High-resolution electron diffraction of reconstituted PhoE porin

PhoE porin has been reconstituted with phospholipid, forming large membrane patches. Electron diffraction shows that the reconstituted PhoE porin forms highly coherent crystalline arrays, giving structural information to a resolution of 3.4 A. The crystal form is of the orthorhombic space group P2(1)2(1)2, with unit cell dimensions a = 150 A and b = 129 A. Images of negatively stained PhoE crystalline patches show that there are four PhoE porin trimers in a unit cell.     

Topology of PhoE porin: the 'eyelet' region

A model for the topology of the PhoE porin has been proposed according to which the polypeptide traverses the outer membrane sixteen times mostly as amphipathic β-sheets, thereby exposing eight loops at the cell surface. Until now, no evidence has been obtained for the surface exposure of the third loop. Recently, the structure of porin of Rhodobacter capsulatus has been determined. The proposed model of PhoE is very similar to the structure of the R capsulatus porin, which has an ‘eyelet’ region, extending into the interior of the pore. The proposed third external loop of PhoE might form a similar ‘eyelet’ region. To determine the location of the predicted third external loop of PhoE, multiple copies of an oligonucleotide linker encoding an antigenic determinant of VP1 protein of foot-and-mouth disease virus (FMDV) were inserted. All hybrid proteins were properly inserted in the outer membrane. The monoclonal antibody MA11, directed against the linear FMDV epitope, was able to bind only to intact cells expressing a hybrid PhoE protein with at least three copies of the FMDV epitope present. Antibiotic sensitivity tests and single-channel conductance measurements revealed that the insertions influenced the channel size. These results are consistent with a location of the third loop of PhoE within the pore channel.     

Three-dimensional electron diffraction of PhoE porin to 2.8 A resolution

A three-dimensional set of electron diffraction intensities of PhoE porin embedded in trehalose extending to 2.8 A resolution has been collected and analyzed. The strongest high-resolution intensities are distributed as a figure of revolution about the z*-axis and are located primarily in a resolution range of 4.5 A to 5.0 A. Within this region, centered near 4.8 A resolution the brightest intensities are clustered about inclination angles of 35 degrees and 0 degrees from the a*, b* plane. This distribution of intensities indicates that the beta-sheet in PhoE porin is arranged to form a cylinder-like structure that contains major populations of beta-sheet strands tilted an average of 35 degrees and 0 degrees with respect to the membrane plane normal. This cylindrical structure has been seen previously in the high-resolution projection map of PhoE as an elliptical ring of high density.     

Osmotic regulation of PhoE porin synthesis in Escherichia coli.

In Escherichia coli, adaptation to hyperosmotic conditions alters the expression of the outer membrane porins OmpF and OmpC. The amount of PhoE porin, which is normally induced by phosphate deprivation, was greatly reduced in cells adapted to high-osmolarity conditions. Osmoregulation of PhoE operated independently of the activity of the PhoR phosphate sensor and did not involve cross-talk from the homologous osmosensor EnvZ. PhoE synthesis was partially restored by additional copies of the positive regulator phoB+ and by the osmoprotectant glycine betaine.     

Structure of PhoE porin in projection at 3.5 A resolution.

The structure of PhoE porin in projection normal to the membrane plane has been determined to a resolution of about 3.5 A by electron crystallographic techniques. The purified protein was reconstituted with lipid to form two-dimensional crystals. High resolution images and electron diffraction patterns of these specimens embedded in trehalose were recorded to obtain respectively the structure factor phase information and the more accurate values of the amplitude. The projection map shows interesting features that are not seen in the earlier map at 6.5 A. Details of the trimeric ring-like structures in our earlier map are now resolved. Each ring-like structure consists of "beads" with interbead spacings of about 4-6 A. These beads are interpreted as the projections of beta-strands along the strands' axes. At the center of the trimeric structure, there is a low density region that we proposed previously to be the location of lipopolysaccharide. Within each ring-like structure, there are complicated features which may play an important role in the size, selectivity, and stability of the channel.     

On the targeting and membrane assembly of the Escherichia coli outer membrane porin, PhoE

Abstract Within gram-negative bacteria such as Escherichia coli , the outer membrane porins provide a relatively non-specific uptake route which is utilised by a wide range of solutes including many antibiotics. Understanding the targeting and membrane assembly of these proteins is therefore of importance and this mini review aims to discuss this process in light of present knowledge.     

