Molecular Basis of Filtering Carbapenems by Porins from β-Lactam-resistant Clinical Strains of Escherichia coli

Integral membrane proteins known as porins are the major pathway by which hydrophilic antibiotics cross the outer mem- brane of Gram-negative bacteria. Single point mutations in porins can decrease the permeability of an antibiotic, either by reduction of channel size or modification of electrostatics in the channel, and thereby confer clinical resistance. Here, we inves- tigate four mutant OmpC proteins from four different clinical isolates of Escherichia coli obtained sequentially from a single patient during a course of antimicrobial chemotherapy. OmpC porin from the first isolate (OmpC20) undergoes three consec- utive and additive substitutions giving rise to OmpC26, OmpC28, and finally OmpC33. The permeability of two zwitte- rionic carbapenems, imipenem and meropenem, measured using liposome permeation assays and single channel electro- physiology differs significantly between OmpC20 and OmpC33. Molecular dynamic simulations show that the antibiotics must pass through the constriction zone of porins with a specific ori- entation, where the antibiotic dipole is aligned along the electric field inside the porin. We identify that changes in the vector of the electric field in the mutated porin, OmpC33, create an addi- tional barrier by “trapping” the antibiotic in an unfavorable ori- entation in the constriction zone that suffers steric hindrance for the reorientation needed for its onward translocation. Iden- tification and understanding the underlying molecular details of such a barrier to translocation will aid in the design of new anti- biotics with improved permeation properties in Gram-negative bacteria

Conformational analysis and in vitro immunomodulatory and insulinotropic properties of the frog skin host-defense peptide rhinophrynin-27 and selected analogs

The study investigates conformational analysis and the in vitro cytokine-mediated immunomodulatory and insulin-releasing activities of rhinophrynin-27 (ELRLPEIARPVPEVLPARLPLPALPRN; RP-27), a proline-arginine-rich peptide first isolated from skin secretions of the Mexican burrowing toad Rhinophrynus dorsalis (Rhinophrynidae). In both water and 50% trifluoroethanol-water, the peptide adopts a polyproline type II helical conformation with a high degree of deviation from the canonical collagen-like folding and a pronounced bend in the molecule at the Glu13 residue. Incubation of mouse peritoneal cells with RP-27 significantly (P < 0.05) inhibited production of the pro-inflammatory cytokines TNF-α and IL-1β and stimulated production of the anti-inflammatory cytokine IL-10. The peptide significantly (P < 0.01) stimulated release of insulin from BRIN-BD11 rat clonal β-cells at concentrations ≥ 1 nM while maintaining the integrity of the plasma membrane and also stimulated insulin release from isolated mouse islets at a concentration of 10−6 M. Increasing the cationicity of RP-27 by substituting glutamic acid residues in the peptide by arginine and increasing hydrophobicity by substituting alanine residues by tryptophan did not result in analogues with increased activity with respect to cytokine production and insulin release. The combination of immunosuppressive and insulinotropic activities together with very low cytotoxicity suggests that RP-27 may represent a template for the development of an agent for use in anti-inflammatory and Type 2 diabetes therapies

VDAC3 as a sensor of oxidative state of the intermembrane space of mitochondria: the putative role of cysteine residue modifications

Voltage-Dependent Anion selective Channels (VDAC) are pore-forming mitochondrial outer membrane proteins. In mammals VDAC3, the least characterized isoform, presents a set of cysteines predicted to be exposed toward the intermembrane space. We find that cysteines in VDAC3 can stay in different oxidation states. This was preliminary observed when, in our experimental conditions, completely lacking any reducing agent, VDAC3 presented a pattern of slightly different electrophoretic mobilities. This observation holds true both for rat liver mitochondrial VDAC3 and for recombinant and refolded human VDAC3. Mass spectroscopy revealed that cysteines 2 and 8 can form a disulfide bridge in native VDAC3. Single or combined site-directed mutagenesis of cysteines 2, 8 and 122 showed that the protein mobility in SDS-PAGE is influenced by the presence of cysteine and by the redox status. In addition, cysteines 2, 8 and 122 are involved in the stability control of the pore as shown by electrophysiology, complementation assays and chemico-physical characterization. Furthermore, a positive correlation between the pore conductance of the mutants and their ability to complement the growth of porin-less yeast mutant cells was found. Our work provides evidence for a complex oxidation pattern of a mitochondrial protein not directly involved in electron transport. The most likely biological meaning of this behavior is to buffer the ROS load and keep track of the redox level in the intermembrane space, eventually signaling it through conformational change

