Inner Membrane Of Gram-negative Bacteria Research Articles

The Structural Features of MlaD Illuminate its Unique Ligand-Transporting Mechanism and Ancestry.

The membrane-associated solute-binding protein (SBP) MlaD of the maintenance of lipid asymmetry (Mla) system has been reported to help the transport of phospholipids (PLs) between the outer and inner membranes of Gram-negative bacteria. Despite the availability of structural information, the molecular mechanism underlying the transport of PLs and the ancestry of the protein MlaD remain unclear. In this study, we report the crystal structures of the periplasmic region of MlaD from Escherichia coli (EcMlaD) at a resolution range of 2.3-3.2 Å. The EcMlaD protomer consists of two distinct regions, viz. N-terminal β-barrel fold consisting of seven strands (referred to as MlaD domain) and C-terminal α-helical domain (HD). The protein EcMlaD oligomerizes to give rise to a homo-hexameric ring with a central channel that is hydrophobic and continuous with a variable diameter. Interestingly, the structural analysis revealed that the HD, instead of the MlaD domain, plays a critical role in determining the oligomeric state of the protein. Based on the analysis of available structural information, we propose a working mechanism of PL transport, viz. "asymmetric protomer movement (APM)". Wherein half of the EcMlaD hexamer would rise in the periplasmic side along with an outward movement of pore loops, resulting in the change of the central channel geometry. Furthermore, this study highlights that, unlike typical SBPs, EcMlaD possesses a fold similar to EF/AMT-type beta(6)-barrel and a unique ancestry. Altogether, the findings firmly establish EcMlaD to be a non-canonical SBP with a unique ligand-transport mechanism.

A Novel In silico Filtration Method for Discovery of Encrypted Antimicrobial Peptides

Background:: Antibacterial resistance has been one of the most important causes of death in the last few decades, necessitating the need to discover new antibiotics. Antimicrobial peptides (AMPs) are among the best candidates due to their broad-spectrum and potent activity against bacteria and low probability of developing resistance against them. Objective:: In this study, we proposed a novel filtration method using knowledge-based approaches to discover encrypted AMPs within a protein sequence Methods:: The encrypted AMPs were selected from a protein sequence, in this case, lactoferrin, based on hydrophobicity, cationicity, alpha-helix structure, helical wheel projection, and binding affinities to gram-negative and positive bacterial membranes. Results:: Six out of 20 potential encrypted AMPs were ultimately selected for further assays. Molecular docking of the selected AMPs with outer and inner membranes of gram-negative bacteria and also gram-positive bacterial membranes showed reasonable binding affinity ranging from ‘-6.7 to -7.5’ and ‘- 4.5 to -5.7’ and ‘-4.6 to -5.7’ kcal/mol, respectively. No toxicity was shown in the candidate AMPs. Conclusion:: According to in silico results, our method succeeded to discover six new encrypted AMPs from human lactoferrin, designated as lactoferrin-derived peptides (LDPs). Further in silico and experimental assays should also be performed to prove the efficiency of our knowledge-based filtration method.

OPRLM: Orientations of proteins in realistic lipid membranes.

A new web tool and a database, OPRLM (Orientations of Proteins in Realistic Lipid Membranes), has been created based on the OPM database and the broadly used GHARMM-GUI toolset. The database provides a complete set of experimental 3D structures of integral and peripheral membrane proteins from the Protein Data Bank that are positioned in planar and curved lipid bilayers using implicit and explicit membrane representations. Membrane systems vary from single-component artificial lipid bilayers to multi-component bilayers with complex lipid composition corresponding to 18 biomembranes with either asymmetric or symmetric distribution of lipid types between two leaflets: 14 “average” eukaryotic cellular and organelle membranes (plasma membranes of mammals, plants, and fungi, endoplasmic reticulum and apparatus Golgi membranes of mammals and fungi, mammalian membranes of endosomes and lysosomes, mitochondrial outer and inner membranes, plant vacuole membranes, thylakoid membranes of plants and cyanobacteria), the outer and the inner membranes of Gram-negative bacteria (E. coli), and “average” cell membranes of Gram-positive bacteria and archaebacteria. All these membrane systems are accessible for public use in CHARMM-GUI Archive (https://www.charmm-gui.org/docs/archive/biomembrane). The web tool allows users of any level of expertise an easy setup of protein-membrane systems for atomic molecular dynamics (MD) simulations in pre-assembled realistic lipid membranes formed by mixtures of explicit lipids. OPRLM is publicly available at https://oprlm.org/.

