Structures of synthetic O-antigen fragments from serotype 2a Shigella flexneri in complex with a protective monoclonal antibody.

The anti-LPS IgG mAb F22-4, raised against Shigella flexneri serotype 2a bacteria, protects against homologous, but not heterologous, challenge in an experimental animal model. We report the crystal structures of complexes formed between Fab F22-4 and two synthetic oligosaccharides, a decasaccharide and a pentadecasaccharide that were previously shown to be both immunogenic and antigenic mimics of the S. flexneri serotype 2a O-antigen. F22-4 binds to an epitope contained within two consecutive 2a serotype pentasaccharide repeat units (RU). Six sugar residues from a contiguous nine-residue segment make direct contacts with the antibody, including the nonreducing rhamnose and both branching glucosyl residues from the two RUs. The glucosyl residue, whose position of attachment to the tetrasaccharide backbone of the RU defines the serotype 2a O-antigen, is critical for recognition by F22-4. Although the complete decasaccharide is visible in the electron density maps, the last four pentadecasaccharide residues from the reducing end, which do not contact the antibody, could not be traced. Although considerable mobility in the free oligosaccharides can thus be expected, the conformational similarity between the individual RUs, both within and between the two complexes, suggests that short-range transient ordering to a helical conformation might occur in solution. Although the observed epitope includes the terminal nonreducing residue, binding to internal epitopes within the polysaccharide chain is not precluded. Our results have implications for vaccine development because they suggest that a minimum of two RUs of synthetic serotype 2a oligosaccharide is required for optimal mimicry of O-Ag epitopes.

Structural and functional studies of FkpA from Escherichia coli, a cis/trans peptidyl-prolyl isomerase with chaperone activity.

The protein FkpA from the periplasm of Escherichia coli exhibits both cis/trans peptidyl-prolyl isomerase (PPIase) and chaperone activities. The crystal structure of the protein has been determined in three different forms: as the full-length native molecule, as a truncated form lacking the last 21 residues, and as the same truncated form in complex with the immunosuppressant ligand, FK506. FkpA is a dimeric molecule in which the 245-residue subunit is divided into two domains. The N-terminal domain includes three helices that are interlaced with those of the other subunit to provide all inter-subunit contacts maintaining the dimeric species. The C-terminal domain, which belongs to the FK506-binding protein (FKBP) family, binds the FK506 ligand. The overall form of the dimer is V-shaped, and the different crystal structures reveal a flexibility in the relative orientation of the two C-terminal domains located at the extremities of the V. The deletion mutant FkpNL, comprising the N-terminal domain only, exists in solution as a mixture of monomeric and dimeric species, and exhibits chaperone activity. By contrast, a deletion mutant comprising the C-terminal domain only is monomeric, and although it shows PPIase activity, it is devoid of chaperone function. These results suggest that the chaperone and catalytic activities reside in the N and C-terminal domains, respectively. Accordingly, the observed mobility of the C-terminal domains of the dimeric molecule could effectively adapt these two independent folding functions of FkpA to polypeptide substrates.

Structure of the Fab fragment from F124, a monoclonal antibody specific for hepatitis B surface antigen.

The crystal structure of the Fab fragment from the monoclonal anti-preS2 antibody F124 (IgG1,kappa) has been solved by molecular replacement and refined at 3.0 A resolution. The Fab crystallizes with two independent molecules in the asymmetric unit. F124 recognizes an epitope contained within the preS2 segment between residues 120 and 132 of the surface antigen of hepatitis B virus. The antibody shows a high affinity for the glycan N-linked to Asn123, but it also cross-reacts with the non-glycosylated peptide fragment 120-132. Although crystallization was performed in the presence of an eightfold excess of the cross-reactive peptide, no evidence for the ligand was found in the antigen-binding site, which is close to a neighbouring molecule in the crystal lattice. The antigen-binding site has a groove-like topology which is modulated with pocket-like cavities. It is characterized by a large number of tyrosine and aspartate residues. The importance of germ-line mutations at the binding site is discussed.

Crystal structure of Urtica dioica agglutinin, a superantigen presented by MHC molecules of class I and class II.

BACKGROUND: Urtica dioica agglutinin (UDA), a monomeric lectin extracted from stinging nettle rhizomes, is specific for saccharides containing N-acetylglucosamine (GlcNAc). The lectin behaves as a superantigen for murine T cells, inducing the exclusive proliferation of Vbeta8.3(+) lymphocytes. UDA is unique among known T cell superantigens because it can be presented by major histocompatibility complex (MHC) molecules of both class I and II. RESULTS: The crystal structure of UDA has been determined in the ligand-free state, and in complex with tri-acetylchitotriose and tetra-acetylchitotetraose at 1.66 A, 1.90 A and 1.40 A resolution, respectively. UDA comprises two hevein-like domains, each with a saccharide-binding site. A serine and three aromatic residues at each site form the principal contacts with the ligand. The N-terminal domain binding site can centre on any residue of a chito-oligosaccharide, whereas that of the C-terminal domain is specific for residues at the nonreducing terminus of the ligand. We have shown previously that oligomers of GlcNAc inhibit the superantigenic activity of UDA and that the lectin binds to glycans on the MHC molecule. We show that UDA also binds to glycans on the T cell receptor (TCR). CONCLUSIONS: The presence of two saccharide-binding sites observed in the structure of UDA suggests that its superantigenic properties arise from the simultaneous fixation of glycans on the TCR and MHC molecules of the T cell and antigen-presenting cell, respectively. The well defined spacing between the two binding sites of UDA is probably a key factor in determining the specificity for Vbeta8.3(+) lymphocytes.

