Orientation of the Escherichia coli outer membrane protein OmpX in phospholipid bilayer membranes determined by solid-State NMR

The solid-state NMR orientation-dependent frequencies measured for membrane proteins in macroscopically oriented lipid bilayers provide precise orientation restraints for structure determination in membranes. Here we show that this information can also be used to supplement crystallographic structural data to establish the orientation of a membrane protein in the membrane. This is achieved by incorporating a few orientation restraints, measured for the Escherichia coli outer membrane protein OmpX in magnetically oriented lipid bilayers (bicelles), in a simulated annealing calculation with the coordinates of the OmpX crystal structure. The (1)H-(15)N dipolar couplings measured for the seven Phe residues of OmpX in oriented bilayers can be assigned by back-calculation of the NMR spectrum from the crystal structure and are sufficient to establish the three-dimensional orientation of the protein in the membrane, while the (15)N chemical shifts provide a measure of cross-validation for the analysis. In C14 lipid bilayers, OmpX adopts a transmembrane orientation with a 7 degrees tilt of its beta-barrel axis relative to the membrane normal, matching the hydrophobic thickness of the barrel with that of the membrane.     

NMR structural studies of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes

The beta-barrels found in the outer membranes of prokaryotic and eukaryotic organisms constitute an important functional class of proteins. Here we present solid-state NMR spectra of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes. We show that OmpX is folded in both glass-supported oriented lipid bilayers and in lipid bicelles that can be magnetically oriented with the membrane plane parallel or perpendicular to the direction of the magnetic field. The presence of resolved peaks in these spectra demonstrates that OmpX undergoes rotational diffusion around an axis perpendicular to the membrane surface. A tightly hydrogen-bonded domain of OmpX resists exchange with D2O for days and is assigned to the transmembrane beta-barrel, while peaks at isotropic resonance frequencies that disappear rapidly in D2O are assigned to the extracellular and periplasmic loops. The two-dimensional 1H/15N separated local field spectra of OmpX have several resolved peaks, and agree well with the spectra calculated from the crystal structure of OmpX rotated with the barrel axis nearly parallel (5 degrees tilt) to the direction of the magnetic field. The data indicate that it will be possible to obtain site-specific resonance assignments and to determine the structure, tilt, and rotation of OmpX in membranes using the solid-state NMR methods that are currently being applied to alpha-helical membrane proteins.     

Methionine Mutations of Outer Membrane Protein X Influence Structural Stability and Beta-Barrel Unfolding

We report the biochemical and biophysical characterization of outer membrane protein X (OmpX), an eight-stranded transmembrane β-barrel from E. coli, and compare the barrel behavior with a mutant devoid of methionine residues. Transmembrane outer membrane proteins of bacterial origin are known to display high tolerance to sequence rearrangements and mutations. Our studies with the triple mutant of OmpX that is devoid of all internal methionine residues (M18L; M21L; M118L) indicate that Met replacement has no influence on the refolding efficiency and structural characteristics of the protein. Surprisingly, the conserved substitution of Met→Leu leads to barrel destabilization and causes a lowering of the unfolding free energy by a factor of ∼8.5 kJ/mol, despite the mutations occurring at the loop regions. We report that the barrel destabilization is accompanied by a loss in cooperativity of unfolding in the presence of chemical denaturants. Furthermore, we are able to detect an unfolding intermediate in the Met-less barrel, whereas the parent protein exhibits a classic two-state unfolding. Thermal denaturation measurements also suggest a greater susceptibility of the OmpX barrel to heat, in the Met-less construct. Our studies reveal that even subtle variations in the extra-membrane region of rigid barrel structures such as OmpX, may bear severe implications on barrel stability. We propose that methionines contribute to efficient barrel structuring and protein-lipid interactions, and are therefore important elements of OmpX stability.     

