Solution structure and activity of the synthetic four-disulfide bond Mediterranean mussel defensin (MGD-1)

MGD-1 is a 39-residue defensin-like peptide isolated from the edible Mediterranean mussel, Mytilus galloprovincialis. This peptide is characterized by the presence of four disulfide bonds. We report here its solid-phase synthesis and an easy way to improve the yield of the four native disulfide bonds. Synthetic and native MGD-1 display similar antibacterial activity, suggesting that the hydroxylation of Trp28 observed in native MGD-1 is not involved in the antimicrobial effect. The three-dimensional solution structure of MGD-1 has been established using (1)H NMR and mainly consists of a helical part (Asn7-Ser16) and two antiparallel beta-strands (Arg20-Cys25 and Cys33-Arg37), together giving rise to the common cystine-stabilized alpha-beta motif frequently observed in scorpion toxins. In MGD-1, the cystine-stabilized alpha-beta motif is stabilized by four disulfide bonds (Cys4-Cys25, Cys10-Cys33, Cys14-Cys35, and Cys21-Cys38), instead of by the three disulfide bonds commonly found in arthropod defensins. Except for the Cys21-Cys38 disulfide bond which is solvent-exposed, the three others belong to the particularly hydrophobic core of the highly constrained structure. Moreover, the C4-P5 amide bond in the cis conformation characterizes the MGD-1 structure. MGD-1 and insect defensin A possess similar bactericidal anti-Gram-positive activity, suggesting that the fourth disulfide bond of MGD-1 is not essential for the biological activity. In agreement with the general features of antibacterial peptides, the MGD-1 and defensin A structures display a typical distribution of positively charged and hydrophobic side chains. The positively charged residues of MGD-1 are located in three clusters. For these two defensin peptides isolated from insects and mollusks, it appears that the rather well conserved location of certain positively charged residues and of the large hydrophobic cluster are enough to generate the bactericidal potency and the Gram-positive specificity.     

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.     

Disulfide bonding arrangements in active forms of the somatomedin B domain of human vitronectin

The N-terminal cysteine-rich somatomedin B (SMB) domain (residues 1-44) of the human glycoprotein vitronectin contains the high-affinity binding sites for plasminogen activator inhibitor-1 (PAI-1) and the urokinase receptor (uPAR). We previously showed that the eight cysteine residues of recombinant SMB (rSMB) are organized into four disulfide bonds in a linear uncrossed pattern (Cys(5)-Cys(9), Cys(19)-Cys(21), Cys(25)-Cys(31), and Cys(32)-Cys(39)). In the present study, we use an alternative method to show that this disulfide bond arrangement remains a major preferred one in solution, and we determine the solution structure of the domain using NMR analysis. The solution structure shows that the four disulfide bonds are tightly packed in the center of the domain, replacing the traditional hydrophobic core expected for a globular protein. The few noncysteine hydrophobic side chains form a cluster on the outside of the domain, providing a distinctive binding surface for the physiological partners PAI-1 and uPAR. The hydrophobic surface consists mainly of side chains from the loop formed by the Cys(25)-Cys(31) disulfide bond, and is surrounded by conserved acidic and basic side chains, which are likely to contribute to the specificity of the intermolecular interactions of this domain. Interestingly, the overall fold of the molecule is compatible with several arrangements of the disulfide bonds. A number of different disulfide bond arrangements were able to satisfy the NMR restraints, and an extensive series of conformational energy calculations performed in explicit solvent confirmed that several disulfide bond arrangements have comparable stabilization energies. An experimental demonstration of the presence of alternative disulfide conformations in active rSMB is provided by the behavior of a mutant in which Asn(14) is replaced by Met. This mutant has the same PAI-1 binding activity as rVN1-51, but its fragmentation pattern following cyanogen bromide treatment is incompatible with the linear uncrossed disulfide arrangement. These results suggest that active forms of the SMB domain may have a number of allowed disulfide bond arrangements as long as the Cys(25)-Cys(31) disulfide bond is preserved.     