OmpC-like porin from Yersinia pseudotuberculosis: Molecular characteristics, physico-chemical and functional properties

Pore-forming protein from the outer membrane of Yersinia pseudotuberculosis cultured at 37°C has been isolated and characterized. Comparative analysis of the primary and three-dimensional structures of this protein and of OmpC porin from E. coli was carried out, functional properties of these proteins have been studied using bilayer lipid membranes (BLM) technique. The degree of homology, molecular mass and pore-forming properties of the isolated porin was found to be closer to those of OmpC porin from E. coli than OmpF porin from Y. pseudotuberculosis. The value of the most probable conductivity of OmpC porin from Y. pseudotuberculosis (0.18 pS) in BLM corresponded to the conductivity of the native trimer of this protein. Using CD spectroscopy, the porins investigated were shown to belong to the β-structured proteins. Data of the primary structure and intrinsic protein fluorescence revealed essential differences in localization and microenvironment of tryptophan residues in the porins investigated. Participation of external loops L2 and L6 in the formation of the antigenic structure of OmpC porin from Y. pseudotuberculosis was demonstrated. On the basis of crystal structure of osmoporin from Klebsiella pneumoniae, three-dimensional models of the monomer and trimer of the Y. pseudotuberculosis porin were obtained. Using Web server AGGRESCAN, the localization of protein structure sites with the increased aggregation capability (hot spots) has been deter-mined. It turned out that some of these zones localize in the region of intramonomeric contacts in the porin trimer; however, a large part of them is located on the external surface of the β-barrel. The process of thermal denaturation has been studied and the melting points of the porins were determined. It was found that significant changes in the microenvironment of the indole fluorophores (especially tryptophan residues of spectral class I) took place in the process of the thermodenaturation of the proteins. These changes preceded the irreversible conformational transition observed for the E. coli porin at 77°C and for the Y. pseudotuberculosis porin at 70°C.     

Demonstration of a folded monomeric form of porin PhoE of Escherichia coli in vivo.

The porins in the outer membranes of gram-negative bacteria are trimeric proteins. A folded monomeric form of the Escherichia coli porin PhoE, with a higher electrophoretic mobility than that of the denatured protein, has recently been detected in in vitro folding studies. To investigate the possible biological significance of the folded monomer, we attempted to detect this form in vivo. After pulse-labeling, folded monomers could be detected by immunoprecipitation. Furthermore, folded monomers were detected in a preparation of mutant PhoE porins, in which the subunit interactions were weakened by a E-66-->R substitution. Together, these results show that the folded monomer is not an in vitro folding artifact but an integral part of the native trimer.     

Dimerization of chemokine receptors and its functional consequences

It became clear over the recent years that most, if not all, G protein-coupled receptors (GPCR) are able to form dimers or higher order oligomers. Chemokine receptors make no exception to this new rule and both homo- and heterodimerization were demonstrated for CC and CXC receptors. Functional analyses demonstrated negative binding cooperativity between the two subunits of a dimer. The consequence is that only one chemokine can bind with high affinity onto a receptor dimer. In the context of receptor activation, this implies that the motions of helical domains triggered by the binding of agonists induce correlated changes in the other protomer. The impact of the chemokine dimerization process in terms of co-receptor function and drug development is discussed.     

E.coli PhoE porin has an opposite voltage-dependence to the homologous OmpF.

We used patch clamp analysis to compare the electrophysiological behavior of two related porins from Escherichia coli, the anion-specific PhoE and the cation-selective OmpF. Outer membrane fractions were obtained from strains expressing just one of these porin types, and the channels were reconstituted into liposomes without prior purification. We show that the orientation of the reconstituted channels is not random and is the same for both PhoE and OmpF. Like cation-selective porins, PhoE shows fast and slow gating to closed levels of various amplitudes, testifying that the channels visit multiple functional states and behave as cooperative entities. The voltage-dependence of PhoE closure is asymmetric, but strikingly, occurs at voltages of inverse polarity from those promoting closures of OmpC and OmpF. Both slow kinetics and inverse voltage-dependence are removed when 70 amino acids from the N-terminal of OmpF are introduced into the homologous region of PhoE. This novel observation regarding the voltage-dependence of the two channel types, along with published results on PhoE and OmpF mutants, allows us to propose a molecular mechanism for voltage sensing and sensor charge movements in bacterial porins. It also offers new cues on the possible physiological relevance in bacteria of this common form of channel modulation.     

