The molecular pathway of enrofloxacin, a fluoroquinolone antibiotic, through the outer membrane channel OmpF of Escherichia coli is investigated. High-resolution ion current fluctuation analysis reveals a strong affinity for enrofloxacin to OmpF, the highest value ever recorded for an antibiotic-channel interaction. A single point mutation in the constriction zone of OmpF, replacing aspartic acid at the 113 position with asparagine (D113N), lowers the affinity to a level comparable to other antibiotics. All-atom molecular dynamics simulations allow rationalizing the translocation pathways: wild-type OmpF has two symmetric binding sites for enrofloxacin located at each channel entry separated by a large energy barrier in the center, which inhibits antibiotic translocation. In this particular case, our simulations suggest that the ion current blockages are caused by molecules occupying either one of these peripheral binding sites. Removal of the negative charge on position 113 removes the central barrier and shifts the two peripheral binding sites to a unique central site, which facilitates translocation. Fluorescence steady-state measurements agree with the different location of binding sites for wild-type OmpF and the mutant. Our results demonstrate how a single-point mutation of the porin, and the resulting intrachannel shift of the affinity site, may substantially modify translocation.

Proteinase3 (PR3) and human neutrophil elastase (HNE) are homologous proteases from the polymorphonuclear neutrophils and have been thought for a long time to have close enzymatic specificity. We have used molecular dynamics simulations to investigate and compare the interactions between different peptides and the two enzymes. The important role played especially by the C-terminal part of the peptides is confirmed. We provide a map of the subsites of PR3 and a description of the interaction scheme for six ligands. The main difference between HNE and PR3 concerns S2, S1', S2', and S3'. The recognition subsites in PR3 are interconnected; in particular, Lys99 participates to a hydrophobic (S4) and a polar (S2) pocket. On the basis of the simulations, we suggest that VADVKDR is a highly specific sequence for PR3; enzymatic assays confirm that it is cleaved by PR3 with a high specificity constant (k(cat)/K(m) = 3,400,000 M(-1) s(-1)) and not by HNE.

Proteinase 3 and neutrophil elastase are serine proteinases of the polymorphonuclear neutrophils, which are considered to have both similar localization and ligand specificity because of their high sequence similarity. However, recent studies indicate that they might have different and yet complementary physiologic roles. Specifically, proteinase 3 has intracellular specific protein substrates resulting in its involvement in the regulation of intracellular functions such as proliferation or apoptosis. It behaves as a peripheral membrane protein and its membrane expression is a risk factor in chronic inflammatory diseases. Moreover, in contrast to human neutrophil elastase, proteinase 3 is the preferred target antigen in Wegener’s granulomatosis, a particular type of vasculitis. We review the structural basis for the different ligand specificities and membrane binding mechanisms of both enzymes, as well as the putative anti‐neutrophil cytoplasm autoantibody epitopes on human neutrophil elastase 3. We also address the differences existing between murine and human enzymes, and their consequences with respect to the development of animal models for the study of human proteinase 3‐related pathologies. By integrating the functional and the structural data, we assemble many pieces of a complicated puzzle to provide a new perspective on the structure–function relationship of human proteinase 3 and its interaction with membrane, partner proteins or cleavable substrates. Hence, precise and meticulous structural studies are essential tools for the rational design of specific proteinase 3 substrates or competitive ligands that modulate its activities.

The biological functions of human neutrophil protease 3 (Pr3) differ from those of neutrophil elastase despite their close structural and functional resemblance. Although both proteases are strongly cationic, their sequences differ mainly in the distribution of charged residues. We have used these differences in electrostatic surface potential in the vicinity of their active site to produce fluorescence resonance energy transfer (FRET) peptide substrates for investigating individual Pr3 subsites. The specificities of subsites S5 to S3 were investigated both kinetically and by molecular dynamic simulations. Subsites S2, S1, and S2 were the main definers of Pr3 specificity. Combinations of results for each subsite were used to deduce a consensus sequence that was complementary to the extended Pr3 active site and was not recognized by elastase. Similar sequences were identified in natural protein substrates such as NFB and p21 that are specifically cleaved by Pr3. FRET peptides derived from these natural sequences were specifically hydrolyzed by Pr3 with specificity constants k cat /K m in the 10 6 M ؊1 s ؊1 range. The consensus Pr3 sequence may also be used to predict cleavage sites within putative protein targets like the proform of interleukin-18, or to develop specific Pr3 peptide-derived inhibitors, because none is available for further studies on the physiopathological function of this protease.Protease 3 (Pr3) 3 was initially described as an elastin-degrading protease whose structural and functional properties are similar to those of human neutrophil elastase (HNE) (1, 2). Pr3 is stored as an active enzyme within the primary granules of human neutrophils, together with HNE and cathepsin G, and is released from activated cells as a free or membrane-bound protease (3, 4). Its three-dimensional structure is very similar to that of HNE, with which its sequence is 57% identical (5). Pr3 and HNE also have extended interaction sites that greatly influence substrate binding and specificity (5). The active sites of the two proteases are also very similar, and both preferentially accommodate small aliphatic residues in their S1 subsite 4 (6, 7). This is why there was no substrate that discriminated between the two proteases until fluorescence resonance energy transfer (FRET) peptides became available. These can be used to study the specificity on both sides of the cleavage site (8, 9). However, kinetic and structural studies have shown that the substrate binding site in Pr3 is more polar, and some subsites are more restrictive than those in HNE (5, 10). This partly explains why Pr3, but not HNE, is not inhibited by the low molecular weight inhibitor SLPI present in the upper airways, even though both proteases are inhibited by the ␣1-protease inhibitor (␣1-Pi) in lung secretions (6,11).Although the primary function of Pr3 and HNE is commonly thought to be the intralysosomal degradation of phagocytized microorganisms, both also act extracellularly to break down matrix proteins (6, 12, 13), release cytokines from their ...