Human defensin 5 disulfide array mutants: disulfide bond deletion attenuates antibacterial activity against Staphylococcus aureus

Human α-defensin 5 (HD5, HD5(ox) to specify the oxidized and disulfide linked form) is a 32-residue cysteine-rich host-defense peptide, expressed and released by small intestinal Paneth cells, that exhibits antibacterial activity against a number of Gram-negative and -positive bacterial strains. To ascertain the contributions of its disulfide array to structure, antimicrobial activity, and proteolytic stability, a series of HD5 double mutant peptides where pairs of cysteine residues corresponding to native disulfide linkages (Cys(3)-Cys(31), Cys(5)-Cys(20), Cys(10)-Cys(30)) were mutated to Ser or Ala residues, overexpressed in E. coli, purified, and characterized. A hexa mutant peptide, HD5[Ser(hexa)], where all six native Cys residues are replaced by Ser residues, was also evaluated. Removal of a single native S-S linkage influences oxidative folding and regioisomerization, antibacterial activity, Gram-negative bacterial membrane permeabilization, and proteolytic stability. Whereas the majority of the HD5 mutant peptides show low micromolar activity against Gram-negative E. coli ATCC 25922 in colony counting assays, the wild-type disulfide array is essential for low micromolar activity against Gram-positive S. aureus ATCC 25923. Removal of a single disulfide bond attenuates the activity observed for HD5(ox) against this Gram-positive bacterial strain. This observation supports the notion that the HD5(ox) mechanism of antibacterial action differs for Gram-negative and Gram-positive species [Wei et al. (2009) J. Biol. Chem. 284, 29180-29192] and that the native disulfide array is a requirement for its activity against S. aureus.     

Determination of disulfide bond assignments and N-glycosylation sites of the human gastrointestinal carcinoma antigen GA733-2 (CO17-1A, EGP, KS1-4, KSA, and Ep-CAM)

The GA733-2 antigen is a cell surface glycoprotein highly expressed on most human gastrointestinal carcinoma and at a lower level on most normal epithelia. It is an unusual cell-cell adhesion protein that does not exhibit any obvious relationship to the four known classes of adhesion molecules. In this study, the disulfide-bonding pattern of the GA733-2 antigen was determined using matrix-assisted laser desorption/ionization mass spectrometry and N-terminal sequencing of purified tryptic peptides treated with 2-[2'-nitrophenylsulfonyl]-3-methyl-3-bromoindolenine or partially reduced and alkylated. Numbering GA733-2 cysteines sequentially from the N terminus, the first three disulfide linkages are Cys1-Cys4, Cys2-Cys6, and Cys3-Cys5, which is a novel pattern for a cysteine-rich domain instead of the expected epidermal growth factor-like disulfide structure. The next three disulfide linkages are Cys7-Cys8, Cys9-Cys10, and Cys11-Cys12, consistent with the recently determined disulfide pattern of the thyroglobulin type 1A domain of insulin-like growth factor-binding proteins 1 and 6. Analysis of glycosylation sites showed that GA733-2 antigen contained N-linked carbohydrate but that no O-linked carbohydrate groups were detected. Of the three potential N-linked glycosylation sites, Asn175 was not glycosylated, whereas Asn88 was completely glycosylated, and Asn51 was partially glycosylated. These data show that the extracellular domain of the GA733-2 antigen consists of three distinct domains; a novel cysteine-rich N-terminal domain (GA733 type 1 motif), a cysteine-rich thyroglobulin type 1A domain (GA733 type 2 motif), and a unique nonglycosylated domain without cysteines (GA733 type 3 motif).     

