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

The solution structure of the heavy chain–only C5-Fc nanobody reveals exposed variable regions that are optimal for COVID-19 antigen interactions

Heavy chain–only antibodies can offer advantages of higher binding affinities, reduced sizes, and higher stabilities than conventional antibodies. To address the challenge of SARS-CoV-2 coronavirus, a llama-derived single-domain nanobody C5 was developed previously that has high COVID-19 virus neutralization potency. The fusion protein C5-Fc comprises two C5 domains attached to a glycosylated Fc region of a human IgG1 antibody and shows therapeutic efficacy in vivo. Here, we have characterized the solution arrangement of the molecule. Two 1443 Da N-linked glycans seen in the mass spectra of C5-Fc were removed and the glycosylated and deglycosylated structures were evaluated. Reduction of C5-Fc with 2-mercaptoethylamine indicated three interchain Cys–Cys disulfide bridges within the hinge. The X-ray and neutron Guinier RG values, which provide information about structural elongation, were similar at 4.1 to 4.2 nm for glycosylated and deglycosylated C5-Fc. To explain these RG values, atomistic scattering modeling based on Monte Carlo simulations resulted in 72,737 and 56,749 physically realistic trial X-ray and neutron structures, respectively. From these, the top 100 best-fit X-ray and neutron models were identified as representative asymmetric solution structures, similar to that of human IgG1, with good R-factors below 2.00%. Both C5 domains were solvent exposed, consistent with the functional effectiveness of C5-Fc. Greater disorder occurred in the Fc region after deglycosylation. Our results clarify the importance of variable and exposed C5 conformations in the therapeutic function of C5-Fc, while the glycans in the Fc region are key for conformational stability in C5-Fc

How Does the VSG Coat of Bloodstream Form African Trypanosomes Interact with External Proteins?

Variations on the statement "the variant surface glycoprotein (VSG) coat that covers the external face of the mammalian bloodstream form of Trypanosoma brucei acts a physical barrier" appear regularly in research articles and reviews. The concept of the impenetrable VSG coat is an attractive one, as it provides a clear model for understanding how a trypanosome population persists; each successive VSG protects the plasma membrane and is immunologically distinct from previous VSGs. What is the evidence that the VSG coat is an impenetrable barrier, and how do antibodies and other extracellular proteins interact with it? In this review, the nature of the extracellular surface of the bloodstream form trypanosome is described, and past experiments that investigated binding of antibodies and lectins to trypanosomes are analysed using knowledge of VSG sequence and structure that was unavailable when the experiments were performed. Epitopes for some VSG monoclonal antibodies are mapped as far as possible from previous experimental data, onto models of VSG structures. The binding of lectins to some, but not to other, VSGs is revisited with more recent knowledge of the location and nature of N-linked oligosaccharides. The conclusions are: (i) Much of the variation observed in earlier experiments can be explained by the identity of the individual VSGs. (ii) Much of an individual VSG is accessible to antibodies, and the barrier that prevents access to the cell surface is probably at the base of the VSG N-terminal domain, approximately 5 nm from the plasma membrane. This second conclusion highlights a gap in our understanding of how the VSG coat works, as several plasma membrane proteins with large extracellular domains are very unlikely to be hidden from host antibodies by VSG.The authors’ lab is funded by the Wellcome Trust (093008/Z10/Z) and the Medical Research Council (MR/L008246/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This is the final version of the article. It was first available from PLOS via http://dx.doi.org/10.1371/journal.ppat.100525

Porins and small-molecule translocation across the outer membrane of Gram-negative bacteria

Gram-negative bacteria and their complex cell envelope, which comprises an outer membrane and an inner membrane, are an important and attractive system for studying the translocation of small molecules across biological membranes. In the outer membrane of Enterobacteriaceae, trimeric porins control the cellular uptake of small molecules, including nutrients and antibacterial agents. The relatively slow porin-mediated passive uptake across the outer membrane and active efflux via efflux pumps in the inner membrane creates a permeability barrier. The synergistic action of outer membrane permeability, efflux pump activities and enzymatic degradation efficiently reduces the intracellular concentrations of small molecules and contributes to the emergence of antibiotic resistance. In this Review, we discuss recent advances in our understanding of the molecular and functional roles of general porins in small-molecule translocation in Enterobacteriaceae and consider the crucial contribution of porins in antibiotic resistance