NMR structural studies of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes

The β-barrels found in the outer membranes of prokaryotic and eukaryotic organisms constitute an important functional class of proteins. Here we present solid-state NMR spectra of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes. We show that OmpX is folded in both glass-supported oriented lipid bilayers and in lipid bicelles that can be magnetically oriented with the membrane plane parallel or perpendicular to the direction of the magnetic field. The presence of resolved peaks in these spectra demonstrates that OmpX undergoes rotational diffusion around an axis perpendicular to the membrane surface. A tightly hydrogen-bonded domain of OmpX resists exchange with D₂O for days and is assigned to the transmembrane β-barrel, while peaks at isotropic resonance frequencies that disappear rapidly in D₂O are assigned to the extracellular and periplasmic loops. The two-dimensional ¹H/¹⁵N separated local field spectra of OmpX have several resolved peaks, and agree well with the spectra calculated from the crystal structure of OmpX rotated with the barrel axis nearly parallel (5° tilt) to the direction of the magnetic field. The data indicate that it will be possible to obtain site-specific resonance assignments and to determine the structure, tilt, and rotation of OmpX in membranes using the solid-state NMR methods that are currently being applied to α-helical membrane proteins.     

NMR structure of the integral membrane protein OmpX

The structure of the integral membrane protein OmpX from Escherichia coli reconstituted in 60 kDa DHPC micelles (OmpX/DHPC) was calculated from 526 NOE upper limit distance constraints. The structure determination was based on complete sequence-specific assignments for the amide protons and the Val, Leu, and Ile(delta1) methyl groups in OmpX, which were selectively protonated on a perdeuterated background. The solution structure of OmpX in the DHPC micelles consists of a well-defined, eight-stranded antiparallel beta-barrel, with successive pairs of beta-strands connected by mobile loops. Several long-range NOEs observed outside of the transmembrane barrel characterize an extension of a four-stranded beta-sheet beyond the height of the barrel. This protruding beta-sheet is believed to be involved in intermolecular interactions responsible for the biological functions of OmpX. The present approach for de novo structure determination should be quite widely applicable to membrane proteins reconstituted in mixed micelles with overall molecular masses up to about 100 kDa, and may also provide a platform for additional functional studies.     

The FadL family: unusual transporters for unusual substrates

The FadL family of proteins is responsible for the transport of hydrophobic compounds across the bacterial outer membrane. Two crystal structures of FadL, the long-chain fatty acid transporter from Escherichia coli, were recently determined, showing a novel fold characterized by the combination of a 14-stranded beta barrel and a "hatch" domain that plugs the barrel. Both crystal forms have several bound detergent molecules in the interior of the protein. This, together with differences between the N-terminal conformations of the FadL structures, has led to the proposal of a transport model that is distinct from those of all other known outer membrane transporters. According to this model, the transport of hydrophobic substrates across the outer membrane, as mediated by FadL family members, is based on diffusion, coupled to spontaneous conformational changes in the hatch domain.     

Expression, two-dimensional crystallization, and three-dimensional reconstruction of the beta8 outer membrane protein Omp21 from Comamonas acidovorans

The Omp21 protein from the proteobacterium Comamonas (Delftia) acidovorans belongs to the recently described beta8 family of outer membrane proteins, characterized by eight antiparallel beta-strands which form a beta-barrel. This family includes virulence proteins, OmpA and OmpX from Escherichia coli, and other related molecules. After we established an expression system, recombinant Omp21 was purified by Ni(2+) chelation affinity chromatography and refolded in situ while bound to resin. The native state of refolded protein was proven by FTIR spectroscopy and monitored with denaturing PAGE (heat modification). Both native and recombinant Omp21 were reconstituted in lipid membranes and crystallized two-dimensionally by controlled dialysis. Recombinant Omp21 crystallized as dimer and formed a p22(1)2(1) lattice with constants of a = 11.1 nm, b = 12.2 nm, gamma = 89.5 degrees. The 3-D structure of negatively stained, recombinant Omp21 was determined at a resolution of 1.8 nm by means of electron crystallography. Comparison with 3-D maps of OmpX and the transmembrane domain of OmpA revealed a high similarity between the mass distribution of exoplasmic loops of Omp21 and OmpA.     

