Mechanism of ligand-gated potassium efflux in bacterial pathogens

Abstract

The undersigned authors wish to note, "The KefFC system of E. coli is maintained in an inactive state by the binding of glutathione (GSH) and is activated by the formation of GSH adducts (GSX), particularly those with bulky substituents. We described two crystal structures with density present in the ligand-binding domain that we interpreted as GSH and GSX. Recently, an independent, experienced crystallographer, who had viewed the structures from our study in a different context, made representations to us that cast doubt on position of the succinimido ring of GSX. We have further reviewed the density maps with the aid of an experienced crystallographer. As a consequence, we believe it is important to draw this altered interpretation of the crystal structures to the attention of readers. In both structures, the density for the backbone of GSH is clear and allows unequivocal assignment of the position of the tripeptide. In PDB coordinate set 3L9X, the density for the succinimido ring is very weak, making interpretation very speculative and the assignment rests on the identity of the ligand added to the crystallization mixture, for which there are two diastereomers in the solutiona possibility that provides some basis for weakening the density. However, in 3L9W there are two anomalies that affect the interpretation of the bound ligand. First, there is no density for the carbon atom attached to the sulfur of GSH and second, there is extra density adjacent to the position of sulfur that could be modelled as a constrained succinimido ring. However, this density could also be water or any other molecule that is trapped in the structure. Thus, while there is good evidence for the peptide, the evidence that it is in the GSH form is uncertain."There are no new data on either the structures or on the gating mechanism. However, we believe that we should be cautious in interpreting the structural data and that the field in general should be made aware of the alternative views of the electron density data. Note that the mutagenesis and spectroscopic data that were presented in the original manuscript are not affected by this alternative interpretation." Tarmo P. The authors note that the following grant should be added to the Acknowledgments: "NIH Grant AG002132." The authors note "The method used for exogenous expression of Ca V 1.2 channels in ref. 32 was incorrectly described as 'viral transduction' in the text. In fact, Yang et al. created transgenic mice with inducible, cardiomyocyte-specific expression of exogenous Ca V 1.2 channels regulated by a tetracycline-inducible promoter. When crossed with a transgenic mouse line expressing doxycycline-regulated reverse transcriptional activator under control of the α-myosin heavy chain protomer, the resulting double transgenic offspring expressed exogenous Ca V 1.2 channels in their cardiac myocytes after treatment with doxycycline. The authors regret the error in describing these methods."www.pnas.org/cgi

KefF, the Regulatory Subunit of the Potassium Efflux System KefC, Shows Quinone Oxidoreductase Activity

“…S4 in the supplemental material). KefF elutes as a homogeneous peak at an elution volume corresponding to a dimeric complex, as expected from the crystal structure and homologous quinone reductases (15,23,35,36). For a soluble protein, KefF shows unusual behavior on SDS gels as, even at moderate loading concentrations, it runs as a diffuse band rather than the sharp bands observed for other proteins on similar SDS-PAGE gels ( Fig.…”

“…After 5 min, either quinones or NEM (0.5 mM final concentration) was added, and the potassium content of the medium was recorded continuously. Rate constants were obtained, using the program Origin 8.0 (Originlab) (35), by fitting exponentials to the change in potassium concentration after addition of an activator.…”

“…A subset of quinones that incorporate an enone moiety are electrophilic and can therefore undergo conjugate addition reactions with nucleophiles (4). GSH, the ligand that binds to KefC (35), spontaneously reacts with these quinones, and multiple additions at the different ortho positions can take place if the quinole is reoxidized to the quinone (22). Benzoquinone and menadione, which belong to this group of GSH-reactive quinones, elicit potassium efflux via KefC (Fig.…”

“…Kef systems are found only in Gram-negative bacteria and have been most extensively studied in Escherichia coli, which has two homologues, KefB and KefC, showing differential sensitivity to electrophiles (12). Recently, we showed that GSH and GSX occupy overlapping binding pockets in KefC but that the additional steric bulk of GSX causes a significant conformational change, which results in activation of KefC (35). In addition, the KTN domain has a nucleotide binding site on a Rossman fold (36), and it has been suggested that KefC is inactivated when NADH is bound at this site (16).…”

Modulation of cardiac Kv currents by Kvbeta2 and pyridine nucleotides.

“…The KefC transport system is inactivated by reduced glutathione, and it is activated by glutathione-S-conjugates. 137 Activation of this transport by glutathione conjugates leads to acidification of the cytosol.…”