Interplay between superantigens and immunoreceptors.

Superantigens (SAGs) cause a massive T-cell proliferation by simultaneously binding to major histocompatibility complex (MHC) class II on antigen-presenting cells and T-cell receptors (TCRs) on T cells. These T-cell mitogens can cause disease in host, such as food poisoning or toxic shock. The best characterized groups of SAGs are the bacterial SAGs secreted by Staphylococcus aureus and Streptococcus pyogenes. Despite a common overall three-dimensional fold of these SAGs, they have been shown to bind to MHC class II in different ways. Recently, it has also been shown that SAGs have individual preferences in their binding to the TCRs. They can interact with various regions of the variable beta-chain of TCRs and at least one SAG seems to bind to the alpha-chain of TCRs. In this review, different subclasses of SAGs are classified based upon their binding mode to MHC class II, and models of trimolecular complexes of MHC-SAG-TCR molecules are described in order to reveal and understand the complexity of SAG-mediated T-cell activation.

Crystal structure of a superantigen bound to MHC class II displays zinc and peptide dependence.

The three-dimensional structure of a bacterial superantigen, Staphylococcus aureus enterotoxin H (SEH), bound to human major histocompatibility complex (MHC) class II (HLA-DR1) has been determined by X-ray crystallography to 2.6 A resolution (1HXY). The superantigen binds on top of HLA-DR1 in a completely different way from earlier co-crystallized superantigens from S.aureus. SEH interacts with high affinity through a zinc ion with the beta1 chain of HLA-DR1 and also with the peptide presented by HLA-DR1. The structure suggests that all superantigens interacting with MHC class II in a zinc-dependent manner present the superantigen in a common way. This suggests a new model for ternary complex formation with the T-cell receptor (TCR), in which a contact between the TCR and the MHC class II is unlikely.

Conformation of desmopressin, an analogue of the peptide hormone vasopressin, in aqueous solution as determined by NMR spectroscopy.

Desmopressin (1-desamino-[DArg8]vasopressin, is a synthetic analogue of the neurohypophyseal peptide hormone vasopressin which has high antidiuretic and antibleeding potency. The structure of desmopressin has been determined in aqueous solution by two-dimensional NMR techniques and molecular dynamics simulations. Both standard and time-averaged distance restraints were used in structure calculations because of the inherent flexibility in small peptides. 21 models calculated with standard restraints were compared with structures refined with time-averaged distance restraints and were found to be good representatives of the conformational ensemble of desmopressin. The macrocyclic ring forms an inverse gamma-turn centered around Gln4. Residues 1 and 2, the disulphide bridge and the three-residue acyclic tail were found to be flexible in solution. Residues 4-6 in the ensemble of calculated structures contain essentially the same backbone conformation as in the crystal structure of pressinoic acid, the cyclic moiety of vasopressin, whereas residues 2-6 superimpose on the NMR-derived conformation of oxytocin bound to neurophysin. The results presented in this work suggest that, in addition to the differences in sequence between desmopressin and vasopressin, differences in conformational and dynamic properties between the two compounds explain their pharmacological differences.

Binding of peptides in solution by the Escherichia coli chaperone PapD as revealed using an inhibition ELISA and NMR spectroscopy.

PapD is the prototype member of a family of periplasmic chaperones which are required for assembly of virulence associated pili in pathogenic, gram-negative bacteria. In the present investigation, an ELISA has been developed for evaluation of compounds as inhibitors of PapD. Synthetic peptides, including an octamer, derived from the C-terminus of the pilus adhesin PapG were able to inhibit PapD in the ELISA. Evaluation of a panel of octapeptides in the ELISA, in combination with NMR studies, showed that the peptides were bound as extended beta-strands by PapD in aqueous solution. The PapD-peptide complex was stabilized by backbone to backbone hydrogen bonds and interactions involving three hydrophobic peptide side chains. This structural information, together with previous crystal structure data, provides a starting point in efforts to design and synthesize compounds which bind to chaperones and interfere with pilus assembly in pathogenic bacteria.

Transferred nuclear Overhauser effect spectroscopy study of a peptide from the PapG pilus subunit bound by the Escherichia coli PapD chaperone.

Interaction of the Escherichia coli PapD chaperone with the synthetic peptide PapG308-314 (Thr-Met-Val-Leu-Ser-Phe-Pro), corresponding to the seven C-terminal residues of the PapG pilus subunit, was studied by transferred nuclear Overhauser effect (TRNOE) spectroscopy. The observation of cross-peaks corresponding to either intraresidue or sequential C(alpha)H/NH and C(beta)H/NH TRNOEs and the absence of sequential NH(i)/NH(i+1) TRNOEs indicate that the peptide binds to PapD in an extended conformation. In addition, line-broadening effects gave information of the peptide's mode of interaction with PapD. These observations were in excellent agreement with a recent crystal structure of a PapG peptide complexed with PapD.

