Thicket and Mesh: How the Outer Membrane Can Resist Tension Imposed by the Cell Wall.

The cell envelope of Gram-negative bacteria is composed of an outer membrane (OM) and an inner membrane (IM) and a peptidoglycan cell wall (CW) between them. Combined with Braun's lipoprotein (Lpp), which connects the OM and the CW, and numerous membrane proteins that exist in both OM and IM, the cell envelope creates a mechanically stable environment that resists various physical and chemical perturbations to the cell, including turgor pressure caused by the solute concentration difference between the cytoplasm of the cell and the extracellular environment. Previous computational studies have explored how individual components (OM, IM, and CW) can resist turgor pressure although combinations of them have been less well studied. To that end, we constructed multiple OM-CW systems, including the Lpp connections with the CW under increasing degrees of strain. The results show that the OM can effectively resist the tension imposed by the CW, shrinking by only 3-5% in area even when the CW is stretched to 2.5× its relaxed area. The area expansion modulus of the system increases with increasing CW strain, although the OM remains a significant contributor to the envelope's mechanical stability. Additionally, we find that when the protein TolC is embedded in the OM, its stiffness increases.

Adaptation of the periplasm to maintain spatial constraints essential for cell envelope processes and cell viability.

The cell envelope of Gram-negative bacteria consists of two membranes surrounding a periplasm and peptidoglycan layer. Molecular machines spanning the cell envelope depend on spatial constraints and load-bearing forces across the cell envelope and surface. The mechanisms dictating spatial constraints across the cell envelope remain incompletely defined. In Escherichia coli, the coiled-coil lipoprotein Lpp contributes the only covalent linkage between the outer membrane and the underlying peptidoglycan layer. Using proteomics, molecular dynamics, and a synthetic lethal screen, we show that lengthening Lpp to the upper limit does not change the spatial constraint but is accommodated by other factors which thereby become essential for viability. Our findings demonstrate E. coli expressing elongated Lpp does not simply enlarge the periplasm in response, but the bacteria accommodate by a combination of tilting Lpp and reducing the amount of the covalent bridge. By genetic screening, we identified all of the genes in E. coli that become essential in order to enact this adaptation, and by quantitative proteomics discovered that very few proteins need to be up- or down-regulated in steady-state levels in order to accommodate the longer Lpp. We observed increased levels of factors determining cell stiffness, a decrease in membrane integrity, an increased membrane vesiculation and a dependance on otherwise non-essential tethers to maintain lipid transport and peptidoglycan biosynthesis. Further this has implications for understanding how spatial constraint across the envelope controls processes such as flagellum-driven motility, cellular signaling, and protein translocation.

Lpp positions peptidoglycan at the AcrA-TolC interface in the AcrAB-TolC multidrug efflux pump.

The multidrug efflux pumps of Gram-negative bacteria are a class of complexes that span the periplasm, coupling both the inner and outer membranes to expel toxic molecules. The best-characterized example of these tripartite pumps is the AcrAB-TolC complex of Escherichia coli. However, how the complex interacts with the peptidoglycan (PG) cell wall, which is anchored to the outer membrane (OM) by Braun's lipoprotein (Lpp), is still largely unknown. In this work, we present molecular dynamics simulations of a complete, atomistic model of the AcrAB-TolC complex with the inner membrane, OM, and PG layers all present. We find that the PG localizes to the junction of AcrA and TolC, in agreement with recent cryo-tomography data. Free-energy calculations reveal that the positioning of PG is determined by the length and conformation of multiple Lpp copies anchoring it to the OM. The distance between the PG and OM measured in cryo-electron microscopy images of wild-type E. coli also agrees with the simulation-derived spacing. Sequence analysis of AcrA suggests a conserved role for interactions with PG in the assembly and stabilization of efflux pumps, one that may extend to other trans-envelope complexes as well.

Carbon dioxide transport across membranes.