Isolation and characterization of disulfide-bonded peptides from the three globular domains of aggregating cartilage proteoglycan

The aggregating cartilage proteoglycan core protein contains two globular domains near the N terminus (G1 and G2) and one near the C terminus (G3). The G1-G3 domains contain 10, 8, and 10 cysteine residues, respectively. The disulfide assignments of the G1 domain have previously been deduced (Neame, P. J., Christner, J. E., and Baker, J. R. (1987) J. Biol. Chem. 262, 17768-17778) as Cys1-Cys2, Cys3-Cys6, Cys4-Cys5, Cys7-Cys10, and Cys8-Cys9, in which the numbers cited after the half-cystine residues are their relative positions from the N terminus. Here we describe a method for the isolation of disulfide-bonded peptides from tryptic digests of bovine nasal cartilage monomer. Sequence analysis of these peptides has allowed us to confirm the pairings previously determined for the G1 domain and to assign a disulfide pattern for the G2 domain of Cys11-Cys14, Cys12-Cys13, Cys15-Cys18, and Cys16-Cys17, in which the Cys15-Cys18 pairing was deduced indirectly. Similarly, for the G3 domain, a pattern of Cys19-Cys20, Cys21-Cys24, Cys22-Cys23, Cys25-Cys27, and Cys26-Cys28 was assigned, in which the Cys22-Cys23 pair was deduced indirectly. The G2 domain therefore contains disulfide bonding which is characteristic of the tandem repeat structures found in the G1 domain and link protein, and the G3 domain contains the three disulfide linkages previously assigned to the family of C-type animal lectins. The method described here, which combines anion-exchange, cation-exchange, and reversed-phase chromatography, should have broad application to the isolation of disulfide-bonded peptides from other heavily glycosylated proteins and proteoglycans.     

Three-dimensional structure in lipid micelles of the pediocin-like antimicrobial peptide curvacin A

The 3D structure of the membrane-permeabilizing 41-mer pediocin-like antimicrobial peptide curvacin A produced by lactic acid bacteria has been studied by NMR spectroscopy. In DPC micelles, the cationic and hydrophilic N-terminal half of the peptide forms an S-shaped beta-sheet-like domain stabilized by a disulfide bridge and a few hydrogen bonds. This domain is followed by two alpha-helices: a hydrophilic 6-mer helix between residues 19 and 24 and an amphiphilic/hydrophobic 11-mer helix between residues 29 and 39. There are two hinges in the peptide, one at residues 16-18 between the N-terminal S-shaped beta-sheet-like structure and the central 6-mer helix and one at residues 26-28 between the central helix and the 11-mer C-terminal helix. The latter helix is the only amphiphilic/hydrophobic part of the peptide and is thus presumably the part that penetrates into the hydrophobic phase of target-cell membranes. The hinge between the two helices may introduce the flexibility that allows the helix to dip into membranes. The helix-hinge-helix structure in the C-terminal half of curvacin A clearly distinguishes this peptide from the other pediocin-like peptides whose structures have been analyzed and suggests that curvacin A along with the structural homologues enterocin P and carnobacteriocin BM1 belong to a subgroup of the pediocin-like family of antimicrobial peptides.     

Solution structure of synthetic penaeidin-4 with structural and functional comparisons with penaeidin-3

Antimicrobial peptide structure has direct implications for the complexity of functions and mechanisms of action. The penaeidin antimicrobial peptide family from shrimp is divided into multiple class designations based on primary structure. The penaeidin classes are not only characterized by variability in primary sequence but also by variation in target specificity and effectiveness. Whereas class 4 exhibits low isoform diversity within species and is highly conserved between species, the primary sequence of penaeidin class 3 is less conserved between species and exhibits considerable isoform diversity within species. All penaeidins, regardless of class or species, are composed of two dramatically different domains: an unconstrained proline-rich domain and a disulfide bond-stabilized cysteine-rich domain. The proline-rich domain varies in length and is generally less conserved, whereas the spacing and specific residue content of the cysteine-rich domain is more conserved. The structure of the synthetic penaeidin class 4 (PEN4-1) from Litopenaeus setiferus was analyzed using several approaches, including chemical mapping of disulfide bonds, circular dichroism analysis of secondary structural characteristics, and complete characterization of the solution structure of the peptide by proton NMR. L. setiferus PEN4-1 was then compared with the previously characterized structure of penaeidin class 3 from Litopenaeus vannamei. Moreover, the specificity of these antimicrobial peptides was examined through direct comparison of activity against a panel of microbes. The penaeidin classes differ in microbial target specificity, which correlates to variability in specific domain sequence. However, the tertiary structure of the cysteine-rich domain and indeed the overall structure of penaeidins are conserved across classes.     

