Recently, we documented that the short, proline-rich antibacterial peptides pyrrhocoricin, drosocin, and apidaecin interact with the bacterial heat shock protein DnaK, and peptide binding to DnaK can be correlated with antimicrobial activity. In the current report we studied the mechanism of action of these peptides and their binding sites to Escherichia coli DnaK. Biologically active pyrrhocoricin made of L-amino acids diminished the ATPase activity of recombinant DnaK. The inactive D-pyrrhocoricin analogue and the membrane-active antibacterial peptide cecropin A or magainin 2 failed to inhibit the DnaK-mediated phosphate release from adenosine 5'-triphosphate (ATP). The effect of pyrrhocoricin on DnaK's other significant biological function, the refolding of misfolded proteins, was studied by assaying the alkaline phosphatase and beta-galactosidase activity of live bacteria. Remarkably, both enzyme activities were reduced upon incubation with L-pyrrhocoricin or drosocin. D-Pyrrhocoricin, magainin 2, or buforin II, an antimicrobial peptide involved in binding to bacterial nucleic acids, had only negligible effect. According to fluorescence polarization and dot blot analysis of synthetic DnaK fragments and labeled pyrrhocoricin analogues, pyrrhocoricin bound with a K(d) of 50.8 microM to the hinge region around the C-terminal helices D and E, at the vicinity of amino acids 583 and 615. Pyrrhocoricin binding was not observed to the homologous DnaK fragment of Staphylococcus aureus, a pyrrhocoricin nonresponsive strain. In line with the lack of ATPase inhibition, drosocin binding appears to be slightly shifted toward the D helix. Our data suggest that drosocin and pyrrhocoricin binding prevents the frequent opening and closing of the multihelical lid over the peptide-binding pocket of DnaK, permanently closes the cavity, and inhibits chaperone-assisted protein folding. The biochemical results were strongly supported by molecular modeling of DnaK-pyrrhocoricin interactions. Due to the prominent sequence variations of procaryotic and eucaryotic DnaK molecules in the multihelical lid region, our findings pave the road for the design of strain-specific antibacterial peptides and peptidomimetics. Far-fetched applications of the species-specific inhibition of chaperone-assisted protein folding include the control of not only bacteria but also fungi, parasites, insects, and perhaps rodents.

Protein carbonylation, one of the most harmful irreversible oxidative protein modifications, is considered as a major hallmark of oxidative stress-related disorders. Protein carbonyl measurements are often performed to assess the extent of oxidative stress in the context of cellular damage, aging and several age-related disorders. A wide variety of analytical techniques are available to detect and quantify protein-bound carbonyls generated by metal-catalyzed oxidation, lipid peroxidation or glycation/glycoxidation. Here we review current analytical approaches for protein carbonyl detection with a special focus on mass spectrometry-based techniques. The utility of several carbonyl-derivatization reagents, enrichment protocols and especially advanced mass spectrometry techniques are compared and discussed in detail. Furthermore, the mechanisms and biology of protein carbonylation are summarized based on recent high-throughput proteomics data.

Proline-rich antimicrobial peptides (PrAMPs) have been investigated and optimized by several research groups and companies as promising lead compounds to treat systemic infections caused by Gram-negative bacteria. PrAMPs, such as apidaecins and oncocins, enter the bacteria and kill them apparently through inhibition of specific targets without a lytic effect on the membranes. Both apidaecins and oncocins were shown to bind with nanomolar dissociation constants to the 70S ribosome. In apidaecins, at least the two C-terminal residues (Arg17 and Leu18) interact strongly with the 70S ribosome, whereas residues Lys3, Tyr6, Leu7, and Arg11 are the major interaction sites in oncocins. Oncocins inhibited protein biosynthesis very efficiently in vitro with half maximal inhibitory concentrations (IC50 values) of 150 to 240 nmol L(-1). The chaperone DnaK is most likely not the main target of PrAMPs but it binds them with lower affinity.

The receptor for advanced glycated end products (RAGE) is a multiligand receptor that is implicated in the pathogenesis of various diseases, including diabetic complications, neurodegenerative disorders, and inflammatory responses. The ability of RAGE to recognize advanced glycated end products (AGEs) formed by nonenzymatic glycoxidation of cellular proteins places RAGE in the category of pattern recognition receptors. The structural mechanism of AGE recognition was an enigma due to the diversity of chemical structures found in AGE-modified proteins. Here, using NMR spectroscopy we showed that the immunoglobulin V-type domain of RAGE is responsible for recognizing various classes of AGEs. Three distinct surfaces of the V domain were identified to mediate AGE-V domain interactions. They are located in the positively charged areas of the V domain. The first interaction surface consists of strand C and loop CC, the second interaction surface consists of strand C, strand F, and loop FG, and the third interaction surface consists of strand A and loop EF. The secondary structure elements of the interaction surfaces exhibit significant flexibility on the ms-s time scale. Despite highly specific AGE-V domain interactions, the binding affinity of AGEs for an isolated V domain is low, ϳ10 M. Using in-cell fluorescence resonance energy transfer we show that RAGE is a constitutive oligomer on the plasma membrane. We propose that constitutive oligomerization of RAGE is responsible for recognizing patterns of AGE-modified proteins with affinities less than 100 nM.

SUMMARY Nonenzymatic protein glycation results in the formation of advanced glycation end products (AGEs) that were implicated in the pathology of diabetes, chronic inflammation, Alzheimer’s disease, and cancer. AGEs mediate their effects primarily through a receptor-dependent pathway in which AGEs bind to a specific cell surface associated receptor, the Receptor for AGEs (RAGE). Nε-carboxy-methyl-lysine (CML) and Nε-carboxy-ethyl-lysine (CEL), constitute two of the major AGE structures found in tissue and blood plasma, and are physiological ligands of RAGE. The solution structure of a CEL containing peptide-RAGE V domain complex reveals that the carboxyethyl moiety fits inside a positively charged cavity of the V domain. Peptide backbone atoms make specific contacts with the V domain. The geometry of the bound CEL peptide is compatible with many CML (CEL) modified sites found in plasma proteins. The structure explains how such patterned ligands as CML (CEL)-proteins bind to RAGE and contribute to RAGE signaling.