The primary virulence factors of many pathogenic bacteria are secreted protein toxins which bind to glycolipid receptors on host cell surfaces. The binding specificities of three such toxins for different glycolipids, mainly from the ganglioside series, were determined by surface plasmon resonance (SPR) using a liposome capture method. Unlike microtiter plate and thin layer chromatography overlay assays, the SPR/liposome methodology allows for real time analysis of toxin binding under conditions that mimic the natural cell surface venue of these interactions and without any requirement for labeling of toxin or receptor. Compared to conventional assays, the liposome technique showed more restricted oligosaccharide specificities for toxin binding. Cholera toxin demonstrated an absolute requirement for terminal galactose and internal sialic acid residues (as in G M1 ) with tolerance for substitution with a second internal sialic acid (as in G D1b ). Escherichia coli heat-labile enterotoxin bound to G M1 and tolerated removal or extension of the internal sialic acid residue (as in asialo-G M1 and G D1b , respectively) but not substitution of the terminal galactose of G M1 . Tetanus toxin showed a requirement for two internal sialic acid residues as in G D1b . Extension of terminal galactose with a single sialic acid was tolerated to some extent. The SPR analyses also yielded rate and affinity constants which are not attainable by conventional assays. Complex binding profiles were observed in that the association and dissociation rate constants varied with toxin:receptor ratios. The sub-nanomolar affinities of cholera toxin and heat-labile enterotoxin for liposome-anchored gangliosides were attributable largely to very slow dissociation rate constants. The SPR/liposome technology should have general applicability in the study of glycolipid-protein interactions and in the evaluation of reagents designed to interfere with these interactions.The protein toxins produced by many pathogenic bacteria are among the best characterized virulence factors. These toxins typically bind to oligosaccharide receptors on host cell surfaces (1). Many belong to the AB 5 family of toxins which are comprised of an enzymatically active and toxic A-subunit and five B-subunits which form the receptor binding portion of the molecule. In most instances the five B-subunits are identical and allow for pentameric attachment to the cell surface receptors. Crystal structures of five AB 5 toxins or their B-pentamers, three complexed with carbohydrate receptors, have been reported. These are cholera toxin (2, 3), Escherichia coli heatlabile toxin (4 -6), shiga toxin (7), shiga-like toxin (8), and pertussis toxin (9). However, this wealth of structural data has not answered all questions relating to the oligosaccharide-binding specificities of these molecules. For example, there is some controversy as to the nature of the functional receptor of LT. Although structurally very similar to CT, LT shows subtle differences in receptor binding specificity (1...

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The extreme pH and protease-rich environment of the upper gastrointestinal tract is a major obstacle facing orally-administered protein therapeutics, including antibodies. Through protein engineering, several Clostridium difficile toxin A-specific heavy chain antibody variable domains (VHHs) were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. Mutant antibodies were compared to their wild-type counterparts with respect to expression yield, non-aggregation status, affinity for toxin A, circular dichroism (CD) structural signatures, thermal stability, protease resistance, and toxin A-neutralizing capacity. The mutant VHHs were found to be well expressed, although with lower yields compared to wild-type counterparts, were non-aggregating monomers, retained low nM affinity for toxin A, albeit the majority showed somewhat reduced affinity compared to wild-type counterparts, and were capable of in vitro toxin A neutralization in cell-based assays. Far-UV and near-UV CD spectroscopy consistently showed shifts in peak intensity and selective peak minima for wild-type and mutant VHH pairs; however, the overall CD profile remained very similar. A significant increase in the thermal unfolding midpoint temperature was observed for all mutants at both neutral and acidic pH. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants. Mutant VHH trypsin resistance was similar to that of wild-type VHHs, although the trypsin resistance of one VHH mutant was significantly reduced. Therefore, the introduction of a second disulfide bond in the hydrophobic core not only increases VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance, with only minor perturbations in target binding affinities. These are all desirable characteristics for the design of protein-based oral therapeutics.

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The plasma membrane is uniquely enriched in phosphatidylserine (PtdSer). This anionic phospholipid is restricted almost exclusively to the inner leaflet of the plasmalemma. Because of their high density, the headgroups of anionic lipids experience electrostatic repulsion that, being exerted asymmetrically, is predicted to favor membrane curvature. We demonstrate that cholesterol limits this repulsion and tendency to curve. Removal of cholesterol or insertion of excess PtdSer increases the charge density of the inner leaflet, generating foci of enhanced charge and curvature where endophilin and synaptojanin are recruited. From these sites emerge tubules that undergo fragmentation, resulting in marked endocytosis of PtdSer. Shielding or reduction of the surface charge or imposition of outward membrane tension minimized invagination and PtdSer endocytosis. We propose that cholesterol associates with PtdSer to form nanodomains where the headgroups of PtdSer are maintained sufficiently separated to limit spontaneous curvature while sheltering the hydrophobic sterol from the aqueous medium.

