The amino acid composition and N-terminal groups of purified elastase show that it is a single peptide chain of 234 residues. 2. The N-terminal sequence is Val-Val-Gly-Gly-Thr-Glu-. 3. The sequences around the four disulphide bridges were determined by using a ;diagonal' electrophoretic technique. 4. These four bridges are homologous with the four common to bovine trypsin and chymotrypsin. 5. Out of 83 residues of the elastase sequence so far determined, 43 are homologous with similar regions of trypsin and chymotrypsin. 6. The evolutionary ancestry of these enzymes is discussed.     

Amino acid sequences around the disulphide bridges and methionine residues of porcine pepsin

1. The amino acid sequences around three disulphide bridges and four methionine residues of porcine pepsin were studied by using diagonal electrophoresis methods. 2. Two of the three disulphide bridges were in small loops of five and six residues. The sequence around one of the two half-cystine residues of the third disulphide bridge had a large number of acidic residues. 3. The sequence of a tetrapeptide containing phosphoserine was also determined. 4. Four unique methionine-containing sequences were constructed. The information is sufficient for the determination of the overlaps in the cyanogen bromide fragments of pepsin. 5. The usefulness of diagonal methods in the study of protein structure, the relative positions of cystinyl and methionyl residues in porcine pepsin and the homology between pepsin and rennin are discussed.     

The disulphide bridges in a cellobiohydrolase and an endoglucanase from Trichoderma reesei.

The positions of the disulphide bridges of the 1,4-beta-glucan cellobiohydrolase (CBH I) of the fungus Trichoderma reesei have been investigated. The results can be summarized as follows. (1) The enzyme contains 12 disulphide bridges and no free cysteine residues. (2) The location of six disulphide bridges have been determined experimentally. (3) The bonding patterns of the two disulphide bridges in the C-terminal region is suggested on the basis of internal homology. (4) The remaining four disulphide bridges are put into two groups, each containing four half-cystine residues where two are adjacent. (5) A repeating bonding pattern is observed along the peptide chain and a non-local disulphide bond with an unusually long separation distance links the N-terminal and the C-terminal region. (6) The disulphide-bonded CNBr peptides of a 1,4-beta-glucan glucanohydrolase (endoglucanase II) from T. reesei have been isolated and a disulphide bonding pattern is suggested on the basis of the sequence homology between the two enzymes.     

Deriving the generic structure of the fibronectin type II domain from the prothrombin Kringle 1 crystal structure.

The proposed homology between the fibronectin type II domain and the Kringle domains of blood clotting and fibrinolytic proteins has been examined in three dimensions by substituting the type II sequence into the bovine prothrombin Kringle 1 tertiary structure, determined by X-ray crystallographical methods at 3.8 A. Structural substitution of aligned amino acids of the type II domains and the Kringle produces a compact chain fold and deletions and insertions in the type II sequence are accommodated within the modelled structure. This confirms the structural homology between the two domains and verifies the sequence alignment and common evolution of the type II and Kringle units. The two structures contain homologous hydrophobic cores, centered around the two disulphide bridges which link conserved beta-type strands. Gross differences between the two domains occur in exterior loops and potential functional sites in these regions of the type II structures as found in fibronectin, Factor XII and seminal fluid protein PDC-109 are proposed. We suggest that the domains evolved from a common ancestral protein comprising the hydrophobic core and disulphide arrangement which later diverged to bind different macromolecules through adaptation of the external loops.     

1H nuclear magnetic resonance study of the solution conformation of an antibacterial protein, sapecin

The solution conformation of an antibacterial protein sapecin has been determined by 1H nuclear magnetic resonance (NMR) and dynamical simulated annealing calculations. It has been shown that the polypeptide fold consists of one flexible loop (residues 4-12), one helix (residues 15-23), and two extended strands (residues 24-31 and 34-40). It was found that the tertiary structure of sapecin is completely different from that of rabbit neutrophil defensin NP-5, which is homologous to sapecin in the amino acid sequences and also has the antibacterial activity. The three-dimensional structure determination has revealed that a basic-residue rich region and the hydrophobic surface face each other on the surface of sapecin.     

The structure of Escherichia coli heat-stable enterotoxin b by nuclear magnetic resonance and circular dichroism.