A Novel Intein-Like Autoproteolytic Mechanism in Autotransporter Proteins

Many virulence factors secreted by pathogenic Gram-negative bacteria are found to be members of the autotransporter protein family. These proteins share a common mechanism by which they exit the periplasm, involving the formation of a 12-stranded β-barrel domain in the outer membrane. The role of this barrel in the secretion of the N-terminal passenger domain is controversial, and no model currently explains satisfactorily the entire body of experimental data. After secretion, some autotransporter barrels autoproteolytically cleave away the passenger, and one crystal structure is known for a barrel of this type in the postcleavage state. Hbp is an autotransporter of the self-cleaving type, which cuts the polypeptide between two absolutely conserved asparagine residues buried within the barrel lumen. Mutation of the first asparagine residue to isosteric aspartic acid prevents proteolysis. Here we present the crystal structure of a truncated Hbp mutant carrying the C-terminal residues of the passenger domain attached to the barrel. This model mimics the state of the protein immediately prior to separation of the passenger and barrel domains, and shows the role of residues in the so-called “linker” between the passenger and β domains. This high-resolution membrane protein crystal structure also reveals the sites of many water molecules within the barrel. The cleavage mechanism shows similarities to those of inteins and some viral proteins, but with a novel means of promoting nucleophilic attack.     

Structural studies of the Cpx pathway activator NlpE on the outer membrane of Escherichia coli

NlpE, an outer membrane lipoprotein, functions during envelope stress responses in Gram-negative bacteria. In Escherichia coli, adhesion to abiotic surfaces has been reported to activate the Cpx pathway in an NlpE-dependent manner. External copper ions are also thought to activate the Cpx pathway mediated by NlpE. We determined the crystal structure of NlpE from E. coli at 2.6 A resolution. The structure showed that NlpE consists of two beta barrel domains. The N-terminal domain resembles the bacterial lipocalin Blc, and the C-terminal domain has an oligonucleotide/oligosaccharide-binding (OB) fold. From both biochemical analyses and the crystal structure, it can be deduced that the cysteine residues in the CXXC motif may be chemically active. Furthermore, two monomers in the asymmetric unit form an unusual 3D domain-swapped dimer. These findings indicate that tertiary and/or quaternary structural instability may be responsible for Cpx pathway activation.     

The crystal structure of the pyoverdine outer membrane receptor FpvA from Pseudomonas aeruginosa at 3.6 angstroms resolution

The pyoverdine outer membrane receptor FpvA from Pseudomonas aeruginosa translocates ferric-pyoverdine across the outer membrane via an energy consuming mechanism that involves the inner membrane energy transducing complex of TonB-ExbB-ExbD and the proton motive force. We solved the crystal structure of FpvA loaded with iron-free pyoverdine at 3.6 angstroms resolution. The pyoverdine receptor is folded in two domains: a transmembrane 22-stranded beta-barrel domain occluded by an N-terminal domain containing a mixed four-stranded beta-sheet (the plug). The beta-strands of the barrel are connected by long extracellular loops and short periplasmic turns. The iron-free pyoverdine is bound at the surface of the receptor in a pocket lined with aromatic residues while the extracellular loops do not completely cover the pyoverdine binding site. The TonB box, which is involved in intermolecular contacts with the TonB protein of the inner membrane, is observed in an extended conformation. Comparison of this first reported structure of an iron-siderophore transporter from a bacterium other than Escherichia coli with the known structures of the E.coli TonB-dependent transporters reveals a high structural homology and suggests that a common sensing mechanism exists for the iron-loading status in all bacterial iron siderophore transporters.     

Molecular dynamics simulations on interaction between bacterial proteins: Implication on pathogenic activities

We perform molecular dynamics simulation studies on interaction between bacterial proteins: an outer‐membrane protein STY3179 and a yfdX protein STY3178 of Salmonella Typhi. STY3179 has been found to be involved in bacterial adhesion and invasion. STY3178 is recently biophysically characterized. It is a soluble protein having antibiotic binding and chaperon activity capabilities. These two proteins co‐occur and are from neighboring gene in Salmonella Typhi‐occurrence of homologs of both STY3178 and STY3179 are identified in many Gram‐negative bacteria. We show using homology modeling, docking followed by molecular dynamics simulation that they can form a stable complex. STY3178 belongs to aqueous phase, while the beta barrel portion of STY3179 remains buried in DPPC bilayer with extra‐cellular loops exposed to water. To understand the molecular basis of interaction between STY3178 and STY3179, we compute the conformational thermodynamics which indicate that these two proteins interact through polar and acidic residues belonging to their interfacial region. Conformational thermodynamics results further reveal instability of certain residues in extra‐cellular loops of STY3179 upon complexation with STY3178 which is an indication for binding with host cell protein laminin.     

Structural predictions of AgfA, the insoluble fimbrial subunit of Salmonella thin aggregative fimbriae.