Crystal structure of a novel antifungal protein distinct with five disulfide bridges from Eucommia ulmoides Oliver at an atomic resolution

EAFP2 is a novel antifungal protein isolated from the bark of the tree Eucommia ulmoides Oliver. It consists of 41 residues and is characterized with a five-disulfide motif and the inhibitory effects on the growth of both cell wall chitin-containing and chitin-free fungi. The crystal structure of EAFP2 at an atomic resolution of 0.84 A has been determined by using Shake-and-Bake direct methods with the program SnB. The phases obtained were of sufficient quality to permit the initial model built automatically and the structural refinement carried out using anisotropic displacement parameters resulted in a final crystallographic R factor of 6.8%. In the resulting structural model, all non-hydrogen protein atoms including an unusual pyroglutamyl acid residue at the N-terminal can fit to the articulated electron densities with one centre and more than 65% of the hydrogen atoms in the protein can be observed as individual peaks in the difference map. The general fold of EAFP2 is composed of a 3(10) helix (Cys3-Arg6), an alpha-helix (Ala27-Cys31) and a three-stranded antiparallel beta-sheet (Cys16-Ser18, Cys23-Ser25, and Cys35-Cys37) and cross-linked by five disulfide bridges. The tertiary structure of EAFP2 can be divided into two structural sectors, A and B. Sector A composed of residues 11-30 adopts a conformation similar to the chitin-binding domain in the hevein-like proteins and features a hydrophobic surface embraced a chitin-binding site (Tyr20, 22, 29, and Ser18). The distinct disulfide bridge Cys7-Cys37 connects the N-terminal ten residues with the C-terminal segment 35-41 to form the sector B, which features a cationic surface distributing all four positively charged residues, Arg6, 9, 36, and 40. Based on these structural features, the possible structural basis of the functional properties of EAFP2 is discussed.     

Structural characterization of the 16-kDa allergen, RA17, in rice seeds. Prediction of the secondary structure and identification of intramolecular disulfide bridges

The 16-kDa rice allergen, RA17, belonging to the alpha-amylase/trypsin inhibitor family was isolated from rice seed and structurally characterized by identifying cystine-containing peptides and predicting the secondary structure and hydrophobic regions. Eight peptides, which constitute three sets of cystine-containing peptides, were purified by HPLC from a thermolytic digest of RA17 and identified by their amino acid sequence and composition, indicating five intramolecular disulfide bridges: Cys34-Cys94, Cys26-(Cys50 or Cys51)-Cys110 and Cys12-(Cys62 or Cys64)-Cys122. Analyses of the CD spectrum and the Chou-Fasman prediction suggested that RA17 had some helical- and sheet-structure regions. Based on these experimental and predicted data, RA17 is proposed to be a globular molecule with a small hydrophobic core having folding restricted by five intramolecular disulfide bridges.     

The structure of the Cys-rich terminal domain of Hydra minicollagen, which is involved in disulfide networks of the nematocyst wall

The minicollagens found in the nematocysts of Hydra constitute a family of invertebrate collagens with unusual properties. They share a common modular architecture with a central collagen sequence ranging from 14 to 16 Gly-X-Y repeats flanked by polyproline/hydroxyproline stretches and short terminal domains that show a conserved cysteine pattern (CXXXCXXXCXXX-CXXXCC). The minicollagen cysteine-rich domains are believed to function in a switch of the disulfide connectivity from intra- to intermolecular bonds during maturation of the capsule wall. The solution structure of the C-terminal fragment including a minicollagen cysteine-rich domain of minicollagen-1 was determined in two independent groups by 1H NMR. The corresponding peptide comprising the last 24 residues of the molecule was produced synthetically and refolded by oxidation under low protein concentrations. Both presented structures are identical in their fold and disulfide connections (Cys2-Cys18, Cys6-Cys14, and Cys10-Cys19) revealing a robust structural motif that is supposed to serve as the polymerization module of the nematocyst capsule.     

