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Abstract Calcium (Ca2+) is well known as a second messenger in eukaryotes, where Ca2+ signaling controls life-sustaining cellular processes. Although bacteria produce the components required for Ca2+ signaling, little is known about the mechanisms of bacterial Ca2+ signaling. Previously, we have identified a putative Ca2+-binding protein EfhP (PA4107) with two canonical EF-hand motifs and reported that EfhP mediates Ca2+ regulation of virulence factors production and infectivity in Pseudomonas aeruginosa, a human pathogen causing life-threatening infections. Here, we show that EfhP selectively binds Ca2+ with 13.7 µM affinity, and that mutations at the +X and −Z positions within each or both EF-hand motifs abolished Ca2+ binding. We also show that the hydrophobicity of EfhP increased in a Ca2+ -dependent manner, however no such response was detected in the mutated proteins. 15 N-NMR showed Ca2+-dependent chemical shifts in EfhP confirming Ca2+-binding triggered structural rearrangements in the protein. Deletion of efhP impaired P. aeruginosa survival in macrophages and virulence in vivo. Disabling EfhP Ca2+ binding abolished Ca2+ induction of pyocyanin production in vitro. These data confirm that EfhP selectively binds Ca2+, which triggers its structural changes required for the Ca2+ regulation of P. aeruginosa virulence, thus establishing the role of EfhP as a Ca2+ sensor.

The molecular chaperone Hsp90 oversees the folding of many proteins associated with cancer progression but existing small-molecule inhibitors of this pathway are not isoform-selective. Here, the authors rationally design an Hsp90 inhibitor that displays high selectivity for the Hsp90β isoform.

Abstract Interleukin-18 (IL-18) is a pleiotropic pro-inflammatory cytokine belonging to the IL-1 superfamily. IL-18 plays an important role in host innate and adaptive immune defense but its aberrant activities are also associated with inflammatory diseases such as rheumatoid arthritis and Crohn's disease. IL-18 activity is modulated in vivo by its naturally occurring antagonist, IL-18 Binding Protein (IL-18BP). Recent crystal structures of human IL-18 (hIL-18) in complex with its antagonists or cognate receptor(s) have revealed a conserved binding interface on hIL-18. Through virtual screening of the National Cancer Institute Diversity Set II and in vitro competitive ELISA we have identified three compounds (NSC201631, NSC80734, and NSC61610) that disrupt hIL-18 binding to the ectromelia virus IL-18BP. Through cell-based bioassay, we show that NSC80734 inhibits IL-18-induced production of IFN-γ in a dose-dependent manner with an EC50 of ~250 nM. Our results and methodology presented here demonstrate the feasibility of developing small molecule inhibitors that specifically target the rather large interface of IL-18 that is involved in extensive protein-protein interactions with both IL-18BP and its cognate receptor(s). Our data therefore provide the basis for an approach by which small molecules can be identified that modulate IL-18 activity.