The structure of the extracellular domains of human interleukin 11α receptor reveals mechanisms of cytokine engagement

Interleukin (IL) 11 activates multiple intracellular signaling pathways by forming a complex with its cell surface α-receptor, IL-11Rα, and the β-subunit receptor, gp130. Dysregulated IL-11 signaling has been implicated in several diseases, including some cancers and fibrosis. Mutations in IL-11Rα that reduce signaling are also associated with hereditary cranial malformations. Here we present the first crystal structure of the extracellular domains of human IL-11Rα and a structure of human IL-11 that reveals previously unresolved detail. Disease-associated mutations in IL-11Rα are generally distal to putative ligand-binding sites. Molecular dynamics simulations showed that specific mutations destabilize IL-11Rα and may have indirect effects on the cytokine-binding region. We show that IL-11 and IL-11Rα form a 1:1 complex with nanomolar affinity and present a model of the complex. Our results suggest that the thermodynamic and structural mechanisms of complex formation between IL-11 and IL-11Rα differ substantially from those previously reported for similar cytokines. This work reveals key determinants of the engagement of IL-11 by IL-11Rα that may be exploited in the development of strategies to modulate formation of the IL-11–IL-11Rα complex.

QStatin, a Selective Inhibitor of Quorum Sensing in Vibrio Species

ABSTRACTPathogenicVibriospecies cause diseases in diverse marine animals reared in aquaculture. Since their pathogenesis, persistence, and survival in marine environments are regulated by quorum sensing (QS), QS interference has attracted attention as a means to control these bacteria in aquatic settings. A few QS inhibitors ofVibriospecies have been reported, but detailed molecular mechanisms are lacking. Here, we identified a novel, potent, and selectiveVibrioQS inhibitor, named QStatin [1-(5-bromothiophene-2-sulfonyl)-1H-pyrazole], which affectsVibrio harveyiLuxR homologues, the well-conserved master transcriptional regulators for QS inVibriospecies. Crystallographic and biochemical analyses showed that QStatin binds tightly to a putative ligand-binding pocket in SmcR, the LuxR homologue inV. vulnificus, and changes the flexibility of the protein, thereby altering its transcription regulatory activity. Transcriptome analysis revealed that QStatin results in SmcR dysfunction, affecting the expression of SmcR regulon required for virulence, motility/chemotaxis, and biofilm dynamics. Notably, QStatin attenuated representative QS-regulated phenotypes in variousVibriospecies, including virulence against the brine shrimp (Artemia franciscana). Together, these results provide molecular insights into the mechanism of action of an effective, sustainable QS inhibitor that is less susceptible to resistance than other antimicrobial agents and useful in controlling the virulence ofVibriospecies in aquacultures.IMPORTANCEYields of aquaculture, such as penaeid shrimp hatcheries, are greatly affected by vibriosis, a disease caused by pathogenicVibrioinfections. Since bacterial cell-to-cell communication, known as quorum sensing (QS), regulates pathogenesis ofVibriospecies in marine environments, QS inhibitors have attracted attention as alternatives to conventional antibiotics in aquatic settings. Here, we used target-based high-throughput screening to identify QStatin, a potent and selective inhibitor ofV. harveyiLuxR homologues, which are well-conserved master QS regulators inVibriospecies. Structural and biochemical analyses revealed that QStatin binds tightly to a putative ligand-binding pocket on SmcR, the LuxR homologue inV. vulnificus, and affects expression of QS-regulated genes. Remarkably, QStatin attenuated diverse QS-regulated phenotypes in variousVibriospecies, including pathogenesis against brine shrimp, with no impact on bacterial viability. Taken together, the results suggest that QStatin may be a sustainable antivibriosis agent useful in aquacultures.

Cloning, expression, purification, crystallization and preliminary crystallographic analysis of the N-terminal domain of serine glutamate repeat A (SgrA) protein fromEnterococcus faecium

Serine glutamate repeat A (SgrA) protein is an LPxTG surface adhesin ofEnterococcus faeciumand is the first bacterial nidogen-binding protein identified to date. It has been suggested that it binds to human nidogen, the extracellular matrix molecule of basal lamina, and plays a key role in the invasion and colonization of eukaryotic host cells. SgrA28–288, having both a putative ligand-binding A domain and repetitive B domain, was expressed inEscherichia coliand purified using Ni-affinity and hydrophobic interaction chromatography. Further, the putative ligand-binding region, rSgrA28–153, was subcloned, overexpressed and purified in both native and selenomethionine-derivative forms. The native rSgrA28–153protein crystallized in the monoclinic space groupP21and diffracted to 3.3 Å resolution using an in-house X-ray source, with unit-cell parametersa= 35.84,b= 56.35,c= 60.20 Å, β = 106.5°.

Identification of a Receptor Subunit and Putative Ligand-Binding Residues Involved in the Bacillus megaterium QM B1551 Spore Germination Response to Glucose

ABSTRACT The molecular basis for the recognition of glucose as a germinant molecule by spores of Bacillus megaterium QM B1551 has been examined. A chromosome-located locus (BMQ_1820, renamed gerWB) is shown to encode a receptor B-protein subunit that interacts with the GerUA and GerUC proteins to form a receptor that is cognate for both glucose and leucine. GerWB represents the third receptor B protein that binds to glucose in this strain. Site-directed mutagenesis (SDM) experiments conducted on charged proline and aromatic residues predicted to reside in the transmembrane domains of a previously characterized receptor B protein, GerVB, reveal the importance to receptor function of a cluster of residues predicted to reside in the middle of the transmembrane 6 (TM6) domain. Reductions in the region of 70- to 165-fold in the apparent affinity of receptors for glucose in which Glu196, Tyr191, and Phe192 are individually replaced by SDM indicate that some or all of these residues may be directly involved in the binding of glucose and perhaps other germinants to the germinant receptor.