Aberrant glycosylation and the overexpression of certain carbohydrate moieties is a consistent feature of cancers, and tumorassociated oligosaccharides are actively investigated as targets for immunotherapy. One of the most common aberrations in glycosylation patterns is the presentation of a single O-linked N-acetylgalactosamine on a threonine or serine residue known as the "Tn antigen." Whereas the ubiquitous nature of Tn antigens on cancers has made them a natural focus of vaccine research, such carbohydrate moieties are not always tumor-specific and have been observed on embryonic and nonmalignant adult tissue. Here we report the structural basis of binding of a complex of a monoclonal antibody (237mAb) with a truly tumor-specific glycopeptide containing the Tn antigen. In contrast to glycopeptide-specific antibodies in complex with simple peptides, 237mAb does not recognize a conformational epitope induced in the peptide by sugar substitution. Instead, 237mAb uses a pocket coded by germ-line genes to completely envelope the carbohydrate moiety itself while interacting with the peptide moiety in a shallow groove. Thus, 237mAb achieves its striking tumor specificity, with no observed physiological cross-reactivity to the unglycosylated peptide or the free glycan, by a combination of multiple weak but specific interactions to both the peptide and to the glycan portions of the antigen.X-ray crystallography | affinity maturation | crystal structure

The kinetics of ligand binding by Se155-4, an antibody specific for the Salmonella serogroup B O-polysaccharide, were studied by surface plasmon resonance. Because trace amounts of oligomers in Fab and singlechain antibody variable domain (scFv) preparations resulted in biphasic binding profiles that were difficult to analyze, all kinetic measurements were performed on purified monomeric fragments and, for certain mutant scFv, dimeric forms. Results obtained with monomeric forms indicated that the relatively low affinity of the antibody was due to rapid dissociation (k off Ϸ 0.25 s ؊1 ). Dimeric forms generally showed off-rates that were approximately 20-fold slower and a 5-fold increase in association rate constants to approximately 2 ؋ 10 5 M ؊1 s ؊1 . Although the association phases for scFv dimers showed good curve fitting to a one component interaction model, the dissociation phases were biphasic, presumably because the availability and accessibility of sites on the antigen always leads to some monovalent attachment. The fast off-rate for dimers was the same as the monomer off-rate. Se155-4 IgG off-rates were very similar to those observed for scFv dimer, whereas the onrate was the same as that obtained with Fab and scFv monomer.The relatively weak affinities that characterize protein-carbohydrate interactions make understanding how biological processes are mediated by oligosaccharides difficult. However, it is becoming evident, particularly for certain plant and animal lectins, that specificity is achieved by a combination of multivalence and the geometry of the subunit arrangement. There is now a considerable amount of data available on structural and energetic aspects of protein-carbohydrate interactions (1, 2). General structural features are the stacking of aromatic side-chains against the sugar rings, the presence of hydrogen bond networks in which the sugar OH groups act as both acceptors and donors, and the coordination of multiple hydrogen bonds by water molecules (1). As for their energetics, titration microcalorimetry has indicated that these interactions are usually enthalpy-driven and that water reorganization, especially desolvation, is a key feature of complexation (2). Kinetics of the interactions are less well known but are obviously important in understanding the subtleties of carbohydrate-mediated biological events. Without consideration of the time factor, analysis of these interactions from a biological standpoint is difficult. The development of surface plasmon resonance techniques has provided an opportunity to explore biomolecular interaction in real time.We have applied SPR 1 technology to the analysis of antigen binding by the antibody Se155-4. The specificity of Se155-4 is dominated by a 3,6-dideoxyhexose, abequose, presented by the lipopolysaccharide O-antigen of Salmonella serogroup B bacteria (3). This polysaccharide repeating unit is built from four hexopyranose units: {32)[␣D-Abe(133)]␣D-Man(134)␣L-Rha(133)␣D-Gal(13}. The structural (4) and energetic (5, 6) aspects of antigen...

The lysosomal cysteine proteinase cathepsin B (EC 3.4.22.1) plays an important role in protein catabolism and has also been implicated in various disease states. The crystal structures of two forms of native recombinant rat cathepsin B have been determined. The overall folding of rat cathepsin B was shown to be very similar to that of the human liver enzyme. The structure of the native enzyme containing an underivatized active site cysteine (Cys29) showed the active enzyme conformation to be similar to that determined previously for the oxidized form. In a second structure Cys29 was derivatized with the reversible blocking reagent pyridyl disulfide. In this structure large side chain conformational changes were observed for the two key catalytic residues Cys29 and His199, demonstrating the potential flexibility of these side chains. In addition the structure of the complex between rat cathepsin B and the inhibitor benzyloxycarbonyl-Arg-Ser(O-Bzl) chloromethylketone was determined. The complex structure showed that very little conformational change occurs in the enzyme upon inhibitor binding. It also allowed visualization of the interaction between the enzyme and inhibitor. In particular the interaction between Glu245 and the P2 Arg residue was clearly demonstrated, and it was found that the benzyl group of the P1 substrate residue occupies a large hydrophobic pocket thought to represent the S'1 subsite. This may have important implications for structure-based design of cathepsin B inhibitors.