Carbon dioxide (CO2) movement across cellular membranes is passive and governed by Fick's law of diffusion. Until recently, we believed that gases cross biological membranes exclusively by dissolving in and then diffusing through membrane lipid. However, the observation that some membranes are CO2 impermeable led to the discovery of a gas molecule moving through a channel; namely, CO2 diffusion through aquaporin-1 (AQP1). Later work demonstrated CO2 diffusion through rhesus (Rh) proteins and NH3 diffusion through both AQPs and Rh proteins. The tetrameric AQPs exhibit differential selectivity for CO2 versus NH3 versus H2O, reflecting physico-chemical differences among the small molecules as well as among the hydrophilic monomeric pores and hydrophobic central pores of various AQPs. Preliminary work suggests that NH3 moves through the monomeric pores of AQP1, whereas CO2 moves through both monomeric and central pores. Initial work on AQP5 indicates that it is possible to create a metal-binding site on the central pore's extracellular face, thereby blocking CO2 movement. The trimeric Rh proteins have monomers with hydrophilic pores surrounding a hydrophobic central pore. Preliminary work on the bacterial Rh homologue AmtB suggests that gas can diffuse through the central pore and three sets of interfacial clefts between monomers. Finally, initial work indicates that CO2 diffuses through the electrogenic Na/HCO3 cotransporter NBCe1. At least in some cells, CO2-permeable proteins could provide important pathways for transmembrane CO2 movements. Such pathways could be amenable to cellular regulation and could become valuable drug targets.

ß-Barrel proteins tether the outer membrane in many Gram-negative bacteria.

Gram-negative bacteria have a cell envelope that comprises an outer membrane (OM), a peptidoglycan (PG) layer and an inner membrane (IM)1. The OM and PG are load-bearing, selectively permeable structures that are stabilized by cooperative interactions between IM and OM proteins2,3. In Escherichia coli, Braun's lipoprotein (Lpp) forms the only covalent tether between the OM and PG and is crucial for cell envelope stability4; however, most other Gram-negative bacteria lack Lpp so it has been assumed that alternative mechanisms of OM stabilization are present5. We used a glycoproteomic analysis of PG to show that ß-barrel OM proteins are covalently attached to PG in several Gram-negative species, including Coxiella burnetii, Agrobacterium tumefaciens and Legionella pneumophila. In C. burnetii, we found that four different types of covalent attachments occur between OM proteins and PG, with tethering of the ß-barrel OM protein BbpA becoming most abundant in the stationary phase and tethering of the lipoprotein LimB similar throughout the cell cycle. Using a genetic approach, we demonstrate that the cell cycle-dependent tethering of BbpA is partly dependent on a developmentally regulated L,D-transpeptidase (Ldt). We use our findings to propose a model of Gram-negative cell envelope stabilization that includes cell cycle control and an expanded role for Ldts in covalently attaching surface proteins to PG.

Outer membrane protein size and LPS O-antigen define protective antibody targeting to the Salmonella surface.

Lipopolysaccharide (LPS) O-antigen (O-Ag) is known to limit antibody binding to surface antigens, although the relationship between antibody, O-Ag and other outer-membrane antigens is poorly understood. Here we report, immunization with the trimeric porin OmpD from Salmonella Typhimurium (STmOmpD) protects against infection. Atomistic molecular dynamics simulations indicate this is because OmpD trimers generate footprints within the O-Ag layer sufficiently sized for a single IgG Fab to access. While STmOmpD differs from its orthologue in S. Enteritidis (SEn) by a single amino-acid residue, immunization with STmOmpD confers minimal protection to SEn. This is due to the OmpD-O-Ag interplay restricting IgG binding, with the pairing of OmpD with its native O-Ag being essential for optimal protection after immunization. Thus, both the chemical and physical structure of O-Ag are key for the presentation of specific epitopes within proteinaceous surface-antigens. This enhances combinatorial antigenic diversity in Gram-negative bacteria, while reducing associated fitness costs.

A Minimal Membrane Metal Transport System: Dynamics and Energetics of mer Proteins.