Solid and Liquid Surface-Supported Bacterial Membrane Mimetics as a Platform for the Functional and Structural Studies of Antimicrobials.

Increasing antibiotic resistance has provoked the urgent need to investigate the interactions of antimicrobials with bacterial membranes. The reasons for emerging antibiotic resistance and innovations in novel therapeutic approaches are highly relevant to the mechanistic interactions between antibiotics and membranes. Due to the dynamic nature, complex compositions, and small sizes of native bacterial membranes, bacterial membrane mimetics have been developed to allow for the in vitro examination of structures, properties, dynamics, and interactions. In this review, three types of model membranes are discussed: monolayers, supported lipid bilayers, and supported asymmetric bilayers; this review highlights their advantages and constraints. From monolayers to asymmetric bilayers, biomimetic bacterial membranes replicate various properties of real bacterial membranes. The typical synthetic methods for fabricating each model membrane are introduced. Depending on the properties of lipids and their biological relevance, various lipid compositions have been used to mimic bacterial membranes. For example, mixtures of phosphatidylethanolamines (PE), phosphatidylglycerols (PG), and cardiolipins (CL) at various molar ratios have been used, approaching actual lipid compositions of Gram-positive bacterial membranes and inner membranes of Gram-negative bacteria. Asymmetric lipid bilayers can be fabricated on solid supports to emulate Gram-negative bacterial outer membranes. To probe the properties of the model bacterial membranes and interactions with antimicrobials, three common characterization techniques, including quartz crystal microbalance with dissipation (QCM-D), surface plasmon resonance (SPR), and neutron reflectometry (NR) are detailed in this review article. Finally, we provide examples showing that the combination of bacterial membrane models and characterization techniques is capable of providing crucial information in the design of new antimicrobials that combat bacterial resistance.

The Ton Motor.

The Ton complex is a molecular motor at the inner membrane of Gram-negative bacteria that uses a proton gradient to apply forces on outer membrane (OM) proteins to permit active transport of nutrients into the periplasmic space. Recently, the structure of the ExbB–ExbD subcomplex was determined in several bacterial species, but the complete structure and stoichiometry of TonB have yet to be determined. The C-terminal end of TonB is known to cross the periplasm and interact with TonB-dependent outer membrane transport proteins with high affinity. Yet despite having significant knowledge of these transport proteins, it is not clear how the Ton motor opens a pathway across the outer membrane for nutrient import. Additionally, the mechanism by which energy is harnessed from the inner membrane subcomplex and transduced to the outer membrane via TonB is not well understood. In this review, we will discuss the gaps in the knowledge about the complete structure of the Ton motor complex and the relationship between ion flow used to generate mechanical work at the outer membrane and the nutrient transport process.

Cryo-EM structure of MsbA in saposin-lipid nanoparticles (Salipro) provides insights into nucleotide coordination.

The ATP-binding cassette transporter MsbA is a lipid flippase, translocating lipid A, glycolipids, and lipopolysaccharides from the inner to the outer leaflet of the inner membrane of Gram-negative bacteria. It has been used as a model system for time-resolved structural studies as several MsbA structures in different states and reconstitution systems (detergent/nanodiscs/peptidiscs) are available. However, due to the limited resolution of the available structures, detailed structural information on the bound nucleotides has remained elusive. Here, we have reconstituted MsbA in saposin A-lipoprotein nanoparticles (Salipro) and determined the structure of ADP-vanadate-bound MsbA by single-particle cryo-electron microscopy to 3.5 Å resolution. This procedure has resulted in significantly improved resolution and enabled us to model all side chains and visualise detailed ADP-vanadate interactions in the nucleotide-binding domains. The approach may be applicable to other dynamic membrane proteins.

MsbA: an ABC transporter paradigm.