Rationalizing the permeation of polar antibiotics into Gram-negative bacteria

The increasing level of antibiotic resistance in Gram-negative bacteria, together with the lack of new potential drug scaffolds in the pipeline, make the problem of infectious diseases a global challenge for modern medicine. The main reason that Gram-negative bacteria are particularly challenging is the presence of an outer cell-protecting membrane, which is not present in Gram-positive species. Such an asymmetric bilayer is a highly effective barrier for polar molecules. Several protein systems are expressed in the outer membrane to control the internal concentration of both nutrients and noxious species, in particular: (i) water-filled channels that modulate the permeation of polar molecules and ions according to concentration gradients, and (ii) efflux pumps to actively expel toxic compounds. Thus, besides expressing specific enzymes for drugs degradation, Gram-negative bacteria can also resist by modulating the influx and efflux of antibiotics, keeping the internal concentration low. However, there are no direct and robust experimental methods capable of measuring the permeability of small molecules, thus severely limiting our knowledge of the molecular mechanisms that ultimately control the permeation of antibiotics through the outer membrane. This is the innovation gap to be filled for Gram-negative bacteria. This review is focused on the permeation of small molecules through porins, considered the main path for the entry of polar antibiotics into Gram-negative bacteria. A fundamental understanding of how these proteins are able to filter small molecules is a prerequisite to design/optimize antibacterials with improved permeation. The level of sophistication of modern molecular modeling algorithms and the advances in new computer hardware has made the simulation of such complex processes possible at the molecular level. In this work we aim to share our experience and perspectives in the context of a multidisciplinary extended collaboration within the IMI-Translocation consortium. The synergistic combination of structural data, in vitro assays and computer simulations has proven to give new insights towards the identification and description of physico–chemical properties modulating permeation. Once similar general rules are identified, we believe that the use of virtual screening techniques will be very helpful in searching for new molecular scaffolds with enhanced permeation, and that molecular modeling will be of fundamental assistance to the optimization stage

Nondestructive Surface Depth Profiles from Angle-Resolved X-ray Photoelectron Spectroscopy Data Using the Maximum Entropy Method. I. A New Protocol

The knowledge of the depth concentration profile of thin-layered Surfaces a few nanometers thick is very important for research and applications in microelectronics, corrosion, wear, and tribology. In-depth profiling methods reported in the literature are either destructive (ion sputtering), based on severe approximations (concentration gradients are not taken into account, and electron inelastic mean free paths (IMFPs) are calculated for electrons traveling throughout pure elemental materials) or limited to relatively simple profiles (less than three components and constant IMFPs). A reconstructed depth profile should be consistent with the angle-resolved X-ray photoelectron spectroscopy (ARXPS) data acquired, but transformation of XPS signal intensities vs emission angle into chemical species concentrations vs depth is an ill-posed mathematical problem which requires inversion of a Laplace transform. The main goal of this work was thus to develop a new, iterative protocol based on the maximum entropy method (MEM) that allows obtaining in-depth concentration profiles of layered surfaces from nondestructive ARXPS measurements. Numerical experiments were performed on a large series of computer generated, ideal, and error-containing ARXPS data from model depthprofiles with lip to four layers and up to eight components. The new algorithm enabled LIS to reconstruct these depth profiles with a maximum uncertainty of +/-20% for layer thickness and of +/-30% for composition of the individual layers. Moreover, the new protocol involves an iterative procedure for calculating the IMFP values of the different components, taking into account the actual depth concentration profile of the sample surface under investigation. The new protocol proved to be more powerful than any of the existing algorithms since it has been successfully applied for reconstructing depth profiles with up to eight components

Human myoglobin: two isoforms that differ at single residue. Their different dynamics suggest distinct and complementary role

In Human up to five different myoglobin isoforms are expressed. Iso-I (∼75-80%) and iso-II (∼15-20%) are the more expressed ones and differ only at the 54th position, K54 and E54 respectively. It has been reported that myoglobin concentration in muscles is higher for high-altitude natives than sea-level populations, and that only iso-II is over-expressed among the myoglobin isoforms. This over-expression is thought to be one of the evolutive adaptation to the high-altitude hypoxic environment. Since it is widely accepted that myoglobin is not only an oxygen storage/deliver system, iso-II over expression has been related to other functions such as NO scavenger and/or nitrite reductase. In this work Molecular Dynamics simulations were applied to study the dynamics of 54K and 54E human myoglobins. Statistical analysis of internal cavities and their interconnections helped to highlight and compare the intrinsic dynamic behavior of these two proteins. Furthermore, the role of the solvent in the mutation proximity has been investigated and it was found that water molecules can quasi-allosterically modulate the dynamics of myoglobin distal region. Important differences have been found especially at the histidine gate, even if the two myoglobin isoforms differ at only one residue that, indeed, is not located at the gate itself. These differences suggest that the two more expressed human myoglobins might have a distinct and complementary role. This would well fit the literature scenario where NO scavenging is though to be important at normoxic condition to retain mitochondrial respiration efficacy, while nitrite reduction is believed to be crucial under hypoxic stress. NO release by myoglobin would decrease mitochondrial oxygen consumption preserving the optimal O2 gradient across the cell but also ensuring sufficient ATP synthesis