Domain Architecture of a Calcium-Permeable AMPA Receptor in a Ligand-Free Conformation

Ligand-gated ion channels couple the free energy of agonist binding to the gating of selective transmembrane ion pores, permitting cells to regulate ion flux in response to external chemical stimuli. However, the stereochemical mechanisms responsible for this coupling remain obscure. In the case of the ionotropic glutamate receptors (iGluRs), the modular nature of receptor subunits has facilitated structural analysis of the N-terminal domain (NTD), and of multiple conformations of the ligand-binding domain (LBD). Recently, the crystallographic structure of an antagonist-bound form of the receptor was determined. However, disulfide trapping of this conformation blocks channel opening, suggesting that channel activation involves additional quaternary packing arrangements. To explore the conformational space available to iGluR channels, we report here a second, clearly distinct domain architecture of homotetrameric, calcium-permeable AMPA receptors, determined by single-particle electron microscopy of untagged and fluorescently tagged constructs in a ligand-free state. It reveals a novel packing of NTD dimers, and a separation of LBD dimers across a central vestibule. In this arrangement, which reconciles diverse functional observations, agonist-induced cleft closure across LBD dimers can be converted into a twisting motion that provides a basis for receptor activation

Prostacyclin: An Inflammatory Paradox

Prostacyclin (PGI2) is a member of the prostaglandin family of bioactive lipids. Its best-characterized role is in the cardiovascular system, where it is released by vascular endothelial cells, serving as a potent vasodilator and inhibitor of platelet aggregation. In recent years, prostacyclin (PGI2) has also been shown to promote differentiation and inhibit proliferation in vascular smooth muscle cells. In addition to these well-described homeostatic roles within the cardiovascular system, prostacyclin (PGI2) also plays an important role as an inflammatory mediator. In this review, we focus on the contribution of prostacyclin (PGI2) as both a pathophysiological mediator and therapeutic agent in three major inflammatory-mediated disease processes, namely rheumatoid arthritis, where it promotes disease progression (“pro-inflammatory”), along with pulmonary vascular disease and atherosclerosis, where it inhibits disease progression (“anti-inflammatory”). The emerging role of prostacyclin (PGI2) in this context provides new opportunities for understanding the complex molecular basis for inflammatory-related diseases, and insights into the development of current and future anti-inflammatory treatments

Structure of the master regulator Rns reveals an inhibitor of enterotoxigenic Escherichia coli virulence regulons

Enteric infections caused by the gram-negative bacteria enterotoxigenic Escherichia coli (ETEC), Vibrio cholerae, Shigella flexneri, and Salmonella enterica are among the most common and affect billions of people each year. These bacteria control expression of virulence factors using a network of transcriptional regulators, some of which are modulated by small molecules as has been shown for ToxT, an AraC family member from V. cholerae. In ETEC the expression of many types of adhesive pili is dependent upon the AraC family member Rns. We present here the 3 Å crystal structure of Rns and show it closely resembles ToxT. Rns crystallized as a dimer via an interface similar to that observed in other dimeric AraC’s. Furthermore, the structure of Rns revealed the presence of a ligand, decanoic acid, that inhibits its activity in a manner similar to the fatty acid mediated inhibition observed for ToxT and the S. enterica homologue HilD. Together, these results support our hypothesis that fatty acids regulate virulence controlling AraC family members in a common manner across a number of enteric pathogens. Furthermore, for the first time this work identifies a small molecule capable of inhibiting the ETEC Rns regulon, providing a basis for development of therapeutics against this deadly human pathogen

Bile Salts And Alkaline Ph Reciprocally Modulate The Interaction Between The Periplasmic Domains Of Vibrio Cholerae Toxr And Toxs

ToxR is a transmembrane transcription factor that is essential for virulence gene expression and human colonization by Vibrio cholerae. ToxR requires its operon partner ToxS, a periplasmic integral membrane protein, for full activity. These two proteins are thought to interact through their respective periplasmic domains, ToxRp and ToxSp. In addition, ToxR is thought to be responsive to various environmental cues, such as bile salts and alkaline pH, but how these factors influence ToxR is not yet understood. Using NMR and reciprocal pull down assays, we present the first direct evidence that ToxR and ToxS physically interact. Furthermore, using NMR and DSF, it was shown that the bile salts cholate and chenodeoxycholate interact with purified ToxRp and destabilize it. Surprisingly, bile salt destabilization of ToxRp enhanced the interaction between ToxRp and ToxSp. In contrast, alkaline pH, which is one of the factors that leads to ToxR proteolysis, decreased the interaction between ToxRp and ToxSp. Taken together, these data suggest a model whereby bile salts or other detergents destabilize ToxR, increasing its interaction with ToxS to promote full ToxR activity. Subsequently, as V. cholerae alkalinizes its environment in late stationary phase, the interaction between the two proteins decreases, allowing ToxR proteolysis to proceed

Structural analysis of the master regulator Rns reveals a small molecule inhibitor of enterotoxigenic Escherichia coli virulence

Abstract Enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrheal disease worldwide and a frequent cause of travelers’ diarrhea. In addition to the production of enterotoxins, studies with human volunteers established ETEC virulence is dependent upon the production of proteinaceous adhesive pili for attaching to the intestinal wall. Although pilins are highly immunogenic, vaccines incorporating them have yet to be proven efficacious. An additional challenge for vaccines is the heterogeneity of ETEC pili, as 20 different pilus types have been identified. However, the expression of a significant number of pilus types is dependent upon Rns, an AraC family transcription factor. Furthermore, Rns also regulates the expression of the virulence factor CexE, an outer membrane coat protein. To determine how Rns functions and is regulated we solved its structure by X-ray crystallography to 3 Å resolution. Rns forms a dimer via its N-terminal domain and its structure is consistent with the dimer binding looped DNA. Our analyses also revealed a fatty acid, decanoic acid, bound within the Rns structure. Although Rns was not known to specifically bind small molecule ligands, biochemical analysis showed decanoic acid specifically stabilized Rns in a dose dependent manner. Lac reporter assays further showed that decanoic acid inhibits Rns function at both activated and repressed promoters. In situ, exogenous decanoic acid inhibited the expression of Rns-dependent CFA/I pili and CexE in different ETEC strains. Thus, our study reveals for the first time a naturally occurring small molecule ligand specifically inhibits Rns activity and potently suppresses the expression of ETEC virulence factors. Our findings provide an alternative approach to vaccines for inhibiting ETEC pathogenesis by using the Rns structure as a framework for rational drug design. Competing Interest Statement The authors have declared no competing interest. Footnotes * Update the abstract and methods