The mer operon in bacteria encodes a set of proteins and enzymes that impart resistance to environmental mercury toxicity by importing Hg2+ and reducing it to volatile Hg(0). Because the reduction occurs in the cytoplasm, mercuric ions must first be transported across the cytoplasmic membrane by one of a few known transporters. MerF is the smallest of these, containing only two transmembrane helices and two pairs of vicinal cysteines that coordinate mercuric ions. In this work, we use molecular dynamics simulations to characterize the dynamics of MerF in its apo and Hg2+ -bound states. We find that the apo state positions one of the cysteine pairs closer to the periplasmic side of the membrane, while in the bound state the same pair approaches the cytoplasmic side. This finding is consistent with the functional requirement of accepting Hg2+ from the periplasmic space, sequestering it on acceptance, and transferring it to the cytoplasm. Conformational changes in the TM helices facilitate the functional interaction of the two cysteine pairs. Free-energy calculations provide a barrier of 16 kcal/mol for the association of the periplasmic Hg2+ -bound protein MerP with MerF and 7 kcal/mol for the subsequent association of MerF's two cysteine pairs. Despite the significant conformational changes required to move the binding site across the membrane, coarse-grained simulations of multiple copies of MerF support the expectation that it functions as a monomer. Our results demonstrate how conformational changes and binding thermodynamics could lead to such a small membrane protein acting as an ion transporter. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.

Tyrosine, cysteine, and proton coupled electron transfer in a ribonucleotide reductase-inspired beta hairpin maquette.

Tyrosine residues act as intermediates in proton coupled electron transfer reactions (PCET) in proteins. For example, in ribonucleotide reductase (RNR), a tyrosyl radical oxidizes an active site cysteine via a 35 Å pathway that contains multiple aromatic groups. When singlet tyrosine is oxidized, the radical becomes a strong acid, and proton transfer reactions, which are coupled with the redox reaction, may be used to control reaction rate. Here, we characterize a tyrosine-containing beta hairpin, Peptide O, which has a cross-strand, noncovalent interaction between its single tyrosine, Y5, and a cysteine (C14). Circular dichroism provides evidence for a thermostable beta-turn. EPR spectroscopy shows that Peptide O forms a neutral tyrosyl radical after UV photolysis at 160 K. Molecular dynamics simulations support a phenolic/SH interaction in the tyrosine singlet and radical states. Differential pulse voltammetry exhibits pH dependence consistent with the formation of a neutral tyrosyl radical and a pKa change in two other residues. A redox-coupled decrease in cysteine pKa from 9 (singlet) to 6.9 (radical) is assigned. At pD 11, picosecond transient absorption spectroscopy after UV photolysis monitors tyrosyl radical recombination via electron transfer (ET). The ET rate in Peptide O is indistinguishable from the ET rates observed in peptides containing a histidine and a cyclohexylalanine (Cha) at position 14. However, at pD 9, the tyrosyl radical decays via PCET, and the decay rate is slowed, when compared to the histidine 14 variant. Notably, the decay rate is accelerated, when compared to the Cha 14 variant. We conclude that redox coupling between tyrosine and cysteine can act as a PCET control mechanism in proteins.

Accelerating Membrane Simulations with Hydrogen Mass Repartitioning.

The time step of atomistic molecular dynamics (MD) simulations is determined by the fastest motions in the system and is typically limited to 2 fs. An increasingly popular approach is to increase the mass of the hydrogen atoms to ∼3 amu and decrease the mass of the parent atom by an equivalent amount. This approach, known as hydrogen-mass repartitioning (HMR), permits time steps up to 4 fs with reasonable simulation stability. While HMR has been applied in many published studies to date, it has not been extensively tested for membrane-containing systems. Here, we compare the results of simulations of a variety of membranes and membrane-protein systems run using a 2 fs time step and a 4 fs time step with HMR. For pure membrane systems, we find almost no difference in structural properties, such as area-per-lipid, electron density profiles, and order parameters, although there are differences in kinetic properties such as the diffusion constant. Conductance through a porin in an applied field, partitioning of a small peptide, hydrogen-bond dynamics, and membrane mixing show very little dependence on HMR and the time step. We also tested a 9 Å cutoff as compared to the standard CHARMM cutoff of 12 Å, finding significant deviations in many properties tested. We conclude that HMR is a valid approach for membrane systems, but a 9 Å cutoff is not.

Structure and Function of Tryptophan-Tyrosine Dyads in Biomimetic ß Hairpins.

