Acad. Surprisingly, the complexes formed by two neutralizing antibodies recognizing TcdA do not show direct interference with the previously identified carbohydrate-binding sites, suggesting that RO-1138452 neutralization of toxin activity may be mediated by mechanisms distinct from steric blockage of receptor binding. A camelid sdAb complex also reveals the molecular structure of the TcdB RBD for the first time, facilitating the crystallization of a strongly negatively charged protein fragment that has resisted previous attempts at crystallization and structure determination. Electrospray ionization mass spectrometry measurements confirm the stoichiometries of sdAbs observed in the crystal structures. These studies indicate how key epitopes in the RBDs from TcdA and TcdB are recognized by sdAbs, providing molecular insights into toxin structure and function and providing for the first time a basis for the design of highly specific toxin-specific therapeutic and diagnostic agents. Keywords: Antibodies, Bacterial Toxins, Mass Spectrometry (MS), Protein-Protein Interactions, X-ray Crystallography Introduction Infection of the large intestine by the obligate anaerobic bacterium is one of the most common and costly hospital-acquired diseases worldwide (1, 2). Although CDI2 is often effectively treated with specific antibiotics, 15C20% of patients suffer recurrent forms of the disease that lack effective treatments. The high economic cost (more than $8 billion/year in the United States alone) and morbidity associated with CDI, as well as the increased prevalence of hypervirulent strains in recent years, underline the urgent need for the development of novel and more effective therapeutics (3, 4). Our approach to develop novel therapeutics has focused on understanding and limiting the pathogenic effects of the two main virulence factors, toxins A and B (TcdA and TcdB) (5, 6). The sequence and three-dimensional structure of TcdA and TcdB reveal a complex, multidomain Rabbit polyclonal to PHF10 architecture in which separate domains are primarily responsible for distinct activities, each of which are essential to the overall pathogenic effects of the toxins (7C9). The three-dimensional arrangement of domains within the toxins has been explored using electron microscopy (10) and small angle x-ray scattering (11), and crystal structures have been determined for several of the domains in isolation (9). The RO-1138452 N-terminal glucosyltransferase domain transfers glucose or RO-1138452 TcdA, the conserved residues mediating packing interactions between adjacent -hairpins differ significantly. Also, the sequences of the LRs in TcdA differ substantially from the LRs in TcdB, even though the sequences of the LRs within each protein are very highly conserved. The effects of these differences on the three-dimensional structure and function of the two toxins have remained poorly understood until the structure below was determined. Some of these structural differences help to explain some of the dramatic functional differences previously reported for the two toxins. Open in a separate window FIGURE 1. Schematic diagram showing the arrangement of SRs (and purified as described previously (12, 13, 24C27). An additional cation exchange chromatography purification step (HiTrap-SP HP column equilibrated in 20 mm Na-HEPES, pH 7.0, 20 mm NaCl, 50 g/liter glycerol and eluted with a 0.02C1 m NaCl gradient in the same buffer) was added to improve the purity of all VHHs. For B39 VHH, 20 mm Na-MOPS, pH 6.5, was used in place of Na-HEPES. Protein concentrations were determined by measuring absorbance at 280 nm, and extinction coefficients were calculated based on amino acid composition using the ExPASy webserver (28). Prior to concentrating protein for crystallization, TcdA-A1 was dialyzed overnight at 4 C against RO-1138452 20 mm Tris-Cl, pH 7.5, 0.15 m NaCl, 0.5 mm EDTA, 30 g/liter glycerol; TcdA-A2 was dialyzed overnight at 4 C against 20 mm Bis-Tris-Cl, pH 6.5, 0.15 m NaCl, 0.5 mm EDTA, 30 g/liter glycerol, 15 g/liter sodium benzenesulfonate; and TcdB-B1 was dialyzed overnight at 4 C against 20 mm Bis-Tris-Cl, pH 6.5, 0.1 m NaCl, 0.5 mm EDTA, 30 g/liter glycerol. Prior to crystallization, VHHs and toxin RBD fragments were mixed in specific molar ratios and diluted into the Tris buffer for the TcdA-A1 complex, the Bis-Tris buffer for TcdA-A2 complexes, and the Bis-Tris buffer without benzenesulfonate for the TcdB-B1 complex. Each mixture was then concentrated using centrifugal filters (10,000 molecular weight cutoff; Millipore) to achieve a final total protein concentration of 5 mg/ml. Protein mixtures were subjected to sparse matrix crystallization screens to identify conditions for crystal growth (see Table 1). Conditions from the initial hits from the sparse matrix screens were optimized to.