Oligomerization of H-pyrophosphatase and its structural and functional consequences

The H⁺-pyrophosphatase (H⁺-PPase) consists of a single polypeptide, containing 16 or 17 transmembrane domains. To determine the higher order oligomeric state of Streptomyces coelicolor H⁺-PPase, we constructed a series of cysteine substitution mutants and expressed them in Escherichia coli. Firstly, we analyzed the formation of disulfide bonds, promoted by copper, in mutants with single cysteine substitutions. 28 of 39 mutants formed disulfide bonds, including S545C, a substitution at the periplasmic side. The formation of intermolecular disulfide bonds suppressed the enzyme activity of several, where the substituted residues were located in the cytosol. Creating disulfide links in the cytosol may interfere with the enzyme's catalytic function. Secondly, we prepared double mutants by introducing second cysteine substitutions into the S545C mutant. These double-cysteine mutants produced cross-linked complexes, estimated to be at least tetramers and possibly hexamers. Thirdly, we co-expressed epitope-tagged, wild type, and inactive mutant H⁺-PPases in E. coli and confirmed the formation of oligomers by co-purifying one subunit using the epitope tag used to label the other. The enzyme activity of these oligomers was markedly suppressed. We propose that H⁺-PPase is present as an oligomer made up of at least two or three sets of dimers.     

Protein modification by aldehydophospholipids and its functional consequences

Phospholipid aldehydes represent a particular subclass of lipid oxidation products. They are chemically reactive and can form Schiff bases with proteins and aminophospholipids. As chemically bound molecular entities they modulate the functional properties of biomolecules in solution and the surface of supramolecular systems including plasma lipoproteins and cell membranes. The lipid-protein and lipid-lipid conjugates may be considered the active primary platforms that are responsible for the biological effects of aldehydophospholipids, e.g. receptor binding, cell signaling, and recognition by the immune system. Despite the fact that aldehydophospholipids are covalently associated, they are subject to exchange between nucleophiles since their imine conjugates are not stable. As a consequence, aldehydophospholipids exist in a dynamic equilibrium between different "states" depending on the lipid and protein environment. Aldehydophospholipids may also contribute to the systemic administration and activity of oxidized phospholipids by inducing release of microparticles by cells. These effects are lipid-specific. Future studies should help clarify the mechanisms and consequences of these membrane-associated effects of "phospholipid stress". This article is part of a Special Issue entitled: Oxidized phospholipids-their properties and interactions with proteins.     

Chemical modification of the anion selectivity of the PhoE porin from the Escherichia coli outer membrane

The PhoE porin of Escherichia coli is induced by phosphate deprivation and when purified, forms moderately anion-selective channels in lipid bilayer membranes. To further investigate the basis of anion selectivity, PhoE was chemically acetylated with acetic anhydride. Acetylation modified the mobility and staining characteristics of the PhoE porin on SDS-polyacrylamide gel electrophoresis but the acetylated protein was still found in its normal trimeric state after solubilization in SDS at low temperatures. Furthermore, the acetylated PhoE porin retained its ability to reconstitute into lipid bilayer membranes and the single channel conductance in 1 M KCl was unaltered. Zero-current potential measurements demonstrated that whereas the native PhoE porin was anion-selective, a 30-40-fold increase in preference for cations upon acetylation resulted in the acetylated PhoE porin being cation-selective. Increasing the pH of KCl solutions bathing lipid bilayer membranes from pH 3 to pH 6 caused symmetrical 4-fold increases in the selectivity of both the native and acetylated PhoE proteins for cations. In contrast, increasing the pH from 7 to 9 caused a 2.5-fold increase in selectivity only for the native PhoE porin. These results suggest that the basis of anion selectivity in the native PhoE porin is fixed protonated amino groups (possibly on lysines) in or near the channel, and furthermore indicate that deprotonated carboxyl groups have a strong influence on ion selectivity.     

Molecular flexibility of Mycobacterium tuberculosis ribosome recycling factor and its functional consequences: An exploration involving mutants

Internal mobility of the two domain molecule of ribosome recycling factor (RRF) is known to be important for its action. Mycobacterium tuberculosis RRF does not complement E. coli for its deficiency of RRF (in the presence of E. coli EF-G alone). Crystal structure had revealed higher rigidity of the M. tuberculosis RRF due to the presence of additional salt bridges between domains. Two inter-domain salt bridges and one between the linker region and the domain containing C-terminal residues were disrupted by appropriate mutations. Except for a C-terminal deletion mutant, all mutants showed RRF activity in E. coli when M. tuberculosis EF-G was also co-expressed. The crystal structures of the point mutants, that of the C-terminal deletion mutant and that of the protein grown in the presence of a detergent, were determined. The increased mobility resulting from the disruption of the salt bridge involving the hinge region allows the appropriate mutant to weakly complement E. coli for its deficiency of RRF even in the absence of simultaneous expression of the mycobacterial EF-G. The loss of activity of the C-terminal deletion mutant appears to be partly due to the rigidification of the molecule consequent to changes in the hinge region.     