Tyrosine-tryptophan (YW) dyads are ubiquitous structural motifs in enzymes and play roles in proton-coupled electron transfer (PCET) and, possibly, protection from oxidative stress. Here, we describe the function of YW dyads in de novo designed 18-mer, ß hairpins. In Peptide M, a YW dyad is formed between W14 and Y5. A UV hypochromic effect and an excitonic Cotton signal are observed, in addition to singlet, excited state (W*) and fluorescence emission spectral shifts. In a second Peptide, Peptide MW, a Y5-W13 dyad is formed diagonally across the strand and distorts the backbone. On a picosecond timescale, the W* excited-state decay kinetics are similar in all peptides but are accelerated relative to amino acids in solution. In Peptide MW, the W* spectrum is consistent with increased conformational flexibility. In Peptide M and MW, the electron paramagnetic resonance spectra obtained after UV photolysis are characteristic of tyrosine and tryptophan radicals at 160 K. Notably, at pH 9, the radical photolysis yield is decreased in Peptide M and MW, compared to that in a tyrosine and tryptophan mixture. This protective effect is not observed at pH 11 and is not observed in peptides containing a tryptophan-histidine dyad or tryptophan alone. The YW dyad protective effect is attributed to an increase in the radical recombination rate. This increase in rate can be facilitated by hydrogen-bonding interactions, which lower the barrier for the PCET reaction at pH 9. These results suggest that the YW dyad structural motif promotes radical quenching under conditions of reactive oxygen stress.

ATP-Dependent Signaling in Simulations of a Revised Model of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily that has uniquely evolved to function as a chloride channel. It binds and hydrolyzes ATP at its nucleotide binding domains to form a pore providing a diffusive pathway within its transmembrane domains. CFTR is the only known protein from the ABC superfamily with channel activity, and its dysfunction causes the disease cystic fibrosis. While much is known about the functional aspects of CFTR, significant gaps remain, such as the structure-function relationship underlying signaling of ATP binding. In the present work, we refined an existing homology model using an intermediate-resolution (9 Å) published cryo-electron microscopy map. The newly derived models have been simulated in equilibrium molecular dynamics simulations for a total of 2.5 µs in multiple ATP-occupancy states. Putative conformational movements connecting ATP binding with pore formation are elucidated and quantified. Additionally, new interdomain interactions between E543, K968, and K1292 have been identified and confirmed experimentally; these interactions may be relevant for signaling ATP binding and hydrolysis to the transmembrane domains and induction of pore opening.

Distribution of mechanical stress in the Escherichia coli cell envelope.

The cell envelope in Gram-negative bacteria comprises two distinct membranes with a cell wall between them. There has been a growing interest in understanding the mechanical adaptation of this cell envelope to the osmotic pressure (or turgor pressure), which is generated by the difference in the concentration of solutes between the cytoplasm and the external environment. However, it remains unexplored how the cell wall, the inner membrane (IM), and the outer membrane (OM) effectively protect the cell from this pressure by bearing the resulting surface tension, thus preventing the formation of inner membrane bulges, abnormal cell morphology, spheroplasts and cell lysis. In this study, we have used molecular dynamics (MD) simulations combined with experiments to resolve how and to what extent models of the IM, OM, and cell wall respond to changes in surface tension. We calculated the area compressibility modulus of all three components in simulations from tension-area isotherms. Experiments on monolayers mimicking individual leaflets of the IM and OM were also used to characterize their compressibility. While the membranes become softer as they expand, the cell wall exhibits significant strain stiffening at moderate to high tensions. We integrate these results into a model of the cell envelope in which the OM and cell wall share the tension at low turgor pressure (0.3 atm) but the tension in the cell wall dominates at high values (>1 atm).

Structural basis for substrate selection by the translocation and assembly module of the ß-barrel assembly machinery.

The assembly of proteins into bacterial outer membranes is a key cellular process that we are only beginning to understand, mediated by the ß-barrel assembly machinery (BAM). Two crucial elements of that machinery are the core BAM complex and the translocation and assembly module (TAM), with each containing a member of the Omp85 superfamily of proteins: BamA in the BAM complex, TamA in the TAM. Here, we used the substrate protein FimD as a model to assess the selectivity of substrate interactions for the TAM relative to those of the BAM complex. A peptide scan revealed that TamA and BamA bind the ß-strands of FimD, and do so selectively. Chemical cross-linking and molecular dynamics are consistent with this interaction taking place between the first and last strand of the TamA barrel domain, providing the first experimental evidence of a lateral gate in TamA: a structural element implicated in membrane protein assembly. We suggest that the lateral gates in TamA and BamA provide different environments for substrates to engage, with the differences observed here beginning to address how the TAM can be more effective than the BAM complex in the folding of some substrate proteins.