Structural characterization of a rhamnose-binding glycoprotein (lectin) from Spanish mackerel (Scomberomorous niphonius) eggs

A rhamnose-binding glycoprotein (lectin), named SML, was isolated from the eggs of Spanish mackerel (Scomberomorous niphonius) by affinity and ion-exchange chromatographies. SML was composed of a non-covalently linked homodimer. The SML subunit was composed of 201 amino acid residues with two tandemly repeated domains, and contained 8 half-Cys residues in each domain, which is highly homologous to the N-terminal lectin domain of calcium-independent alpha-latrotoxin receptor in mammalian brains. Each domain has the same disulfide bonding pattern; Cys10-Cys40, Cys20-Cys99, Cys54-Cys86 and Cys67-Cys73 were located in the N-terminal domain, and Cys108-Cys138, Cys117-Cys195, Cys152-Cys182 and Cys163-Cys169 were in the C-terminal domain. SML was N-glycosylated at Asn168 in the C-terminal domain. The structure of the sugar chain was determined to be NeuAc-Galbeta1-4GlcNAcbeta1-2Manalpha1-6-(NeuAc-Galbeta1-4GlcNAcbeta1-2Manalpha1-3)Manbeta1-4GlcNAcbeta1-4GlcNAc-Asn.     

Positions of disulfide bonds in rye (Secale cereale) seed chitinase-a

The positions of disulfide bonds of rye seed chitinase-a (RSC-a) were identified by the isolation of disulfide-containing peptides produced with enzymatic and/or chemical cleavages of RSC-a, followed by sequencing them. An unequivocal assignment of disulfide bonds in this enzyme was as follows: Cys3-Cysl8, Cys12-Cys24, Cys15-Cys42, Cys17-Cys31, and Cys35-Cys39 in the chitin-binding domain (CB domain), Cys82-Cys144, Cys156-Cys164, and Cys282-Cys295 in the catalytic domain (Cat domain), and Cys263 was a free form.     

Localization of the intrachain disulfide bonds of the envelope glycoprotein 71 from Friend murine leukemia virus.

Envelope glycoprotein 71 from Friend murine leukemia virus was purified to homogeneity by reversed-phase HPLC. It could be shown that all 20 cysteine residues of the molecule are linked by disulfide bonds. After complete tryptic digestion, peptides containing cystine were identified by comparison of the reversed-phase HPLC profile of the digest with that of a reduced aliquot which had been subjected to affinity chromatography on thiol-Sepharose. The locations of the 10 disulfide bonds were determined by isolation, further digestion and analysis of peptides containing cystine. The first cysteine residue of the sequence (Cys46) was shown to be coupled to the sixth (Cys98), leading to a large loop containing four additional cysteine residues. Computer model building and energy calculations led to the assignment of Cys72 to Cys87 and Cys73 to Cys83. The following four cysteine residues of the sequence also constitute a structural unit, with Cys121 bonded to Cys141 and Cys133 to Cys146, and the last two cysteine residues in the amino-terminal domain of glycoprotein 71 form a small loop (Cys178 to Cys184). The first two cysteine residues of the carboxy-terminal domain produce a very small hydrophobic loop (Cys312-Cys315). Cys361 is bound to Cys373, Cys342 to Cys396 and Cys403 to Cys416. A model for the folding pattern of the viral glycoprotein is proposed.     