The unusually stable and multifunctional, thin aggregative fimbriae common to all Salmonella spp. are principally polymers of the fimbrin subunit, AgfA. AgfA of Salmonella enteritidis consists of two domains: a protease-sensitive, 22 amino acid residue N-terminal region and a protease-resistant, 109 residue C-terminal core. The unusual amino acid sequence of the AgfA core region comprises two-, five- and tenfold internal sequence homology patterns reflected in five conserved, 18-residue tandem repeats. These repeats have the consensus sequence, Sx5QxGx2NxAx3Q and are linked together by four or five residues, (x)xAx2. The predicted secondary structure for this unusual arrangement of tandem repeats in AgfA indicates mainly extended conformation with the beta strands linked by four to six residues. Candidate proteins of known structure with motifs of alternating beta strands and short loops were selected from folds described in SCOP as a source of coordinates for AgfA model construction. Three all-beta class motifs selected from the Serratia marcescens metalloprotease, myelin P2 protein or vitelline membrane outer protein I were used for initial AgfA homology build-up procedures ultimately resulting in three structural models; beta barrel, beta prism and parallel beta helix. The beta barrel model is a compact, albeit irregular structure, with the beta strands arranged in two antiparallel beta sheet faces. The beta prism model does not reflect the 5 or 10-fold symmetry of the AgfA primary sequence. However, the favored, parallel beta helix model is a compact coil of ten helically arranged beta strands forming two parallel beta sheet faces. This arrangement predicts a regular, potentially stable, C-terminal core region consistent with the observed tandem repeat sequences, protease-resistance and strong tendency of this fimbrin to oligomerize and aggregate. Positional conservation of amino acid residues in AgfA and the Escherichia coli AgfA homologue, CsgA, provides strong support for this model. The parallel beta helix model of AgfA offers an interesting solution to a multifunctional fimbrin molecular surface having solvent exposed areas, regions for major and minor subunit interactions as well as fiber-fiber interactions common to many bacterial fimbriae.     

Juxtamembrane tryptophans have distinct roles in defining the OmpX barrel–micelle boundary and facilitating protein–micelle association

Defining the span of the transmembrane region, a key requirement to ensure correct folding, stability and function of bacterial outer membrane β-barrels, is assisted by the amphipathic property of tryptophan. We demonstrate the unique and distinctive properties of the interface Trp76 and Trp140 of outer membrane protein X, and map their positional relevance to the refolding process, barrel formation and the resulting stability in dodecylphosphocholine micelles. The solvent-exposed Trp76 displays a rigid interfacial localization, whereas Trp140 is relatively micelle-solvated and contributes to barrel folding and global OmpX stability. Kinetic contribution to OmpX stability is influenced by the two tryptophans. Differential associations of the indoles with the detergent milieu therefore contribute to micelle-assisted β-barrel folding and concomitant OmpX stability.     

Structure of outer membrane protein A transmembrane domain by NMR spectroscopy

We have determined the three-dimensional fold of the 19 kDa (177 residues) transmembrane domain of the outer membrane protein A of Escherichia coli in dodecylphosphocholine (DPC) micelles in solution using heteronuclear NMR. The structure consists of an eight-stranded beta-barrel connected by tight turns on the periplasmic side and larger mobile loops on the extracellular side. The solution structure of the barrel in DPC micelles is similar to that in n-octyltetraoxyethylene (C(8)E(4)) micelles determined by X-ray diffraction. Moreover, data from NMR dynamic experiments reveal a gradient of conformational flexibility in the structure that may contribute to the membrane channel function of this protein.     

Evaluation of a structural model of Pseudomonas aeruginosa outer membrane protein OprM, an efflux component involved in intrinsic antibiotic resistance