Existence and purification of porin heterotrimers of Escherichia coli K12 OmpC, OmpF, and PhoE proteins

Porin is a trimeric membrane protein that functions as a diffusion pore in the outer membrane of Escherichia coli. We report the existence and purification of porin heterotrimers between the ompC, ompF, and phoE porin gene products. Separation was achieved using a high resolution anion exchange column. The amount of each heterotrimer species present depended on the level of expression of the subunits and was consistent with random mixing of trimer subunits. A strong effect of bacterial lipopolysaccharide on the chromatography of porin was also detected. These results imply that assembly of porin trimers occurs between subunits synthesized on different polysomes and that subunit contacts between the porin subunits occur in conserved regions of the primary sequence.     

Prediction of the general structure of OmpF and PhoE from the sequence and structure of porin from Rhodobacter capsulatus. Orientation of porin in the membrane.

By comparing the hydrophilicity profiles and sequences of porin from Rhodobacter capsulatus with those of OmpF and PhoE from Escherichia coli, a set of insertions and deletions for alignment of the sequences has been deduced. With this alignment a similar folding of OmpF and PhoE has been predicted as found by X-ray structure analysis of porin from Rhodobacter capsulates. Furthermore, the orientation of the porin trimer in the outer membrane was inferred from topological data on PhoE. According to this result a single channel of approx. 30 A diameter starts at the outer surface. Near the middle of the outer membrane bilayer this channel branches out into three separate channels, each running within a single porin monomer to the periplasmic surface.     

The pho-controlled outer membrane porin PhoE does not contain specific binding sites for phosphate or polyphosphates

Purified PhoE-porins were reconstituted into black lipid bilayer membranes, and the selectivity and size of the reconstituted pores were determined. Addition of polyphosphates influenced the internal charge situation of the pore resulting in a shift from anion to cation selectivity. However, the pore size as judged from single channel conductances was not influenced by the addition of polyphosphates. A strong inhibition of the pore conductance only occurred when Mg2+ was also present in the aqueous phase. The inhibition of the pore function is presumably caused by the formation of a chelate between the divalent cation and the polyphosphate. Nevertheless, neither this inhibition nor the selectivity shift are specific to phosphate, because both effects can be mimicked by other polyvalent anions such as citrate. Inhibition of the PhoE pore function by polyphosphate in in vivo experiments confirmed the results of in vitro experiments that polyphosphate is only able to affect the permeability of the outer membrane toward beta-lactam antibiotics if Mg2+ is present. The outcome of the in vivo and the in vitro experiments are consistent with the assumption that the PhoE-porins do not contain a specific binding site for phosphate or polyphosphates but are anion selective because of an excess of positively charged amino acids inside or at the surface of the pore.     

Induction of the PhoE porin by NaCI as the basis for salt-induced acid sensitivity in Escherichia coli

Z. LAZIM, T.J. HUMPHREY AND R.J. ROWBURY. 1996. Organisms grown in low salt broth (LSB) are acid resistant but become sensitive on growth for 30-60 min with 300 mmol 1⁻¹ added NaCl. Salt-induced acid sensitivity only occurs in relA⁺ strains and sensitization is abolished by glucose, this catabolite repression effect being reversed by cAMP. The finding that sensitization did not occur in a phoE strain but did occur in a phoE⁺ derivative of it suggested that the response might result from PhoE induction, since PhoE acts as the major outer membrane (OM) proton pore under most conditions. In agreement with this, low-salt broth (LSB)-grown cells of a chromosomally lac⁻ strain carrying pJP102 ( phoE-lacZ ) produced low levels of β-galactosidase but growth with added NaCl led to rapid and appreciable induction. Also, a phoA mutant carrying a phoE-phoA fusion produced little alkaline phosphatase after growth in LSB but much more in LSB with added NaCl. Increased β-galactosidase synthesis (in phoE-lacZ strains) in the presence of NaCl was abolished by glucose, this effect being reversible by cAMP, and there was more NaCl-induced synthesis of this enzyme in relA⁺ strains.
Accordingly, it appears that addition of NaCl to LSB leads to acid sensitivity because it induces synthesis of the OM proton pore PhoE.     

Global and regional brain metabolic scaling and its functional consequences

Background  

Information processing in the brain requires large amounts of metabolic energy, the spatial distribution of which is highly heterogeneous, reflecting the complex activity patterns in the mammalian brain.