ATP-binding cassette (ABC) transporters play an important role in various cellular processes. They display a similar architecture and share a mechanism which couples ATP hydrolysis to substrate transport. However, in the light of current data and recent experimental progress, this protein superfamily appears as multifaceted as their broad substrate range. Among the prokaryotic ABC transporters, MsbA can serve as a paradigm for research in this field. It is located in the inner membrane of Gram-negative bacteria and functions as a floppase for the lipopolysaccharide (LPS) precursor core-LPS, which is involved in the biogenesis of the bacterial outer membrane. While MsbA shows high similarity to eukaryotic ABC transporters, its expression in Gram-negative bacteria makes it conveniently accessible for many experimental approaches from spectroscopy to 3D structure determination. As an essential protein for bacterial membrane integrity, MsbA has also become an attractive target for the development of novel antibiotics. Furthermore, it serves as a model for multidrug efflux pumps. Here we provide an overview of recent findings and their relevance to the field, highlight the potential of methods such as solid-state NMR and EPR spectroscopy and provide a perspective for future work.

Structure and Stoichiometry of the Ton Molecular Motor.

The Ton complex is a molecular motor that uses the proton gradient at the inner membrane of Gram-negative bacteria to generate force and movement, which are transmitted to transporters at the outer membrane, allowing the entry of nutrients into the periplasmic space. Despite decades of investigation and the recent flurry of structures being reported by X-ray crystallography and cryoEM, the mode of action of the Ton molecular motor has remained elusive, and the precise stoichiometry of its subunits is still a matter of debate. This review summarizes the latest findings on the Ton system by presenting the recently reported structures and related reports on the stoichiometry of the fully assembled complex.

Inner Membrane Translocases and Insertases.

The inner membrane of Gram-negative bacteria is a ~6nm thick phospholipid bilayer. It forms a semi-permeable barrier between the cytoplasm and periplasm allowing only regulated export and import of ions, sugar polymers, DNA and proteins. Inner membrane proteins, embedded via hydrophobic transmembrane α-helices, play an essential role in this regulated trafficking: they mediate insertion into the membrane (insertases) or complete crossing of the membrane (translocases) or both. The Gram-negative inner membrane is equipped with a variety of different insertases and translocases. Many of them are specialized, taking care of the export of only a few protein substrates, while others have more general roles. Here, we focus on the three general export/insertion pathways, the secretory (Sec) pathway, YidC and the twin-arginine translocation (TAT) pathway, focusing closely on the Escherichia coli (E. coli) paradigm. We only briefly mention dedicated export pathways found in different Gram-negative bacteria. The Sec system deals with the majority of exported proteins and functions both as a translocase for secretory proteins and an insertase for membrane proteins. The insertase YidC assists the Sec system or operates independently on membrane protein clients. Sec and YidC, in common with most export pathways, require their protein clients to be in soluble non-folded states to fit through the translocation channels and grooves. The TAT pathway is an exception, as it translocates folded proteins, some loaded with prosthetic groups.

Wzx flippases exhibiting complex O-unit preferences require a new model for Wzx-substrate interactions.

The Wzx flippase is a critical component of the O‐antigen biosynthesis pathway, being responsible for the translocation of oligosaccharide O units across the inner membrane in Gram‐negative bacteria. Recent studies have shown that Wzx has a strong preference for its cognate O unit, but the types of O‐unit structural variance that a given Wzx can accommodate are poorly understood. In this study, we identified two Yersinia pseudotuberculosis Wzx that can distinguish between different terminal dideoxyhexose sugars on a common O‐unit main‐chain, despite both being able to translocate several other structurally‐divergent O units. We also identified other Y. pseudotuberculosis Wzx that can translocate a structurally divergent foreign O unit with high efficiency, and thus exhibit an apparently relaxed substrate preference. It now appears that Wzx substrate preference is more complex than previously suggested, and that not all O‐unit residues are equally important determinants of translocation efficiency. We propose a new “Structure‐Specific Triggering” model in which Wzx translocation proceeds at a low level for a wide variety of substrates, with high‐frequency translocation only being triggered by Wzx interacting with one or more preferred O‐unit structural elements found on its cognate O unit(s).

Structural basis for dual-mode inhibition of the ABC transporter MsbA.