Solid-phase synthesis and conformational studies of helper T cell immunogenic peptides that carry carbohydrate haptens linked to serine.

N alpha-Fmoc serine and its corresponding pentafluorophenyl ester were glycosylated with the 1,2-trans peracetates of the disaccharides galabiose and cellobiose. Complete stereoselectivity and 52-75% yields were obtained under boron trifluoride etherate promotion. Lower yields and loss of stereoselectivity were obtained when thioglycosides, trichloroacetimidates or glycosyl bromides were employed as glycosyl donors. The glycosylated building blocks were used in solid-phase synthesis of derivatives of a helper T cell immunogenic peptide consisting of amino acids 52-61 from hen-egg lysozyme. 1H-NMR spectroscopy in DMSO-d6 showed that the peptide moiety of the glycopeptides assumed random conformations which were not influenced by glycosylation at different positions.

Structure of a cyclic peptide with a catalytic triad, determined by computer simulation and NMR spectroscopy.

We report the design of a cyclic, eight-residue peptide that possesses the catalytic triad residues of the serine proteases. A manually built model has been relaxed by 0.3 ns of molecular dynamics simulation at room temperature, during which no major changes occurred in the peptide. The molecule has been synthesised and purified. Two-dimensional NMR spectroscopy provided 35 distance and 7 torsion angle constraints, which were used to determine the three-dimensional structure. The experimental conformation agrees with the predicted one at the beta-turn, but deviates in the arrangement of the disulphide bridge that closes the backbone to a ring. A 1.2 ns simulation at 600 K provided extended sampling of conformation space. The disulphide bridge reoriented into the experimental arrangement, producing a minimum backbone rmsd from the experimental conformation of 0.8 A. At a later stage in the simulation, a transition at Ser3 produced more pronounced high-temperature behaviour. The peptide hydrolyses p-nitrophenyl acetate about nine times faster than free histidine.

Structural features of a polypeptide carrier promoting secretion of a beta-lactamase fusion protein in yeast.

Escherichia coli beta-lactamase was secreted into the culture medium of Saccharomyces cerevisiae in biologically active form, when fused to the C-terminus of the hsp150 delta-carrier. The hsp150 delta-carrier is an N-terminal fragment of the yeast hsp150 protein, having a signal peptide and consisting mostly of a 19 amino acid peptide repeated 11 times in tandem. Here we expressed the hsp150 delta-carrier fragment alone in S. cerevisiae. Apparently due to a positional effect of the gene insertion, large amounts of the hsp150 delta-carrier were synthesized. About half of the de novo synthesized carrier molecules were secreted into the culture medium, the rest remaining mostly in the pre-Golgi compartment. The extensively O-glycosylated carrier fragment was purified from the culture medium under non-denaturing conditions. Circular dichroism spectroscopy showed that it had no regular secondary structure. Nuclear magnetic resonance spectroscopy showed that a non-glycosylated synthetic peptide, the consensus sequence of the repetitive 19 amino acid peptide, also lacked secondary structure. The unstructured carrier polypeptide may facilitate proper folding and secretion of heterologous proteins attached to it.

Glycosylated peptide hormones: pharmacological properties and conformational studies of analogues of [1-desamino,8-D-arginine]vasopressin.

Two analogues of the antidiuretic drug [1-desamino,8-D-arginine]vasopressin (DDAVP), which have a glycosylated serine at position 4, have been prepared by Fmoc solid phase peptide synthesis. The glycosylated analogues had significantly higher bioavailabilities than the nonglycosylated [D-Tyr2,Ser4]DDAVP and DDAVP on intraintestinal administration in rat. The improved bioavailability resulted from an increased absorption from the small intestine and most likely from an increased stability toward enzymatic degradation, whereas plasma clearance was either unaffected or slightly increased by the glycosylation. The glycosylated analogues displayed only very low agonistic and antagonistic activities at the vasopressin V2-receptor. Conformational studies performed by 1H NMR spectroscopy did not reveal any major influence from glycosylation on the conformation of the peptide backbone. The lack of receptor binding displayed by the analogues is therefore most likely explained by steric repulsion between the carbohydrate moiety and the vasopressin receptor which prevents receptor binding.

Solid-phase synthesis and conformational studies of glycosylated derivatives of helper-T-cell immunogenic peptides from hen-egg lysozyme.

3-Mercaptopropionic acid and N alpha-Fmoc serine, both with unprotected carboxyl groups, were stereospecifically glycosylated in 62-82% yields, using saccharide 1,2-trans peracetates and Lewis acid catalysis. The resulting glycosylated building blocks were used in the synthesis of derivatives of helper-T-cell stimulating peptides, with the carbohydrate moiety located at the amino terminus, or internally in the peptide chain. 1H NMR spectroscopy in Me2SO-d6 showed that the glycopeptides assumed random conformations, which were not influenced by the glycosylation or by single substitutions of amino acids in the peptide moiety.