Global landscape of cell envelope protein complexes in Escherichia coli.

Bacterial cell envelope protein (CEP) complexes mediate a range of processes, including membrane assembly, antibiotic resistance and metabolic coordination. However, only limited characterization of relevant macromolecules has been reported to date. Here we present a proteomic survey of 1,347 CEPs encompassing 90% inner- and outer-membrane and periplasmic proteins of Escherichia coli. After extraction with non-denaturing detergents, we affinity-purified 785 endogenously tagged CEPs and identified stably associated polypeptides by precision mass spectrometry. The resulting high-quality physical interaction network, comprising 77% of targeted CEPs, revealed many previously uncharacterized heteromeric complexes. We found that the secretion of autotransporters requires translocation and the assembly module TamB to nucleate proper folding from periplasm to cell surface through a cooperative mechanism involving the ß-barrel assembly machinery. We also establish that an ABC transporter of unknown function, YadH, together with the Mla system preserves outer membrane lipid asymmetry. This E. coli CEP 'interactome' provides insights into the functional landscape governing CE systems essential to bacterial growth, metabolism and drug resistance.

Evidence for a molecular diode-based mechanism in a multispecific ATP-binding cassette (ABC) exporter: SER-1368 as a gatekeeping residue in the yeast multidrug transporter Pdr5.

ATP-binding cassette multidrug efflux pumps transport a wide range of substrates. Current models suggest that a drug binds relatively tightly to a transport site in the transmembrane domains when the protein is in the closed inward facing conformation. Upon binding of ATP, the transporter can switch to an outward facing (drug off or drug releasing) structure of lower affinity. ATP hydrolysis is critically important for remodeling the drug-binding site to facilitate drug release and to reset the transporter for a new transport cycle. We characterized the novel phenotype of an S1368A mutant that lies in the putative drug-binding pocket of the yeast multidrug transporter Pdr5. This substitution created broad, severe drug hypersensitivity, although drug binding, ATP hydrolysis, and intradomain signaling were indistinguishable from the wild-type control. Several different rhodamine 6G efflux and accumulation assays yielded evidence consistent with the possibility that Ser-1368 prevents reentry of the excluded drug.

The deviant ATP-binding site of the multidrug efflux pump Pdr5 plays an active role in the transport cycle.

Pdr5 is the founding member of a large subfamily of evolutionarily distinct, clinically important fungal ABC transporters containing a characteristic, deviant ATP-binding site with altered Walker A, Walker B, Signature (C-loop), and Q-loop residues. In contrast to these motifs, the D-loops of the two ATP-binding sites have similar sequences, including a completely conserved aspartate residue. Alanine substitution mutants in the deviant Walker A and Signature motifs retain significant, albeit reduced, ATPase activity and drug resistance. The D-loop residue mutants D340A and D1042A showed a striking reduction in plasma membrane transporter levels. The D1042N mutation localized properly had nearly WT ATPase activity but was defective in transport and was profoundly hypersensitive to Pdr5 substrates. Therefore, there was a strong uncoupling of ATPase activity and drug efflux. Taken together, the properties of the mutants suggest an additional, critical intradomain signaling role for deviant ATP-binding sites.

Connecting membrane fluidity and surface charge to pore-forming antimicrobial peptides resistance by an ANN-based predictive model.

Efficiency of antibacterial chemotherapy is gradually more challenged by the emergence of pathogenic strains exhibiting high levels of antibiotic resistance. Pore-forming antimicrobial peptides (PF-AMPs) such as alamethicin (Alm) are therefore in the focus of extensive research efforts. In the present study, an artificial neural network (ANN)-based quantitative structure-activity relationship (SAR) modeling of membrane phospholipids vs. PF-AMPs, in context to membrane fluidity and surface charge, was carried out. We observed that the potency of PF-AMPs depends on the fatty acyl chain and polar head group of phospholipids. Alm showed surface interactions with zwitterionic phospholipids however could penetrate deeper inside the hydrophobic core of anionic membranes. Here, the resistance developed in bacterial cells was coupled to membrane fluidity and surface charge, and simultaneously, these principles could be applied for combating resistance against PF-AMPs. The correlation coefficient between observed CR and predicted CR using ANN was found to be 0.757. Thus, ANN could be used as a reliable modeling method for predicting CR, given the structure of the biomimetic membrane in terms of membrane fluidity and surface charge. Fully explored mechanisms of resistance, a forward modeling step in the design cycle of AMPs, can be cross-linked to the inward modeling using ANN to complete the peptide design cycle. The SAR between membrane phospholipids and PF-AMPs could furnish valuable information regarding their design to provide us efficacious peptides against premier pathogens. So far, this is the only report available to predict and quantify interactions of PF-AMPs with membrane phospholipids.