Substrate-mediated stabilization of a tetrameric drug target reveals Achilles heel in anthrax.

Bacillus anthracis is a gram-positive spore-forming bacterium that causes anthrax. With the increased threat of anthrax in biowarfare, there is an urgent need to characterize new antimicrobial targets from B. anthracis. One such target is dihydrodipicolinate synthase (DHDPS), which catalyzes the committed step in the pathway yielding meso-diaminopimelate and lysine. In this study, we employed CD spectroscopy to demonstrate that the thermostability of DHDPS from B. anthracis (Ba-DHDPS) is significantly enhanced in the presence of the substrate, pyruvate. Analytical ultracentrifugation studies show that the tetramer-dimer dissociation constant of the enzyme is 3-fold tighter in the presence of pyruvate compared with the apo form. To examine the significance of this substrate-mediated stabilization phenomenon, a dimeric mutant of Ba-DHDPS (L170E/G191E) was generated and shown to have markedly reduced activity compared with the wild-type tetramer. This demonstrates that the substrate, pyruvate, stabilizes the active form of the enzyme. We next determined the high resolution (2.15 A) crystal structure of Ba-DHDPS in complex with pyruvate (3HIJ) and compared this to the apo structure (1XL9). Structural analyses show that there is a significant (91 A(2)) increase in buried surface area at the tetramerization interface of the pyruvate-bound structure. This study describes a new mechanism for stabilization of the active oligomeric form of an antibiotic target from B. anthracis and reveals an "Achilles heel" that can be exploited in structure-based drug design.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of dihydrodipicolinate synthase from Bacillus anthracis in the presence of pyruvate.

Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step in the lysine-biosynthesis pathway in bacteria, plants and some fungi. In this study, the expression of DHDPS from Bacillus anthracis (Ba-DHDPS) and the purification of the recombinant enzyme in the absence and presence of the substrate pyruvate are described. It is shown that DHDPS from B. anthracis purified in the presence of pyruvate yields greater amounts of recombinant enzyme with more than 20-fold greater specific activity compared with the enzyme purified in the absence of substrate. It was therefore sought to crystallize Ba-DHDPS in the presence of the substrate. Pyruvate was soaked into crystals of Ba-DHDPS prepared in 0.2 M sodium fluoride, 20%(w/v) PEG 3350 and 0.1 M bis-tris propane pH 8.0. Preliminary X-ray diffraction data of the recombinant enzyme soaked with pyruvate at a resolution of 2.15 A are presented. The pending crystal structure of the pyruvate-bound form of Ba-DHDPS will provide insight into the function and stability of this essential bacterial enzyme.

Construction, crystal structure and application of a recombinant protein that lacks the collagen-like region of BclA from Bacillus anthracis spores.

Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by an exosporium, which consists of a basal layer surrounded by a nap of hair-like filaments. The major structural component of the filaments is called BclA, which comprises a central collagen-like region (CLR) and a globular C-terminal domain. Here, the entire CLR coding sequence of BclA was removed, and the resulting protein (tBclA) produced in Escherichia coli. The crystallographic structure of tBclA was determined to 1.35 A resolution, and consists of an all-beta structure with a TNF-like jelly fold topology (12 beta-strands which form 2 beta-sheets of five strands each) consistent with previous studies on wild-type BclA. These globular domains are tightly packed into trimeric structures (surface shape complementarity; S (c) = 0.83), demonstrating that formation of the core structure of BclA is independent of the anchoring collagen-like region. A polyclonal antibody raised against tBclA recognized B. anthracis spores directly, and showed little cross-reactivity (<10%) with the spores of the closely related species Bacillus cereus and Bacillus thuringiensis, when compared to two other polyclonal antibodies raised against B. anthracis spore extracts and inactivated spores. The tBclA protein was used to purify a pool of specific antibodies from bovine colostrum whey samples from cows inoculated with the Sterne strain anthrax vaccine, which also showed reactivity with B. anthracis spores. Together, these results demonstrate that tBclA provides a safer and more effective way to the production and purification of antibodies with high binding affinity for B. anthracis spores. Biotechnol. Bioeng. 2008;99: 774-782. (c) 2007 Wiley Periodicals, Inc.

Biofunctionalized surfactant mesophases as polyvalent inhibitors of cholera toxin.

The cubic lyotropic mesophase composed of the ganglioside G(M1) and the synthetic surfactant phytantriol has been employed as a scaffold to prepare a polyvalent inhibitor of cholera toxin (CT). Surfactant mixtures containing up to 20% (w/w) G(M1)/phytantriol afforded a hydrated insoluble gel-like material, which retained an inverse cubic phase (Q) structure in excess water and at 22 degrees C, as confirmed using small-angle X-ray scattering. The G(M1)-functionalized mesophases bind up to 98.8% of CT from solution containing 100 ng/mL of CT with a dissociation constant (K(d)) of 67-73 nM. The estimated IC50 values for the mesophase systems were 0.1-0.27 microM. The inhibitory effect of the mesophases may be enhanced through the high internal surface area of the inverse cubic phase in addition to the optimal self-orientation of G(M1) ligand to match the distribution of binding sites on the toxin surface. As a result, polyvalent inhibitor materials manufactured using these mesophase structures do not require precise control of ligand distribution, which offers advantages with respect to complexity of synthesis and formulation when compared to more rigid and conformationally restricted materials. This approach provides a route to a unique class of polyvalent inhibitors, which should be broadly applicable to a range of bacterial and plant toxins.

Monoclonal antibodies to ricin: in vitro inhibition of toxicity and utility as diagnostic reagents.

Monoclonal antibodies (MAbs) against ricin toxin (RT) and its subunits were produced in mice. The MAbs were initially selected based upon the ability to either bind ricin or the individual subunits in a solid-phase enzyme-linked immunosorbent assay (ELISA). Several candidates were selected for further evaluation, including their ability to inhibit ricin intoxication in vitro and their utility as immunodiagnostic reagents. Although their ability to capture antigen when bound to the solid phase was poor, some MAbs demonstrated potential utility as detection reagents in solid-phase immunoassays. Several MAbs were also able to inhibit ricin-mediated eukaryotic cell cytotoxicity in vitro. These MAbs may prove useful for preventing and/or treating ricin intoxication.

Expression of Campylobacter hyoilei lipo-oligosaccharide (LOS) antigens in Escherichia coli.

Campylobacter spp. are well recognized as primary pathogens in animals and in people. To isolate and define the genetic regions encoding major surface antigens of Campylobacter hyoilei, genomic DNA of the type strain of the species, RMIT-32A, was cloned into a cosmid vector, pLA2917, in Escherichia coli and the resulting genomic library was screened using antiserum raised to the parent C. hyoilei strain. Six cosmid clones were found to express a series of immunoreactive bands in the 15-25 kDa range. These bands were proteinase K-resistant and were found in the LPS fraction of the cells, suggesting that the recombinant cosmids expressed C. hyoilei lipo-oligosaccharide (LOS) antigen(s). The minimum DNA insert size required for expression of C. hyoilei LOS antigen(s) in E. coli was 11.8 kb. This region was subcloned into the plasmid vector pBR322. The partial sequencing of the 11.8 kb region showed that it contains two ORFs, designated rfbF and rfbP, showing homology with the rfbF gene from Serratia marcescens and the rfbP gene from Salmonella typhimurium. Both genes are involved in LPS synthesis. The region also contained a sequence homologous to the rfaC gene of E. coli and Sal. typhimurium which is involved in core oligosaccharide synthesis.