The heat-stable enterotoxin b (STb) is secreted by enterotoxigenic Escherichia coli that cause secretory diarrhea in animals and humans. It is a 48-amino acid peptide containing two disulfide bridges, between residues 10 and 48 and 21 and 36, which are crucial for its biological activity. Here, we report the solution structure of STb determined by two- and three-dimensional NMR methods. Approximate interproton distances derived from NOE data were used to construct structures of STb using distance-geometry and simulated annealing procedures. The NMR-derived structure shows that STb is helical between residues 10 and 22 and residues 38 and 44. The helical structure in the region 10-22 is amphipathic and exposes several polar residues to the solvent, some of which have been shown to be important in determining the toxicity of STb. The hydrophobic residues on the opposite face of this helix make contacts with the hydrophobic residues of the C-terminal helix. The loop region between residues 21 and 36 has another cluster of hydrophobic residues and exposes Arg 29 and Asp 30, which have been shown to be important for intestinal secretory activity. CD studies show that reduction of disulfide bridges results in a dramatic loss of structure, which correlates with loss of function. Reduced STb adopts a predominantly random-coil conformation. Chromatographic measurements of concentrations of native, fully reduced, and single-disulfide species in equilibrium mixtures of STb in redox buffers indicate that the formation of the two disulfide bonds in STb is only moderately cooperative. Similar measurements in the presence of 8 M urea suggest that the native secondary structure significantly stabilizes the disulfide bonds.     

Three-dimensional structure of a complement control protein module in solution.

The complement control protein (CCP) modules (also known as short consensus repeats) are defined by a consensus sequence within a stretch of about 60 amino acid residues. These modules have been identified more than 140 times in over 20 proteins, including 12 proteins of the complement system. The solution structure of the 16th CCP module from human complement factor H has been determined by a combination of 2-dimensional nuclear magnetic resonance spectroscopy and restrained simulated annealing. In all, 548 structurally important nuclear Overhauser enhancement cross-peaks were quantified as distance restraints and, together with 41 experimentally measured angle restraints, were incorporated into a simulated annealing protocol to determine a family of closely related structures that satisfied the experimental observations. The CCP structure is shown to be based on a beta-sandwich arrangement; one face made up of three beta-strands hydrogen-bonded to form a triple-stranded region at its centre and the other face formed from two separate beta-strands. Both faces of the molecule contribute highly conserved hydrophobic side-chains to a compact core. The regions between the beta-strands are composed of both well-defined turns and less well-defined loops. Analysis of CCP sequence alignments, in light of the determined structure, reveals a high degree of conservation amongst residues of obvious structural importance, while almost all insertions, deletions or replacements observed in the known sequences are found in the less well-defined loop regions. On the basis of these observations it is postulated that models of other CCP modules that are based on the structure presented here will be accurate. Certain families of CCP modules differ from the consensus in that they contain extra cysteine residues. As a test of structural consensus, the extra disulphide bridges are shown to be easily accommodated within the determined CCP model.     

The disulphide bridges of human cystatin C (γ-trace) and chicken cystatin

Human cystatin C, a powerful physiological protein inhibitor of cathepsins B, H and L, contains two disulphide bridges, at least one of which is essential to its inhibitory activity. The positions of these bridges in the single polypeptide chain of the protein have been determined by diagonal paper electrophoresis. The cysteine residues at positions 73 and 83 form one bridge, and those at positions 97 and 117 form the other. In the homologous cystatin of chicken egg-white, the disulphides are Cys 71–Cys 81, and Cys 95–Cys 115.     

Optimal molecular structures of prion AGAAAAGA amyloid fibrils formatted by simulated annealing

To date, there is little structural data available on the AGAAAAGA palindrome in the hydrophobic region (113–120) of prion proteins, although many experimental studies have shown that this region has amyloid fibril forming properties. This region belongs to the N-terminal unstructured region (1–123) of prions, the structure of which has proved hard to determine using NMR or X-ray crystallography. This paper reports the successful construction of three amyloid fibril models for this region. The models were formatted by standard simulated annealing using suitable templates from the Protein Data Bank, and were refined using several traditional optimization methods within AMBER. Because the NMR or X-ray structure of the hydrophobic region AGAAAAGA of prion proteins has not yet been determined, these models can be used as a reference for experimental studies on this region. The results presented here confirm standard simulated annealing as an effective tool in molecular modeling. The three constructed models for amyloid fibrils may be useful in furthering the goals of medicinal chemistry in this field.     

