Rational design of novel peptidic DnaK ligands.

The hsp70 chaperone DnaK from E. coli plays a major role in cellular stress response and is involved in assisted protein folding in vivo. By screening a combinatorial peptide library, we identified several DnaK-specific peptide ligands with nanomolar affinities, which are able to inhibit the secondary amide peptide bond cis/trans isomerase (APIase) activity of DnaK, as well as DnaK/DnaJ/GrpE-assisted refolding of firefly luciferase. Our designed DnaK inhibitors have the capability to penetrate E. coli cells and feature a high protease resistance. Once inside the cell, they physically target DnaK. NMR-based (1)H/(15)N-HSQC experiments furthermore confirmed that the designed peptidic ligands all bind in an identical manner to the conventional peptide-binding site of DnaK. The subsequent blocking of DnaK function apparently results in the observed antibacterial effects on E. coli cells, with minimum inhibitory concentrations in the range of 100 microM.

Allosteric coupling between the lid and interdomain linker in DnaK revealed by inhibitor binding studies.

The molecular chaperone DnaK assists protein folding and refolding, translocation across membranes, and regulation of the heat shock response. In Escherichia coli, the protein is a target for insect-derived antimicrobial peptides, pyrrhocoricins. We present here the X-ray crystallographic analysis of the E. coli DnaK substrate-binding domain in complex with pyrrhocoricin-derived peptide inhibitors. The structures show that pyrrhocoricins act as site-specific, dual-mode (competitive and allosteric) inhibitors, occupying the substrate-binding tunnel and disrupting the latch between the lid and the beta-sandwich. Our structural analysis revealed an allosteric coupling between the movements of the lid and the interdomain linker, identifying a previously unknown mechanism of the lid-mediated regulation of the chaperone cycle.

Rotavirus VP6 as an Adjuvant for Bivalent Norovirus Vaccine Produced in Nicotiana benthamiana.

Rotaviruses (RVs) and noroviruses (NoVs) are major causes of childhood acute gastroenteritis. During development of a combination vaccine based on NoV virus-like particles (VLP) and RV VP6 produced in baculovirus expression system in insect cells, a dual role of VP6 as a vaccine antigen and an adjuvant for NoV-specific immune responses was discovered. Here the VP6 adjuvant effect on bivalent GI.4 and GII.4-2006a NoV VLPs produced in Nicotiana benthamiana was investigated. BALB/c mice were immunized intradermally with suboptimal (0.3 µg) dose of each NoV VLP alone or combined with 10 µg of VP6, or equal doses of NoV VLPs and VP6 (1 µg/antigen). NoV-specific serum IgG antibodies and their blocking activity were analyzed using vaccine-homologous and heterologous NoV VLPs. Immunization with 0.3 µg NoV VLPs alone was insufficient to induce NoV-specific immune responses, but with co-administration of 10 µg of VP6, antibodies against vaccine-derived and heterologous NoV genotypes were generated. Furthermore, corresponding adjuvant effect of VP6 was observed with 1 µg dose. Efficient uptake and presentation of VP6 by dendritic cells was demonstrated in vitro. These results show that adjuvant effect of VP6 on bivalent NoV VLP vaccine is independent of the cell source used for vaccine production.

A novel Hsp70 inhibitor prevents cell intoxication with the actin ADP-ribosylating Clostridium perfringens iota toxin.

Hsp70 family proteins are folding helper proteins involved in a wide variety of cellular pathways. Members of this family interact with key factors in signal transduction, transcription, cell-cycle control, and stress response. Here, we developed the first Hsp70 low molecular weight inhibitor specifically targeting the peptide binding site of human Hsp70. After demonstrating that the inhibitor modulates the Hsp70 function in the cell, we used the inhibitor to show for the first time that the stress-inducible chaperone Hsp70 functions as molecular component for entry of a bacterial protein toxin into mammalian cells. Pharmacological inhibition of Hsp70 protected cells from intoxication with the binary actin ADP-ribosylating iota toxin from Clostridium perfringens, the prototype of a family of enterotoxins from pathogenic Clostridia and inhibited translocation of its enzyme component across cell membranes into the cytosol. This finding offers a starting point for novel therapeutic strategies against certain bacterial toxins.

Fatty acyl benzamido antibacterials based on inhibition of DnaK-catalyzed protein folding.

We have reported that the hsp70 chaperone DnaK from Escherichia coli might assist protein folding by catalyzing the cis/trans isomerization of secondary amide peptide bonds in unfolded or partially folded proteins. In this study a series of fatty acylated benzamido inhibitors of the cis/trans isomerase activity of DnaK was developed and tested for antibacterial effects in E. coli MC4100 cells. N(alpha)-[Tetradecanoyl-(4-aminomethylbenzoyl)]-l-asparagine is the most effective antibacterial with a minimal inhibitory concentration of 100 +/- 20 microg/ml. The compounds were shown to compete with fluorophore-labeled sigma(32)-derived peptide for the peptide binding site of DnaK and to increase the fraction of aggregated proteins in heat-shocked bacteria. Despite its inability to serve as a folding helper in vivo a DnaK-inhibitor complex was still able to sequester an unfolded protein in vitro. Structure activity relationships revealed a distinct dependence of DnaK-assisted refolding of luciferase on the fatty acyl chain length, whereas the minimal inhibitory concentration was most sensitive to the structural nature of the benzamido core. We conclude that the isomerase activity of DnaK is a major survival factor in the heat shock response of bacteria and that small molecule inhibitors can lead to functional inactivation of DnaK and thus will display antibacterial activity.