Selectivity of tryptophan residues in mediating photolysis of disulfide bridges in goat alpha-lactalbumin

Goat alpha-lactalbumin (GLA) contains four tryptophan (Trp) residues and four disulfide bonds. Illumination with near-UV light results in the cleavage of disulfide bridges and in the formation of free thiols. To obtain information about the reaction products, the illuminated protein was carbamidomethylated and digested with trypsin and the peptides were analyzed by mass spectrometry. Peptides containing Cys120Cam, Cys61Cam, or Cys91Cam were detected, as well as two peptides containing a new Cys-Lys cross-link. In one, Cys6 was cross-linked to Lys122, while the cross-link in the second was either a Cys91-Lys79 or Cys73-Lys93 cross-link; however, the exact linkage could not be defined. The results demonstrate photolytic cleavage of the Cys6-Cys120, Cys61-Cys77, and Cys73-Cys91 disulfide bonds. While photolysis of Cys6-Cys120 and Cys73-Cys91 disulfide bonds in GLA has been reported, cleavage of the Cys61-Cys77 disulfide bonds has not been previously detected. To examine the contribution of the individual Trp residues, we constructed the GLA mutants, W26F, W60F, W104F, and W118F, by replacing single Trp residues with phenylalanine (Phe). The substitution of each Trp residue led to less thiol production compared to that for wild-type GLA, showing that each Trp residue in GLA contributed to the photolytic cleavage of disulfide bridges. The specificity was expressed by the nature of the reaction products. No cleavage of the Cys6-Cys120 disulfide bridge was detected when the W26F mutant was illuminated, and no cleavage of the Cys73-Cys91 disulfide bridge was seen following illumination of W26F or W104F. In contrast, Cys61Cam, resulting from the cleavage of the Cys61-Cys77 disulfide bridge, was found following illumination of any of the mutants.     

The disulfide bridge pattern of snake venom disintegrins, flavoridin and echistatin.

Flavoridin and echistatin, isolated from the venom of Trimeresurus flavoviridis and Echis carinatus, respectively, belong to the disintegrin family of integrin beta 1 and beta 3 inhibitors of low molecular weight RGD-containing, cysteine-rich peptides. Since disulfide bonds are critical for expression of biological activity, we sought to determine their location in these two proteins. In flavoridin, direct evidence for the existence of linkage between Cys4-Cys19 and between Cys45 and Cys64 was obtained by analysis of proteolytic products, and indirect evidence suggests links between Cys6-Cys14 and Cys13-Cys36. In echistatin, links between Cys8-Cys37 and Cys20-Cys39 were identified by direct chemical analysis.     

Structural and functional properties of a novel serine protease inhibiting peptide family in arthropods

Recently, several arthropod peptides that belong to a new serine protease inhibitor family were discovered. Three members (HI, PMP-D2=LMCI-1 and PMP-C=LMCI-2) were isolated from the migratory locust, Locusta migratoria. Five additional members (SGPI-1-5) were identified in the desert locust Schistocerca gregaria, and a heterodimeric serine protease inhibitor (pacifastin) was isolated from the hemolymph of the crayfish Pacifastacus leniusculus. The light chain of pacifastin constitutes the inhibitory subunit that has nine cysteine-rich domains (PLDs) that are homologous with the locust inhibitors. These locust inhibitors and PLDs share a conserved array of six cysteine residues (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-4)-Cys-Thr-Xaa(3)-Cys), which are involved in an identical disulfide bridge pattern (Cys(1)-Cys(4), Cys(2)-Cys(6), Cys(3)-Cys(5)). The solution structures of LMCI-1 and LMCI-2 showed a similar, compact, globular folding, which is unique within the group of the small 'canonical' inhibitors. Moreover, the reactive site, including the P1-P'1 bond was thoroughly investigated by means of synthetic variants. However, the biological function(s) of the locust inhibitors is (are) not fully understood. LMCI-1 and LMCI-2 were shown to inhibit the endogenous proteolytic activating cascade of prophenoloxidase. Northern blot analysis indicated that the genes encoding the SGPI precursors are differentially expressed in a time-, stage- and hormone-dependent manner.     