We use a multiscale approach, combining molecular dynamics simulations with metadynamics, to simulate the translocation of ampicillin through OmpF from Escherichia coli (E. coli). In-depth analysis has allowed us to reveal the complete picture of the translocation process in terms of both energetics and physicochemical properties. We have demonstrated the existence of a unique affinity site at the constriction region, accessible from both sides and defined by specific pore-antibiotic interactions. By providing optimal binding, the constriction region works like an enzyme toward the permeation of ampicillin. We find reduction in entropy to be compensated by enthalpic contributions from a favorable network of interactions (hydrogen bonds and hydrophobic contacts) which is also mediated by two slow water molecules bridging the antibiotic-pore interactions. Finally, as ampicillin assumes a preferential value for a torsional angle when at the constriction region, we investigated the consequence of the conformational preorganization of ampicillin toward its translocation. As a whole, our analysis opens the way to chemical modifications of antibiotics to allow improving uptake through porins contributing to combat bacterial resistance.

Our aim in this study was to provide an atomic description of ampicillin translocation through OmpF, the major outer membrane channel in Escherichia coli and main entry point for beta-lactam antibiotics. By applying metadynamics simulations, we also obtained the energy barriers along the diffusion pathway. We then studied the effect of mutations that affect the charge and size at the channel constriction zone, and found that in comparison to the wild-type, much lower energy barriers are required for translocation. The expected higher translocation rates were confirmed on the macroscopic scale by liposome-swelling assays. A microscopic view on the millisecond timescale was obtained by analysis of temperature-dependent ion current fluctuations in the presence of ampicillin and provide the enthalpic part of the energy barrier. By studying antibiotic translocation over various timescales and length scales, we were able to discern its molecular mechanism and rate-limiting interactions, and draw biologically relevant conclusions that may help in the design of drugs with enhanced permeation rates.

Gram-negative bacteria are protected by an outer membrane barrier, and to reach their periplasmic target, penicillins have to diffuse through outer membrane porins such as OmpF. Here we propose a structure-dynamics-based strategy for improving such antibiotic uptake. Using a variety of experiments (high-resolution single channel recording, Minimum Inhibitory Concentration (MIC), liposome swelling assay) and accelerated molecular simulations, we decipher the subtle balance of interactions governing ampicillin diffusion through the porin OmpF. This suggests mutagenesis of a hot spot residue of OmpF for which additional simulations reveal drastic changes in the molecular and energetic pathway of ampicillin's diffusion. Inverting the problem, we predict and describe how benzylpenicillin diffuses with a lower effective energy barrier by interacting differently with OmpF. The thorough comparison between the theoretical predictions and the three independent experiments, which were set up to measure the kinetics of transport and biological activity, gives insights on how to combine such different investigation techniques with the aim of providing complementary validation. Our study illustrates the importance of microscopic interactions at the constriction region of the biological channel to control the antibiotic flux through it. We conclude by providing a complete inventory of the channel and antibiotic hot spots and discuss the implications in terms of antibacterial screening and design.

In mammals, the olfactory epithelium secretes odorantbinding proteins (OBPs), which are lipocalins found freely dissolved in the mucus layer protecting the olfactory neurons. OBPs may act as passive transporters of predominantly hydrophobic odorant molecules across the aqueous mucus layer, or they may play a more active role in which the olfactory neuronal receptor recognizes the OBP-ligand complex. To better understand the molecular events accompanying the initial steps in the olfaction process, we have performed molecular dynamics studies of rat and pig OBPs with the odorant molecule thymol. These calculations provide an atomic level description of conformational changes and pathway intermediates that remain difficult to study directly. A series of eight independent molecular dynamics trajectories of rat OBP permitted the observation of a consensus pathway for ligand unbinding and the calculation of the potential of mean force (PMF) along this path. Titration microcalorimetry confirmed the specific binding of thymol to this protein with a strong hydrophobic component. In both rat and pig OBPs we observed lipocalin strand pair opening in the presence of ligand, consistent with potential roles of these proteins in olfactive receptor recognition.