SUMMARY Quorum-sensing bacteria communicate via small molecules called autoinducers to coordinate collective behaviors. Because quorum sensing controls virulence factor expression in many clinically relevant pathogens, membrane-permeable quorum sensing antagonists that prevent population-wide expression of virulence genes offer a potential route to novel antibacterial therapeutics. Here, we report a strategy for inhibiting quorum-sensing receptors of the widespread LuxR family. Structure-function studies with natural and synthetic ligands demonstrate that the dimeric LuxR-type transcription factor CviR from Chromobacterium violaceum is potently antagonized by molecules that bind in place of the native acylated homoserine lactone autoinducer, provided that they stabilize a closed conformation. In such conformations, each of the two DNA-binding domains interacts with the ligand-binding domain of the opposing monomer. Consequently, the DNA-binding helices are held apart by ~60 Å, twice the ~30 Å separation required for operator binding. This approach may represent a general strategy for the inhibition of multi-domain proteins.

Bacteria can communicate via diffusible signal molecules they generate and release to coordinate their behavior in response to the environment. Signal molecule concentration is often proportional to bacterial population density, and when this reaches a critical concentration, reflecting a bacterial quorum, specific behaviors including virulence, symbiosis, and horizontal gene transfer are activated. Quorum-sensing regulation in many Gramnegative bacteria involves acylated homoserine lactone signals that are perceived through binding to LuxR-type, acylated-homoserine-lactone-responsive transcription factors. Bacteria of the rhizobial group employ the LuxR-type transcriptional activator TraR in quorum sensing, and its activity is further regulated through interactions with the TraM antiactivator. In this study, we have crystallographically determined the 3D structure of the TraR-TraM antiactivation complex from Rhizobium sp. strain NGR234. Unexpectedly, the antiactivator TraM binds to TraR at a site distinct from its DNA-binding motif and induces an allosteric conformational change in the protein, thereby preventing DNA binding. Structural analysis reveals a highly conserved TraR-TraM interface and suggests a mechanism for antiactivation complex formation. This structure may inform alternative strategies to control quorumsensing-regulated microbial activity including amelioration of infectious disease and antibiotic resistance. In addition, the structural basis of antiactivation presents a regulatory interaction that provides general insights relevant to the field of transcription regulation and signal transduction.crystal structure ͉ TraR-TraM complex ͉ allosteric mechanism ͉ protein-protein interaction ͉ signal transduction

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Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen and a primary cause of pneumonia and urinary tract infections in intensive care units (1, 2). Among the most vulnerable individuals are those with immunodeficiency, cystic fibrosis, or severe burns or who require mechanical ventilation. A major virulence determinant of P. aeruginosa is a type III secretion system (T3SS). 2 The T3SS encodes a multiprotein complex, called an injectisome, which functions by injecting or translocating effector proteins directly into the cytoplasm of eukaryotic host cells. The T3SS of P. aeruginosa is used to translocate four known effectors that subvert signal transduction pathways to inhibit phagocytosis and elicit cytotoxicity toward host cells (3, 4).Expression of the P. aeruginosa T3SS is highly regulated and under the direct transcriptional control of ExsA, a member of the AraC family of transcriptional activators (5). Environmental signals, including contact of P. aeruginosa with host cells and Ca 2ϩ -limiting growth conditions, induce ExsA-dependent transcription of the T3SS (6, 7). Although the mechanism of induction by host cell contact is unclear, Ca 2ϩ depletion regulates T3SS gene transcription by inducing type III secretory activity (8). Transcription of T3SS genes is intimately coupled to type III secretory activity through a regulatory cascade that governs ExsA activity. This regulatory cascade consists of three interacting proteins (ExsD, ExsC, and ExsE). ExsD functions as an anti-activator by directly binding to and inhibiting ExsA-dependent transcription (8). ExsC functions as an anti-anti-activator by binding to and antagonizing ExsD activity (9). Finally, ExsE is an inhibitor of ExsC activity and a secreted substrate of the T3SS (10, 11). Dasgupta et al. (9) have proposed the following model to account for transcriptional induction of T3SS genes by Ca 2ϩ depletion. Under Ca 2ϩ replete conditions, ExsE is retained in the cytoplasm because of the lack of type III secretory activity and forms a complex with ExsC. The sequestration of ExsC by ExsE allows for the binding of the anti-activator ExsD to ExsA, thereby blocking transcription. In response to Ca 2ϩ depletion, however, ExsE is secreted into the extracellular milieu. The corresponding decrease in intracellular ExsE levels releases ExsC, which then sequesters ExsD and thereby makes ExsA available to activate transcription of T3SS genes.A common feature of proteins secreted by the T3SS is the requirement for chaperone activity (12). ExsC has biochemical characteristics of a type III-specific chaperone and is required for ExsE stability within the cytoplasm and for ExsE secretion (9, 10). T3SS-specific chaperones fall into one of three classes based on substrate specificity. Class I, II, and III chaperones facilitate secretion of effectors, components of the transloca-

Mineralocorticoid receptor (MR) antagonists continue to be a prevalent area of research in the pharmaceutical industry. Herein we report the discovery of various spirooxindole and dibenzoxazepine constructs as potent MR antagonists. SAR analysis of our spirooxindole hit led to highly potent compounds containing polar solubilizing groups, which interact with the helix-11 region of the MR ligand binding domain (LBD). Various dibenzoxazepine moieties were also prepared in an effort to replace a known dibenzoxepane system which interacts with the hydrophobic region of the MR LBD. In addition, an X-ray crystal structure was obtained from a highly potent compound which was shown to exhibit both partial agonist and antagonist modes of action against MR.