Crystal structure of the DsbB-DsbA complex reveals a mechanism of disulfide bond generation

Oxidation of cysteine pairs to disulfide requires cellular factors present in the bacterial periplasmic space. DsbB is an E. coli membrane protein that oxidizes DsbA, a periplasmic dithiol oxidase. To gain insight into disulfide bond formation, we determined the crystal structure of the DsbB-DsbA complex at 3.7 A resolution. The structure of DsbB revealed four transmembrane helices and one short horizontal helix juxtaposed with Cys130 in the mobile periplasmic loop. Whereas DsbB in the resting state contains a Cys104-Cys130 disulfide, Cys104 in the binary complex is engaged in the intermolecular disulfide bond and captured by the hydrophobic groove of DsbA, resulting in separation from Cys130. This cysteine relocation prevents the backward resolution of the complex and allows Cys130 to approach and activate the disulfide-generating reaction center composed of Cys41, Cys44, Arg48, and ubiquinone. We propose that DsbB is converted by its specific substrate, DsbA, to a superoxidizing enzyme, capable of oxidizing this extremely oxidizing oxidase.     

Functional structure of the somatomedin B domain of vitronectin

The N-terminal somatomedin B domain (SMB) of vitronectin binds PAI-1 and the urokinase receptor with high affinity and regulates tumor cell adhesion and migration. We have shown previously in the crystal structure of the PAI-1/SMB complex that SMB, a peptide of 51 residues, is folded as a compact cysteine knot of four pairs of crossed disulfide bonds. However, the physiological significance of this structure was questioned by other groups, who disputed the disulfide bonding shown in the crystal structure (Cys5-Cys21, Cys9-Cys39, Cys19-Cys32, Cys25-Cys31), notably claiming that the first disulfide is Cys5-Cys9 rather than the Cys5-Cys21 bonding shown in the structure. To test if the claimed Cys5-Cys9 bond does exist in the SMB domain of plasma vitronectin, we purified mouse and rat plasma vitronectin that have a Met (hence cleavable by cyanogen bromide) at residue 14, and also prepared recombinant human SMB variants from insect cells with residues Asn14 or Leu24 mutated to Met. HPLC and mass spectrometry analysis showed that, after cyanogen bromide digestion, all the fragments of the SMB derived from mouse or rat vitronectin or the recombinant SMB mutants are still linked together by disulfides, and the N-terminal peptide (residue 1-14 or 1-24) can only be released when the disulfide bonds are broken. This clearly demonstrates that Cys5 and Cys9 of SMB do not form a disulfide bond in vivo, and together with other structural evidence confirms that the only functional structure of the SMB domain of plasma vitronectin is that seen in its crystallographic complex with PAI-1.     

Local interactions and the role of the 6-120 disulfide bond in alpha-lactalbumin: implications for formation of the molten globule state

Molten globule states are partially folded states of proteins which are compact and contain a high degree of secondary structure but which lack many of the fixed tertiary interactions associated with the native state. A set of peptides has been prepared in order to probe the role of local interactions in the vicinity of the Cys(6)-Cys(120) disulfide bond in stabilizing the molten globule state of human alpha-lactalbumin. Peptides derived from the N-terminal and C-terminal regions of human alpha-lactalbumin have been analyzed using nuclear magnetic resonance, circular dichroism, fluorescence spectroscopy and sedimentation equilibrium experiments. A peptide corresponding to the first helical region in the native protein, residues 1-13, is only slightly helical in isolation. Extending the peptide to include residues 14-18 results in a modest increase in helicity. A peptide derived from the C-terminal 12 residues, residues 112-123, is predominantly unstructured. Crosslinking the N- and C-terminal peptides by the native disulfide bond results in almost no increase in structure and there is no evidence for any significant cooperative structure formation over the range of pH 2.2-11.7. These results demonstrate that there is very little enhancement of local structure due to the formation of the Cys(6)-Cys(120) disulfide bond. This is in striking contrast to peptides derived from the region of the Cys(28)-Cys(111) disulfide.