The outer membrane protein OprM of Pseudomonas aeruginosa is involved in intrinsic and mutational multiple-antibiotic resistance as part of two resistance-nodulation-division efflux systems. The crystal structure of TolC, a homologous protein in Escherichia coli, was recently published (V. Koronakis, A. Sharff, E. Koronakis, B. Luisl, and C. Hughes, Nature 405:914-919, 2000), demonstrating a distinctive architecture comprising outer membrane beta-barrel and periplasmic helical-barrel structures, which assemble differently from the common beta-barrel-only conformation of porins. Based on their sequence similarity, a similar content of alpha-helical and beta-sheet structure determined by circular dichroism spectroscopy, and our observation that OprM, like TolC, reconstitutes channels in planar bilayer membranes, OprM and TolC were considered to be structurally homologous, and a model of OprM was constructed by threading its sequence to the TolC crystal structure. Residues thought to be important for the TolC structure were conserved in space in this OprM model. Analyses of deletion mutants and previously isolated insertion mutants of OprM in the context of this model allowed us to propose roles for different protein domains. Our data indicate that the helical barrel of the protein is critical for both the function and the integrity of the protein, while a C-terminal domain localized around the equatorial plane of this helical barrel is dispensable. Extracellular loops appear to play a lesser role in substrate specificity for this efflux protein compared to classical porins, and there appears to be a correlation between the change in antimicrobial activity for OprM mutants and the pore size. Our model and channel formation studies support the "iris" mechanism of action for TolC and permit us now to form more focused hypotheses about the functional domains of OprM and its related family of efflux proteins.     

Solution NMR mapping of water-accessible residues in the transmembrane β-barrel of OmpX

The atomic structure of OmpX, the smallest member of the bacterial outer membrane protein family, has been previously established by X-ray crystallography and NMR spectroscopy. In apparent conflict with electrophysiological studies, the lumen of its transmembrane β-barrel appears too tightly packed with amino acid side chains to let any solute flow through. In the present study, high-resolution solution NMR spectra were obtained of OmpX kept water-soluble by either amphipol A8-35 or the detergent dihexanoylphosphatidylcholine. Hydrogen/deuterium exchange measurements performed after prolonged equilibration show that, whatever the surfactant used, some of the amide protons of the membrane-spanning region exchange much more readily than others, which likely reflects the dynamics of the barrel.     

Membrane protein dynamics in different environments: simulation study of the outer membrane protein X in a lipid bilayer and in a micelle

The bacterial outer membrane protein OmpX from Escherichia coli has been investigated by molecular dynamics simulations when embedded in a phospholipid bilayer and as a protein-micelle aggregate. The resulting simulation trajectories were analysed in terms of structural and dynamic properties of the membrane protein. In agreement with experimental observations, highest relative stability was found for the β-barrel region that is embedded in the lipophilic phase, whereas an extracellular protruding β-sheet, which is a unique structural feature of OmpX that supposedly plays an important role in cell adhesion and invasion, shows larger structure fluctuations. Additionally, we investigated water permeation into the core of the β-barrel protein, which contains a tight salt-bridge and hydrogen-bond network, so that extensive water flux is unlikely. Differences between the bilayer and the micellar system were observed in the length of the barrel and its position inside the lipid environment, and in the protein interactions with the hydrophilic part of the lipids near the lipid/water interface. Those variations suggest that micelles and other detergent environments might not offer a wholly membrane-like milieu to promote adoption of the physiological conformational state by OmpX.     

Gene duplication of the eight-stranded beta-barrel OmpX produces a functional pore: a scenario for the evolution of transmembrane beta-barrels

The repeating unit of outer membrane beta-barrels from Gram-negative bacteria is the beta-hairpin, and representatives of this protein family always have an even strand number between eight and 22. Two dominant structural forms have eight and 16 strands, respectively, suggesting gene duplication as a possible mechanism for their evolution. We duplicated the sequence of OmpX, an eight-stranded beta-barrel protein of known structure, and obtained a beta-barrel, designated Omp2X, which can fold in vitro and in vivo. Using single-channel conductance measurements and PEG exclusion assays, we found that Omp2X has a pore size similar to that of OmpC, a natural 16-stranded barrel. Fusions of the homologous proteins OmpX, OmpA and OmpW were able to fold in vitro in all combinations tested, revealing that the general propensity to form a beta-barrel is sufficient to evolve larger barrels by simple genetic events.     

Projection structure of the monomeric porin OmpG at 6 A resolution

The Escherichia coli porin OmpG, which acts as an efficient unspecific channel for mono-, di- and trisaccharides, has been purified and crystallized in two dimensions. Projection maps of two different crystal forms of OmpG at 6 A resolution show that the protein has a beta-barrel structure characteristic for outer membrane proteins, and that it does not form trimers, unlike most other porins such as OmpF and OmpC, but appears in monomeric form. The size of the barrel is approximately 2.5 nm, indicating that OmpG may consist of 14 beta-strands. The projection map suggests that the channel is restricted by internal loops.