The three-dimensional structure of the cytoplasmic domains of EpsF from the type 2 secretion system of Vibrio cholerae.

The type 2 secretion system (T2SS), a multi-protein machinery that spans both the inner and the outer membranes of Gram-negative bacteria, is used for the secretion of several critically important proteins across the outer membrane. Here we report the crystal structure of the N-terminal cytoplasmic domain of EpsF, an inner membrane spanning T2SS protein from Vibrio cholerae. This domain consists of a bundle of six anti-parallel helices and adopts a fold that has not been described before. The long C-terminal helix alpha6 protrudes from the body of the domain and most likely continues as the first transmembrane helix of EpsF. Two N-terminal EpsF domains form a tight dimer with a conserved interface, suggesting that the observed dimer occurs in the T2SS of many bacteria. Two calcium binding sites are present in the dimer interface with ligands provided for each site by both subunits. Based on this new structure, sequence comparisons of EpsF homologs and localization studies of GFP fused with EpsF, we propose that the second cytoplasmic domain of EpsF adopts a similar fold as the first cytoplasmic domain and that full-length EpsF, and its T2SS homologs, have a three-transmembrane helix topology.

Multivalent drug design and inhibition of cholera toxin by specific and transient protein-ligand interactions.

Multivalent inhibitors of the cholera toxin B pentamer are potential therapeutic drugs for treating cholera and serve as models for demonstrating multivalent ligand effects through a structure-based approach. A crucial yet often overlooked aspect of multivalent drug design is the length, rigidity and chemical composition of the linker used to connect multiple binding moieties. To specifically study the role of chemical linkers in multivalent ligand design, we have synthesized a series of compounds with one and two binding motifs connected by several different linkers. These compounds have affinity for and potency against the cholera toxin B pentamer despite the fact that none can simultaneously bind two toxin receptor sites. Results from saturation transfer difference NMR reveal transient, non-specific interactions between the cholera toxin and linker groups contribute significantly to overall binding affinity of monovalent compounds. However, the same random protein-ligand interactions do not appear to affect binding of bivalent molecules. Moreover, the binding affinities and potencies of these 'non-spanning' bivalent ligands appear to be wholly independent of linker length. Our detailed analysis identifies multiple effects that account for the improved inhibitory potencies of bivalent ligands and suggest approaches to further improve the activity of this class of compounds.

Structural biology and structure-based inhibitor design of cholera toxin and heat-labile enterotoxin.

Structural biology studies on cholera toxin and the closely related heat-labile enterotoxin from enterotoxigenic Escherichia coli over the past decade have shed light on the mechanism of toxin action at molecular and atomic levels. Also, components of the extracellular protein secretion apparatus that translocate the toxins across the outer membrane are being investigated. At the same time, structure-based design has led to various classes of compounds targeting different toxin sites, including highly potent multivalent inhibitors that block the toxin receptor-binding process.

Nonspanning bivalent ligands as improved surface receptor binding inhibitors of the cholera toxin B pentamer.

A series of bivalent ligands of varying length were synthesized to inhibit the receptor-binding process of cholera toxin. Competitive surface receptor binding assays showed that significant potency gains relative to the constituent monovalent ligands were achieved independently from the ability of the extended bivalent ligands to span binding sites within the toxin pentamer. Several models that could account for the unexpected improvement in IC(50) values are examined, taking into account crystallographic analysis of each ligand in complex with the toxin pentamer. Evidence is presented that steric blocking at the receptor binding surface may play a role. The results of our study suggest that the use of relatively short, "nonspanning" bivalent ligands, or monovalent ligands of similar topology and bulk may be an effective way of blocking the interaction of multimeric proteins with their cell surface receptors.

3,5-Substituted phenyl galactosides as leads in designing effective cholera toxin antagonists; synthesis and crystallographic studies.

With the aim of developing high-affinity mono and multivalent antagonists of cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) we are using the galactose portion of the natural receptor ganglioside GM1 as an anchoring fragment in structure-based inhibitor design efforts. In order to establish a better structure-activity relationship for guiding these studies, we designed and prepared a small focused library of twenty 3,5-substituted phenylgalactosides based on two previous leads. The compounds were tested for their ability to block CTB(5) binding to immobilized ganglioside receptor and compared to the two previous leads. The crystal structures of the most promising compounds bound to either CTB(5) or LTB(5) were then determined in order to understand the basis for affinity differences. The most potent new compound yielded a six-fold improvement over our benchmark lead m-nitrophenyl-alpha-d-galactopyranoside (MNPG), and a two-fold improvement in IC(50) over a newer MNPG derivative. These results support the notion that the m-nitrophenyl moiety of MNPG and its derivatives is an important element to retain in future optimization efforts. Additionally, a consensus binding-pocket for the alkylmorpholine or piperazine moiety present in all of the designed antagonists was established as an important area of the GM1 binding site to target in future work.