The movement of core-lipopolysaccharide across the inner membrane of Gram-negative bacteria is catalysed by an essential ATP-binding cassette transporter, MsbA. Recent structures of MsbA and related transporters have provided insights into the molecular basis of active lipid transport; however, structural information about their pharmacological modulation remains limited. Here we report the 2.9 Å resolution structure of MsbA in complex with G907, a selective small-molecule antagonist with bactericidal activity, revealing an unprecedented mechanism of ABC transporter inhibition. G907 traps MsbA in an inward-facing, lipopolysaccharide-bound conformation by wedging into an architecturally conserved transmembrane pocket. A second allosteric mechanism of antagonism occurs through structural and functional uncoupling of the nucleotide-binding domains. This study establishes a framework for the selective modulation of ABC transporters and provides rational avenues for the design of new antibiotics and other therapeutics targeting this protein family.

Targeting and Insertion of Membrane Proteins.

The insertion and assembly of proteins into the inner membrane of bacteria are crucial for many cellular processes, including cellular respiration, signal transduction, and ion and pH homeostasis. This process requires efficient membrane targeting and insertion of proteins into the lipid bilayer in their correct orientation and proper conformation. Playing center stage in these events are the targeting components, signal recognition particle (SRP) and the SRP receptor FtsY, as well as the insertion components, the Sec translocon and the YidC insertase. Here, we will discuss new insights provided from the recent high-resolution structures of these proteins. In addition, we will review the mechanism by which a variety of proteins with different topologies are inserted into the inner membrane of Gram-negative bacteria. Finally, we report on the energetics of this process and provide information on how membrane insertion occurs in Gram-positive bacteria and Archaea. It should be noted that most of what we know about membrane protein assembly in bacteria is based on studies conducted in Escherichia coli.

Activity-Related Conformational Changes in d,d-Carboxypeptidases Revealed by In Vivo Periplasmic Förster Resonance Energy Transfer Assay in Escherichia coli.

ABSTRACTOne of the mechanisms of β-lactam antibiotic resistance requires the activity of d,d-carboxypeptidases (d,d-CPases) involved in peptidoglycan (PG) synthesis, making them putative targets for new antibiotic development. The activity of PG-synthesizing enzymes is often correlated with their association with other proteins. The PG layer is maintained in the periplasm between the two membranes of the Gram-negative cell envelope. Because no methods existed to detect in vivo interactions in this compartment, we have developed and validated a Förster resonance energy transfer assay. Using the fluorescent-protein donor-acceptor pair mNeonGreen-mCherry, periplasmic protein interactions were detected in fixed and in living bacteria, in single samples or in plate reader 96-well format. We show that the d,d-CPases PBP5, PBP6a, and PBP6b of Escherichia coli change dimer conformation between resting and active states. Complementation studies and changes in localization suggest that these d,d-CPases are not redundant but that their balanced activity is required for robust PG synthesis.

Modulation of Activity of Ultrashort Lipopeptides toward Negatively Charged Model Lipid Films.

Because of the increasing resistance of pathogens to commonly used antibiotics, there is an urgent need to find alternative antimicrobial compounds with different mechanisms of action. Among them, lipopeptides are recognized as promising candidates. In this work, the Langmuir technique and atomic force microscopy were employed to investigate the interactions of two novel lipopeptides with negatively charged phospholipid membranes, which served as a simplified model of inner membrane of Gram-negative bacteria. Lipid films contained phosphatidylethanolamine and phosphatidylglycerol extracts from E. coli bacteria. Lipopeptides were composed of palmitoyl chain covalently coupled to N-terminus of peptide with Trp-Lys-Leu-Lys amino acid sequence and the conformation of third residue was either d-Leu or l-Leu. It was found that chirality of leucine strongly affects interfacial behavior of these compounds, which was ascribed to the difference in effective size of the peptide portion of the molecules. Although the lipopeptides were the same in terms of amino acid sequence, charge, and identity of lipophilic chain, the experiments revealed that the barrier for their insertion into the lipid membrane is significantly different. Namely, it was lower for lipopeptide containing d-Leu residue. We have also found that insertion of the lipopeptides into the model membranes strongly alters lateral distribution of the membrane components and leads to its substantial fluidization. The dynamics of reorganization was noticeably faster in the presence of lipopeptide with smaller size of peptide moiety, i.e., containing d-Leu. It proves that effective size of the peptide headgroup is an important factor determining lipopeptide activity toward the lipid membranes.