Redox-Driven Conformational Dynamics in a Photosystem-II-Inspired ß-Hairpin Maquette Determined through Spectroscopy and Simulation.

Tyrosine-based radical transfer plays an important role in photosynthesis, respiration, and DNA synthesis. Radical transfer can occur either by electron transfer (ET) or proton coupled electron transfer (PCET), depending on the pH. Reversible conformational changes in the surrounding protein matrix may control reactivity of radical intermediates. De novo designed Peptide A is a synthetic 18 amino-acid ß-hairpin, which contains a single tyrosine (Y5) and carries out a kinetically significant PCET reaction between Y5 and a cross-strand histidine (H14). In Peptide A, amide II' (CN) changes are observed in the UV resonance Raman (UVRR) spectrum, associated with tyrosine ET and PCET; these bands were attributed previously to a reversible change in secondary structure. Here, we use molecular dynamics simulations to define this conformational change in Peptide A and its H14-to-cyclohexylalanine variant, Peptide C. Three different Y5 charge states, tyrosine (YH), tyrosinate (Y-), and neutral tyrosyl radical (Y·), are considered. The simulations show that Peptide A-YH and A-Y- retain secondary structure and noncovalent interactions, whereas A-Y· is unstable. In contrast, both Peptide C-Y- and Peptide C-Y· are unstable, due to the loss of the Y5-H14 π-π interaction. These simulations are consistent with previous UVRR experimental results on the two ß-hairpins. Furthermore, they demonstrate the ability of simulations using fixed-charge force fields to accurately capture redox-linked conformational dynamics in a ß-strand peptide.

Living on the edge: Simulations of bacterial outer-membrane proteins.

Gram-negative bacteria are distinguished in part by a second, outer membrane surrounding them. This membrane is distinct from others, possessing an outer leaflet composed not of typical phospholipids but rather large, highly charged molecules known as lipopolysaccharides. Therefore, modeling the structure and dynamics of proteins embedded in the outer membrane requires careful consideration of their native environment. In this review, we examine how simulations of such outer-membrane proteins have evolved over the last two decades, culminating most recently in detailed, highly accurate atomistic models of the outer membrane. We also draw attention to how the simulations have coupled with experiments to produce novel insights unattainable through a single approach. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.

Water distribution in dentin matrices: bound vs. unbound water.

OBJECTIVE: This work measured the amount of bound versus unbound water in completely-demineralized dentin. METHODS: Dentin beams prepared from extracted human teeth were completely demineralized, rinsed and dried to constant mass. They were rehydrated in 41% relative humidity (RH), while gravimetrically measuring their mass increase until the first plateau was reached at 0.064 (vacuum) or 0.116 gH2O/g dry mass (Drierite). The specimens were then exposed to 60% RH until attaining the second plateau at 0.220 (vacuum) or 0.191 gH2O/g dry mass (Drierite), and subsequently exposed to 99% RH until attaining the third plateau at 0.493 (vacuum) or 0.401 gH2O/g dry mass (Drierite). RESULTS: Exposure of the first layer of bound water to 0% RH for 5 min produced a -0.3% loss of bound water; in the second layer of bound water it caused a -3.3% loss of bound water; in the third layer it caused a -6% loss of bound water. Immersion in 100% ethanol or acetone for 5 min produced a 2.8 and 1.9% loss of bound water from the first layer, respectively; it caused a -4 and -7% loss of bound water in the second layer, respectively; and a -17 and -23% loss of bound water in the third layer. Bound water represented 21-25% of total dentin water. Chemical dehydration of water-saturated dentin with ethanol/acetone for 1 min only removed between 25 and 35% of unbound water, respectively. SIGNIFICANCE: Attempts to remove bound water by evaporation were not very successful. Chemical dehydration with 100% acetone was more successful than 100% ethanol especially the third layer of bound water. Since unbound water represents between 75 and 79% of total matrix water, the more such water can be removed, the more resin can be infiltrated.