No abstract

Quorum sensing (QS) is a population density-dependent regulatory system in bacteria that couples gene expression to cell density through accumulation of diffusible signaling molecules. Pectobacteria are causal agents of soft rot disease in a range of economically important crops. They rely on QS to coordinate their main virulence factor, production of plant cell wall degrading enzymes (PCWDEs). Plants have evolved an array of antimicrobial compounds to anticipate and cope with pathogens, of which essential oils (EOs) are widely recognized. Here, volatile EOs, carvacrol and eugenol, were shown to specifically interfere with QS, the master regulator of virulence in pectobacteria, resulting in strong inhibition of QS genes, biofilm formation and PCWDEs, thereby leading to impaired infection. Accumulation of the signal molecule N-acylhomoserine lactone declined upon treatment with EOs, suggesting direct interaction of EOs with either homoserine lactone synthase (ExpI) or with the regulatory protein (ExpR). Homology models of both proteins were constructed and docking simulations were performed to test the above hypotheses. The resulting binding modes and docking scores of carvacrol and eugenol support potential binding to ExpI/ExpR, with stronger interactions than previously known inhibitors of both proteins. The results demonstrate the potential involvement of phytochemicals in the control of Pectobacterium.

The pKa of the catalytic His57 N(epsilon)H in the tetrahedral complex (TC) of chymotrypsin with trifluoromethyl ketone inhibitors is 4-5 units higher relative to the free enzyme (FE). Such stable TC's, formed with transition state (TS) analog inhibitors, are topologically similar to the catalytic TS. Thus, analysis of this pKa shift may shed light on the role of water solvation in the general base catalysis by histidine. We applied our QM/SCRF(VS) approach to study this shift. The method enables explicit quantum mechanical DFT calculations of large molecular clusters that simulate chemical reactions at the active site (AS) of water solvated enzymes. We derived an analytical expression for the pKa dependence on the degree of water exposure of the ionizable group, and on the total charge in the enzyme AS, Q(A) and Q(B), when the target ionizable functional group (His57 in this study) is in the acidic (A) and basic (B) forms, respectively. Q2(B) > Q2(A) both in the FE and in the TC of chymotrypsin. Therefore, water solvation decreases the relative stability of the protonated histidine in both. Ligand binding reduces the degree of water solvation of the imidazole ring, and consequently elevates the histidine pKa. Thus, the binding of the ligand plays a triggering role that switches on the cascade of catalytic reactions in serine proteases.

Adult polyglucosan body disease (APBD) is a late-onset disease caused by intracellular accumulation of polyglucosan bodies, formed due to glycogen-branching enzyme (GBE) deficiency. To find a treatment for APBD, we screened 1,700 FDA-approved compounds in fibroblasts derived from APBD-modeling GBE1-knockin mice. Capitalizing on fluorescent periodic acid-Schiff reagent, which interacts with polyglucosans in the cell, this screen discovered that the flavoring agent guaiacol can lower polyglucosans, a result also confirmed in APBD patient fibroblasts. Biochemical assays showed that guaiacol lowers basal and glucose 6-phosphate-stimulated glycogen synthase (GYS) activity. Guaiacol also increased inactivating GYS1 phosphorylation and phosphorylation of the master activator of catabolism, AMP-dependent protein kinase. Guaiacol treatment in the APBD mouse model rescued grip strength and shorter lifespan. These treatments had no adverse effects except making the mice slightly hyperglycemic, possibly due to the reduced liver glycogen levels. In addition, treatment corrected penile prolapse in aged GBE1-knockin mice. Guaiacol's curative effects can be explained by its reduction of polyglucosans in peripheral nerve, liver, and heart, despite a short half-life of up to 60 minutes in most tissues. Our results form the basis to use guaiacol as a treatment and prepare for the clinical trials in APBD.

We focus on dimeric DNA-binding proteins from two well-studied families: orthodox type II restriction endonucleases (REs) and transcription factors (TFs). Interactions of the protein's recognition sites with the DNA and, particularly, the contribution of each of the monomers to one-dimensional (1D) sliding along nonspecific DNA were studied using computational tools. Coarse-grained molecular dynamics simulations of DNA scanning by various TFs and REs provide insights into how the symmetry of a homodimer can be broken while they nonspecifically interact with DNA. The characteristics of protein sliding along DNA, such as the average sliding length, partitioning between 1D and 3D search, and the one-dimensional diffusion coefficient D1, strongly depend on the salt concentration, which in turn affects the probability of the two monomers adopting a cooperative symmetric sliding mechanism. Indeed, we demonstrate that maximal DNA search efficiency is achieved when the protein adopts an asymmetric search mode in which one monomer slides while its partner hops. We find that proteins classified as TFs have a higher affinity for the DNA, longer sliding lengths, and an increased probability of symmetric sliding in comparison with REs. Moreover, TFs can perform their biological function over a much wider range of salt concentrations than REs. Our results demonstrate that the different biological functions of DNA-binding proteins are related to the different nonspecific DNA search mechanisms they adopt.