Abstract 396: Acrolein Modification Weakens the Antimicrobial Activity of Apolipoprotein A1

Human apolipoprotein A-I (apoA-I) has been show to exhibit antimicrobial activity by neutralizing lipopolysaccharides and destabilizing inner membranes of gram-negative bacteria. Previous studies showed that acrolein, a highly reactive αβ unsaturated aldehyde generated in cigarette smoking, modifies ε-amino side chains of lysine residues in apoA-I. The current study investigated the effect of acrolein exposure on the structure and antimicrobial activity of apoA-I. Incubation of apoA-I with acrolein using a 1:20 molar ratio, acrolein modification was evident by the appearance of apoA-I oligomers due to intermolecular crosslinking. Increase of the acrolein to protein ratio resulted in heavily cross-linked apoA-I, with protein bands appearing at 63, 98, and 126 kDa. The presence of acrolein-modified lysines in the oligomers was verified through Western blot analysis using mab5F6 antibody that specifically detects acrolein modified lysine residues in proteins. The structural changes of modified apoA-I was analyzed using circular dichroism. The α-helical content of acrolein-modified apoA-I was not significantly different from the unmodified protein. However, the midpoint of guanidine-induced denaturation increased from 0.97 to 1.50 M guanidine upon modification, indicating a significant increase in protein stability. This suggests that while modification did not alter the secondary structure, the protein fold was altered due to cross-linking. To measure the effect of acrolein modification on the interaction with bacterial membranes, binding experiments were performed with phosphatidylglycerol entrapped with calcein. This showed that the percentage of calcein released by apoA-I decreased from 87.5 ± 2.3 % to 4.7 ± 0.13 % when the protein was modified by acrolein. Thus acrolein-modified apoA-I binds phosphatidylglycerol less effectively, possibly due to loss of electrostatic interactions with anionic phospholipid vesicles. In addition, binding of apoA-I to lipopolysaccharides was significantly weaker when the protein was modified by acrolein. These results suggest that apoA-I modification by acrolein results in a protein with a decreased ability to bind to bacterial membranes and is thus less potent as an antimicrobial protein.

Fluorescence and Absorbance Spectroscopy Methods to Study Membrane Perturbations by Antimicrobial Host Defense Peptides.

Antimicrobial peptides (AMPs) are currently intensely studied because of their potential as new bactericidal and bacteriostatic agents. The mechanism of action of numerous AMPs involves the permeabilization of bacterial membranes. Several methods have been developed to study peptide-membrane interactions; in particular optical spectroscopy methods are widely used. The intrinsic fluorescence properties of the Trp indole ring in Trp-containing AMPs can be exploited by measuring the fluorescence blue shift and acrylamide-induced fluorescence quenching. One important aspect of such studies is the use of distinct models of the bacterial membrane, in most cases large unilamellar vesicles (LUVs) with different, yet well-defined, phospholipid compositions. Deploying LUVs that are preloaded with fluorescent dyes, such as calcein, also allows for the study of vesicle permeabilization by AMPs. In addition, experiments using genetically engineered live Escherichia coli cells can be used to distinguish between the effects of AMPs on the outer and inner membranes of gram-negative bacteria. In combination, these methods can provide a detailed insight into the mode of action of AMPs.

Antimicrobial Peptide Potency is Facilitated by Greater Conformational Flexibility when Binding to Gram-negative Bacterial Inner Membranes.

The interaction of antimicrobial peptides (AMPs) with the inner membrane of Gram-negative bacteria is a key determinant of their abilities to exert diverse bactericidal effects. Here we present a molecular level understanding of the initial target membrane interaction for two cationic α-helical AMPs that share structural similarities but have a ten-fold difference in antibacterial potency towards Gram-negative bacteria. The binding and insertion from solution of pleurocidin or magainin 2 to membranes representing the inner membrane of Gram-negative bacteria, comprising a mixture of 128 anionic and 384 zwitterionic lipids, is monitored over 100 ns in all atom molecular dynamics simulations. The effects of the membrane interaction on both the peptide and lipid constituents are considered and compared with new and published experimental data obtained in the steady state. While both magainin 2 and pleurocidin are capable of disrupting bacterial membranes, the greater potency of pleurocidin is linked to its ability to penetrate within the bacterial cell. We show that pleurocidin displays much greater conformational flexibility when compared with magainin 2, resists self-association at the membrane surface and penetrates further into the hydrophobic core of the lipid bilayer. Conformational flexibility is therefore revealed as a key feature required of apparently α-helical cationic AMPs for enhanced antibacterial potency.

