In this study, we utilize Protein Residue Networks (PRNs), constructed using Local Spatial Pattern (LSP) alignment, to explore the dynamic behavior of Catabolite Activator Protein (CAP) upon the sequential binding of cAMP. We employed the Degree Centrality of these PRNs to investigate protein dynamics on a sub-nanosecond time scale, hypothesizing that it would reflect changes in CAP\'s entropy related to its thermal motions. We show that the binding of the first cAMP led to an increase in stability in the Cyclic-Nucleotide Binding Domain A (CNBD-A) and destabilization in CNBD-B, agreeing with previous reports explaining the negative cooperativity of cAMP binding in terms of an entropy-driven allostery. LSP-based PRNs also allow for the study of Betweenness Centrality, another graph-theoretical characteristic of PRNs, providing insights into global residue connectivity within CAP. Using this approach, we were able to correctly identify amino acids that were shown to be critical in mediating allosteric interactions in CAP. The agreement between our studies and previous experimental reports validates our method, particularly with respect to the reliability of Degree Centrality as a proxy for entropy related to protein thermal dynamics. Because LSP-based PRNs can be easily extended to include dynamics of small organic molecules, polynucleotides, or other allosteric proteins, the methods presented here mark a significant advancement in the field, positioning them as vital tools for a fast, cost-effective, and accurate analysis of entropy-driven allostery and identification of allosteric hotspots.

We present a rare comparison of structures of the same protein but generated by different potentials. We used four popular water potentials (SPC, TIP3P, TIP4P, TIP5P) in conjunction with the equally popular ff99SB. However, the ff12SB protein potential was used with TI3P only. Simulations (60 ns) were run on the catabolite activator protein (CAP), which is a textbook case of allosteric interaction. Overall, all potentials generated largely similar structures but failed to reproduce a crucial structural feature determined by NMR experiment. This example shows the need to develop next-generation potentials. Graphical abstract Catabolite activator protein.

Pseudomonas aeruginosa is an opportunistic pathogen that presents a complex regulatory network called \'quorum-sensing\', which is responsible for the transcription of genes coding for several traits implicated in its pathogenicity. Strain 148 is a dolphin isolate that has been shown to produce quorum-sensing-regulated virulence traits and to be virulent in a mouse model, despite the fact that it contains a 20-kbp deletion that eliminates from the chromosome the lasR gene and the lasI promoter. LasR is a key quorum-sensing transcriptional regulator that, when coupled with the autoinducer 3-oxo-dodecanoyl homoserine lactone (3O-C12-HSL) produced by LasI, activates transcription of genes coding for some virulence-associated traits such as elastase, lasI, rhlI and rhlR. RhlR is also a key quorum-sensing transcriptional regulator that, when interacting with the autoinducer butanoyl homoserine lactone (C4-HSL) that is produced by the synthase RhlI, activates the genes involved in the synthesis of some virulence-associated traits, as rhamnolipids and pyocyanin. We describe that in P. aeruginosa 148, the LasR/3O-C12-HSL-independent rhlR transcriptional activation is due to the release of the negative effect of Vfr (a CRP-ortholog) caused by the insertion of an IS element in vfr, and that rhlI transcription is driven from the rhlR promoter, forming the rhlR-I operon.

During the past two decades, the phenomenon of hormesis has gained increasing recognition in environmental and toxicological communities. However, the mechanistic understanding of hormesis, to date, is extremely limited. Herein is proposed a novel parametric model with a mechanistic basis and two model-based parameters for hormesis that was successfully applied to the hormetic dose-response observed in the chronic toxicity of sulfonamides on Photobacterium phosphoreum. On the basis of the methods of molecular docking and quantitative structure-activity relationships (QSARs), we proposed a mechanistic hypothesis for hormesis that introduces for the first time the concept of quorum sensing in toxicological studies and explains the mechanism at the level of the receptors. The mechanistic hypothesis stated that (1) specific target binding like interaction with LuxR may contribute to transcriptional activation leading to enhanced luciferase activity at low dose exposure of sulfonamides, and (2) as the dose of sulfonamides increases, more sulfonamides competitively bind to dihydropteroate synthase, which inhibit the biosynthesis of folic acid and thus provoke toxicity. This mechanistic hypothesis, which explains both the dose-dependent and time-dependent features of hormesis, could give new insight into the mechanistic study of hormesis.

The sigma(E) (extracytoplasmic stress response sigma factor in Escherichia coli) signaling system of Gram-negative bacteria plays an essential role in the maintenance of the extracytoplasmic compartment. Upon induction of this system, approximately 100 genes are up-regulated. The majority of these genes encode proteins that participate in the synthesis, assembly, and homeostasis of outer membrane proteins and lipopolysaccharides. A second aspect of the sigma(E) response is a regulatory loop that prevents expression of the major porins. Misfolding or overproduction of most of these porins is sufficient to trigger the envelope stress response. Recent work indicates that small Hfq-binding RNAs play a major role in maintaining envelope homeostasis and, so far, two sigma(E)-dependent small noncoding RNAs (sRNAs), MicA and RybB, have been shown to facilitate rapid removal of multiple omp transcripts in response to elevated activity of the alternative sigma factor. Here we report the identification of the sRNA (CyaR, cyclic AMP-activated RNA) that promotes decay of the ompX mRNA. The transcription of the cyaR gene is stringently controlled by cAMP-cAMP receptor protein and, unexpectedly, cyaR expression is also up-regulated, directly or indirectly, by sigma(E). In addition, this work identified MicA as a factor that cooperates in the negative control of ompX expression. The conservation of CyaR, MicA, RybB, and their targets suggests that the omp mRNA-sRNA regulatory network is an integral part of the envelope stress response in many enterobacteria.