MOMP from Campylobacter jejuni Is a Trimer of 18-Stranded β-Barrel Monomers with a Ca²⁺ Ion Bound at the Constriction Zone

The Gram-negative organism Campylobacter jejuni is the major cause of food poisoning. Unlike Escherichia coli, which has two major porins, OmpC and OmpF, C. jejuni has one, termed major outer membrane protein (MOMP) through which nutrients and antibiotics transit. We report the 2.1-Å crystal structure of C. jejuni MOMP expressed in E. coli and a lower resolution but otherwise identical structure purified directly from C. jejuni. The 2.1-Å resolution structure of recombinant MOMP showed that although the protein has timeric arrangement similar to OmpC, it is an 18-stranded, not 16-stranded, β-barrel. The structure has identified a Ca²⁺ bound at the constriction zone, which is functionally significant as suggested by molecular dynamics and single-channel experiments. The water-filled channel of MOMP has a narrow constriction zone, and single-molecule studies show a monomeric conductivity of 0.7 ± 0.2 nS and a trimeric conductance of 2.2 ± 0.2 nS. The ion neutralizes negative charges at the constriction zone, reducing the transverse electric field and reversing ion selectivity. Modeling of the transit of ciprofloxacin, an antibiotic of choice for treating Campylobacter infection, through the pore of MOMP reveals a trajectory that is dependent upon the presence metal ion

Structure of a lectin from Canavalia gladiata seeds: new structural insights for old molecules

<p>Abstract</p> <p>Background</p> <p>Lectins are mainly described as simple carbohydrate-binding proteins. Previous studies have tried to identify other binding sites, which possible recognize plant hormones, secondary metabolites, and isolated amino acid residues. We report the crystal structure of a lectin isolated from <it>Canavalia gladiata </it>seeds (CGL), describing a new binding pocket, which may be related to pathogen resistance activity in ConA-like lectins; a site where a non-protein amino-acid, α-aminobutyric acid (Abu), is bound.</p> <p>Results</p> <p>The overall structure of native CGL and complexed with α-methyl-mannoside and Abu have been refined at 2.3 Å and 2.31 Å resolution, respectively. Analysis of the electron density maps of the CGL structure shows clearly the presence of Abu, which was confirmed by mass spectrometry.</p> <p>Conclusion</p> <p>The presence of Abu in a plant lectin structure strongly indicates the ability of lectins on carrying secondary metabolites. Comparison of the amino acids composing the site with other legume lectins revealed that this site is conserved, providing an evidence of the biological relevance of this site. This new action of lectins strengthens their role in defense mechanisms in plants.</p

Recovery after spinal cord relapse in multiple sclerosis is predicted by radial diffusivity

Background: The aim of this study was to determine whether the diffusion tensor-derived radial diffusivity and axial diffusivity, measured in the cortico-spinal tract in the cervical cord, predict clinical recovery after a cord relapse in patients with multiple sclerosis, and change over time

Identification of the Schistosoma mansoni TNF-Alpha Receptor Gene and the Effect of Human TNF-Alpha on the Parasite Gene Expression Profile

Schistosoma mansoni is the major causative agent of schistosomiasis in the Americas. This parasite takes advantage of host signaling molecules such as cytokines and hormones to complete its development inside the host. Tumor necrosis factor-alpha (TNF-α) is one of the most important host cytokines involved in the inflammatory response. When cercariae, the infective stage, penetrates the human skin the release of TNF-α is started. In this work the authors describe the complete sequence of a possible TNF-α receptor in S. mansoni and detect that the receptor is most highly expressed in cercariae among all life cycle stages. Aiming to mimic the situation at the site of skin penetration, cercariae were mechanically transformed in vitro into schistosomula and exposed to human TNF-α. Exposure of early-developing schistosomula to the human hormone caused a large-scale change in the expression of parasite genes. Exposure of adult worms to human TNF-α caused gene expression changes as well, and the set of parasite altered genes in the adult parasite was different from that of schistosomula. This work increases the number of known signaling pathways of the parasite, and opens new perspectives into understanding the molecular components of TNF-α response as well as into possibly interfering with parasite–host interaction