Identification of a Novel N-Acetylmuramic Acid Transporter in Tannerella forsythia.

In this study, we report the identification of a novel transporter for peptidoglycan amino sugar N-acetylmuramic acid (MurNAc) in the periodontal pathogen T. forsythia It has been known since the late 1980s that T. forsythia is a MurNAc auxotroph relying on environmental sources for this essential sugar. Most sugar transporters, and the MurNAc transporter MurP in particular, require a PTS phosphorelay to drive the uptake and concurrent phosphorylation of the sugar through the inner membrane in Gram-negative bacteria. Our study uncovered a novel type of PTS-independent MurNAc transporter, and although so far, it seems to be unique to T. forsythia, it may be present in a range of bacteria both of the oral cavity and gut, especially of the phylum Bacteroidetes.

ArnT: Structure and Mechanism of the Aminoarabinose Transferase Responsible for Resistance to Polymyxin-Class Antibiotics

Polymyxins are cationic peptide antibiotics, once discarded due to toxicity concerns, that have been revived in the clinic and are widely used to treat multi-drug resistant (MDR) infections. Resistance to polymyxin-class antibiotics is a growing concern, as a result, one that can critically impair our ability to combat MDR infections.ArnT (4-amino-4-deoxy-L-arabinose transferase) is an integral lipid-to-lipid glycosyltransferase that modifies lipid A, the lipid component of bacterial lipopolysaccharide (LPS). ArnT is located in the inner membrane of Gram-negative bacteria and catalyzes the transfer of a modified arabinose moiety from undecaprenyl phosphate to lipid A. The addition of the arabinose moiety causes a charge modification of the bacterial outer membrane, limiting its interactions with cationic peptides, and enabling bacteria to develop resistance to polymyxin-class antibiotics and natural antimicrobial peptides.Here we report crystal structures of ArnT from a gram-negative bacterium, alone and in complex with the lipid carrier undecaprenyl phosphate, at 2.8 and 3.2A resolution, respectively. ArnT consists of a transmembrane domain with thirteen transmembrane helices and a periplasmic soluble domain. The overall fold is reminiscent of protein glycosyltransferases from bacteria and archaea, but ArnT possesses unique features that are related to its function as a lipid glycosyltransferase.Notably, we identify cavities for both lipidic substrates, accessible to the membrane environment, that converge at the active site. We observe a significant coil-to-helix structural transition upon binding of undecaprenyl phosphate that stabilizes the carrier lipid near the active site and expands the lipid A cavity, likely enabling subsequent binding of lipid A. Finally, we utilize a polymyxin resistance assay to investigate the role of particular residues in light of the structures, and propose a model for catalysis by ArnT family enzymes.

Lipopolysaccharide transport to the cell surface: biosynthesis and extraction from the inner membrane

The cell surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS). The network of charges and sugars provided by the dense packing of LPS molecules in the outer leaflet of the outer membrane interferes with the entry of hydrophobic compounds into the cell, including many antibiotics. In addition, LPS can be recognized by the immune system and plays a crucial role in many interactions between bacteria and their animal hosts. LPS is synthesized in the inner membrane of Gram-negative bacteria, so it must be transported across their cell envelope to assemble at the cell surface. Over the past two decades, much of the research on LPS biogenesis has focused on the discovery and understanding of Lpt, a multi-protein complex that spans the cell envelope and functions to transport LPS from the inner membrane to the outer membrane. This paper focuses on the early steps of the transport of LPS by the Lpt machinery: the extraction of LPS from the inner membrane. The accompanying paper (May JM, Sherman DJ, Simpson BW, Ruiz N, Kahne D. 2015 Phil. Trans. R. Soc. B 370, 20150027. (doi:10.1098/rstb.2015.0027)) describes the subsequent steps as LPS travels through the periplasm and the outer membrane to its final destination at the cell surface.