A proton nuclear magnetic resonance study of the conformation of bovine anaphylatoxin C5a in solution

The solution conformation of bovine anaphylatoxin C5a has been investigated by nuclear magnetic resonance (NMR) spectroscopy. The 1H-NMR spectrum is assigned in a sequential manner using a variety of two-dimensional NMR techniques. A qualitative interpretation of the short range nuclear Overhauser enhancement data involving the NH, C alpha H and C beta H protons suggests that C5a has four helices comprising residues 5-11, 15-25, 33-39 and 46-61, and is composed of a globular head (residues 5-61) and a C-terminal tail. The polypeptide fold was determined by hybrid distance geometry-dynamical simulated annealing calculations on the basis of 203 approximate interproton distance restraints, 22 distance restraints for 11 intrahelical hydrogen bonds (identified on the basis of the pattern of short range NOEs and slowly exchanging backbone amide protons) and restraints for the 3 disulfide bridges. The overall polypeptide fold is similar to that of the sequence related human recombinant anaphylatoxin C5a [(1988) Proteins 3, 139-145].     

Solution structure of the third TB domain from LTBP1 provides insight into assembly of the large latent complex that sequesters latent TGF-beta

Almost all TGF-beta is secreted as part of a large latent complex. This complex is formed from three molecules, a latent transforming growth factor-beta binding protein (LTBP), which plays roles in targeting and activation, a latency associated peptide (LAP), which regulates latency, and the TGF-beta cytokine. LAP is the TGF-beta pro-peptide that is cleaved intracellularly prior to secretion, and TGF-beta binds non-covalently to LAP. Formation of the large latent complex is important for the efficient secretion of TGF-beta. Previous studies have revealed that the LTBP-LAP interaction is mediated by intracellular exchange of a single disulphide bond within the third, and only the third, TB domain (TB3) with LAP. We have previously reported the structure of a homologous TB domain from fibrillin-1. However, TB3 contains a two amino acid insertion, not found in fibrillin-1 TB domains, which is not amenable to molecular modelling. In order to clarify the basis of TB domain function, we have determined the solution NMR structure of TB3(LTBP1). Comparison with the fibrillin-1 TB domain reveals that the two-residue insertion is associated with a significant increase in solvent accessibility of one of the disulphide bonds (linking the second and sixth cysteine residues). Site-directed mutagenesis and NMR studies indicate that this is the only disulphide bond that can be removed without perturbing the TB domain fold. Furthermore, a ring of negatively charged residues has been identified that surrounds this disulphide bond. Homology modelling suggests that the surface properties of TB3 domains from different LTBP isoforms correlate with binding activities. This research provides testable hypotheses regarding the molecular basis of complex formation between LTBPs and LAPs.     

Solution conformation of endothelin, a potent vaso-constricting bicyclic peptide. A combined use of 1H NMR spectroscopy and distance geometry calculations

The solution structure of endothelin-1, a newly discovered potent bicyclic peptide vaso-constrictor agent, has been investigated using 1H NMR conformational constraints and distance geometry calculations. The conformation is constrained by two disulphide bridges between Cys1-Cys15 and Cys3-Cys11 but the NMR data and computed conformers show additional helical structure between residues Leu6 and Cys11. Our results are compared with previous conflicting reports on the solution conformation of this peptide.     

The primary structure of a major polypeptide component from the venom of Naja melanoleuca.

The venom of the forest cobra, Naja melanoleuca, contains a number of homologous polypeptides containing between 60 and 71 amino acid resides. The primary structure of a major component (approx. 10% by weight of the crude venom) has been determined unambiguously. The molecule contians 61 amino acid residues and four disulphide bridges. It has not effect on neuromuscular transmission or the excitatory or inhibitory responses to acetylcholine of molluscan neurons. The molecule is similar to, but not identical with, the so-called cytotoxins VII2 and VII3 isolated, by others, from the same venom but reported to be minor components.