Solution NMR studies of J-domain co-chaperones and bacterial outer membrane permeability

The ubiquitous chaperone Hsp70 is a major player in guiding and controlling cellular protein folding processes. It is ATP-dependent and co-chaperoned by individual members of the diverse class of J-domain proteins (JDPs). By synergistic action, these co-chaperones mediate a plethora of versatile functions such as protein maturation, disaggregation, protein translocation and degradation. JDPs bind client proteins and transfer them to Hsp70 while simultaneously stimulating the intrinsically low ATP hydrolysis rate of Hsp70 with their highly conserved J-domain. Here, we compare the structural and functional characteristics of the conserved GF-region of two JDPs from the endoplasmic reticulum (ER), ERdj3 and ERdj4, using NMR spectroscopy. We demonstrate that the GF-region of ERdj3 and ERdj4 forms an alpha-helix (helix 5) as has been observed for cytoplasmic DNAJB1. However, in stark contrast to their cytoplasmic counterpart, the helix 5 of ERdj3 and ERdj4 populates a conformational equilibrium of the folded helix docked to the J-domain and an undocked disordered conformation. Interestingly, helix 5 occupies the same binding interface involved in binding to the ER-resident Hsp70 BiP during the stimulation of ATP hydrolysis. We then show that the binding of helix 5 to the J-domain is characterized by different binding strengths which results in distinct functional consequences for the interaction of ERdj3 and ERdj4 with BiP. While for ERdj4 helix 5 binds strongly to the J-domain and thereby inhibits the interaction with BiP, it has no impact on the functional synergy between BiP and ERdj3. Therefore, we conclude that the GF-region confers specificity with respect to BiP binding among these two ER-resident JDPs.
Pseudomonas aeruginosa can cause severe infections and constitutes a substantial challenge for human health due to its resistance to antibiotics and disinfectants. An important factor for this intrinsic antibiotic resistance is the remarkably low permeability of the outer membrane of P. aeruginosa with respect to the uptake of nutrients and drugs. Translocation of molecules across the outer membrane is tightly regulated by a large number of specific porins. Unlike other Gram-negative bacteria, P. aeruginosa simultaneously achieves a high metabolic versatility and a particularly low outer membrane permeability. Herein, we address this paradox by investigating the role of these porins in antibiotic and nutrient uptake. We established an NMR based assay in combination with 4 genetically modified P. aeruginosa strains that allowed us to measure substrate consumption of individual porins under in vivo conditions. Our data reveal, that except for the porin OprD, the porins do not constitute an entry for antibiotics into the cell. Systematic in vivo NMR spectroscopy-based measurements of the translocatome of the outer membrane porins reveal promiscuous overlapping substrate profiles for 14 tested porins suggesting specificity by exclusion rather than by selective import of substances. Surprisingly, we find that positively charged and hydrophobic substrates can pass the outer membrane in a porin-independent manner. In contrast, the specific porins are required for the transport of molecules with two or more carboxylate groups. These findings elucidate the role of different porins and the outer membrane in antibiotics permeation and provide new insights to improve and develop new antimicrobial compounds for the treatment of infectious diseases.