Crystal structure of a dominant B-cell epitope from the preS2 region of hepatitis B virus in the form of an inserted peptide segment in maltodextrin-binding protein.

We report here the crystal structure of MalE-B363, a recombinant construction of maltodextrin-binding protein bearing a dominant B-cell epitope sequence from the preS2 region of the hepatitis B surface antigen. The inserted peptide sequence, which replaces the seven carboxy-terminal residues of maltodextrin-binding protein, carries the 14 amino acid residue epitope contained between residues 132 and 145 from the preS2 region. The epitope sequence is flanked on either side by additional residues that result from the genetically engineered insertion, bringing the total length of the foreign peptide to 26 amino acid residues. The hybrid protein has been previously shown to be recognised by monoclonal antibodies elicited by the native viral antigen. Three independent molecules of MalE-B363 are present in the asymmetric unit of the crystal. All 14 epitope residues could be traced for one molecule, ten epitope residues had significant electron density for the second, but no density was visible for the epitope of the third. The conformation of the amino-terminal segment of the epitope from Gln132(e) to Gly138(e) is similar in the two molecules of MalE-B363 for which the foreign peptide could be traced. Moreover, the conformation of a smaller segment, comprising residues Asp133(e) to Arg137(e), is similar to that present in the previously determined crystal structure of MalE-B133, another insertion/deletion mutant of maltodextrin-binding protein bearing the preS2 epitope. The presence of a common structural motif for the same sequence in disparate molecular environments suggests that this conformation might be present also in the native viral antigen. This could provide a structural basis to explain the cross-reactivity of anti-preS2 monoclonal antibodies with these hybrid proteins.

Maltodextrin-binding protein hybrids carrying epitopes from the preS2 region of hepatitis B virus: expression, antibody-binding and preliminary crystallographic studies.

Five hybrid constructions of maltodextrin-binding protein (MBP), each containing an inserted epitope(s) from the preS2 region of the envelope proteins of hepatitis B virus (HBV), have been expressed. The anti-preS2 monoclonal antibody S2.3 was shown to cross-react with the MBP hybrid constructions, demonstrating that the epitopes presented by these recombinant proteins mimic the antigenic behaviour of the native viral antigen. In addition, all five hybrid proteins have been crystallized. Preliminary structural solutions obtained by molecular replacement indicate that the native conformation of MBP is preserved in the hybrid constructions despite the significant length of the epitope insertions.

Crystal structure of a recombinant form of the maltodextrin-binding protein carrying an inserted sequence of a B-cell epitope from the preS2 region of hepatitis B virus.

We report the crystal structure of MalE-B133, a recombinant form of the maltodextrin-binding protein (MBP) of Escherichia coli carrying an inserted amino-acid sequence of a B-cell epitope from the preS2 region of the hepatitis B virus (HBV). The structure was determined by molecular replacement methods and refined to 2.7 A resolution. MalE-B133 is an insertion/deletion mutant of MBP in which residues from positions 134 to 142, an external alpha helix in the wild-type structure, are replaced by a foreign peptide segment of 19 amino acids. The inserted residues correspond to the preS2 sequence from positions 132 to 145 and five flanking residues that arise from the creation of restriction sites. The conformation of the recombinant protein, excluding the inserted segment, closely resembles that of wild-type MBP in the closed maltose-bound form. MalE-B133 was shown by previous studies to display certain immunogenic and antigenic properties of the hepatitis B surface antigen (HBsAg), which contains the preS2 region. The crystal structure reveals the conformation of the first nine epitope residues (preS2 positions 132 to 140) exposed on the surface of the molecule. The remaining five epitope residues (preS2 positions 141 to 145) are not visible in electron density maps. The path of the polypeptide chain in the visible portion of the insert differs from that of the deleted segment in the structure of wild-type MBP, displaying a helical conformation at positions 134 to 140 (preS2 sequence numbering). A tripeptide (Asp-Pro-Arg) at the N terminus of the helix forms a stable structural motif that may be implicated in the cross-reactivity of anti-HBsAg antibodies with the hybrid protein.

Structural patterns at residue positions 9, 18, 67 and 82 in the VH framework regions of human and murine immunoglobulins.

VH framework regions of human and mouse immunoglobulins display three characteristic patterns in the conformation of the main polypeptide chain and side-chains at residue positions 9, 18, 67 and 82. These structural patterns are associated with the amino acid sequence at positions 9 and 67. Human and murine VH sequences show a strong correlation between the occurrence of Gly at position 9 and Phe at position 67 in VH subgroup III, and the frequent occurrence of Pro, Ala or Ser at position 9 and a non-aromatic residue at position 67 in other VH subgroups. Variations in VH framework segments have been shown to be of importance in procedures to humanize monoclonal murine antibodies and may be involved in the conformation of epitopes recognized by anti-VH antibodies. The structural patterns described here can be expected to influence the results of rotation and translation search functions in the crystallographic structure determination of Fab and Fv fragments by the molecular replacement method.