Β-barrel Outer Membrane Proteins Research Articles

C-terminal amino acid residues of the trimeric autotransporter adhesin YadA of Yersinia enterocolitica are decisive for its recognition and assembly by BamA

The Bam complex is a highly conserved multiprotein machine essential for the assembly of β-barrel outer membrane proteins. It is composed of the essential outer membrane protein BamA and four outer membrane associated lipoproteins BamB-E. The Yersinia enterocolitica Adhesin A (YadA) is the prototype of trimeric auotransporter adhesins (TAAs), consisting of a head, stalk and a β-barrel membrane anchor. To investigate the role of BamA in biogenesis of TAAs, we expressed YadA in a BamA-depleted strain of Escherichia coli, which resulted in degradation of YadA. Yeast-two-hybrid experiments and immunofluorescence studies revealed that BamA and YadA interact directly and colocalize. As BamA recognizes the C-terminus of OMPs, we exchanged the nine most C-terminal amino acids of YadA. Substitution of the amino acids in position 1, 3 or 5 from the C-terminus with glycine resulted in DegP-dependent degradation of YadA. Despite degradation all YadA proteins assembled in the outer membrane. In summary we demonstrate that (i) BamA is essential for biogenesis of the TAA YadA, (ii) BamA interacts directly with YadA, (iii) the C-terminal amino acid motif of YadA is important for the BamA-dependent assembly and differs slightly compared with other OMPs, and (iv) BamA and YadA colocalize.

Dissection of β-barrel outer membrane protein assembly pathways through characterizing BamA POTRA 1 mutants of Escherichia coli

BamA of Escherichia coli is an essential component of the hetero-oligomeric machinery that mediates β-barrel outer membrane protein (OMP) assembly. The C- and N-termini of BamA fold into trans-membrane β-barrel and five soluble POTRA domains respectively. Detailed characterization of BamA POTRA 1 missense and deletion mutants revealed two competing OMP assembly pathways, one of which is followed by the archetypal trimeric β-barrel OMPs, OmpF and LamB, and is dependent on POTRA 1. Interestingly, our data suggest that BamA also requires its POTRA 1 domain for proper assembly. The second pathway is independent of POTRA 1 and is exemplified by TolC. Site-specific cross-linking analysis revealed that the POTRA 1 domain of BamA interacts with SurA, a periplasmic chaperone required for the assembly of OmpF and LamB, but not that of TolC and BamA. The data suggest that SurA and BamA POTRA 1 domain function in concert to assist folding and assembly of most β-barrel OMPs except for TolC, which folds into a unique soluble α-helical barrel and an OM-anchored β-barrel. The two assembly pathways finally merge at some step beyond POTRA 1 but presumably before membrane insertion, which is thought to be catalysed by the trans-membrane β-barrel domain of BamA.

The role of SurA factor in outer membrane protein transport and virulence

The Escherichia coli periplasmic chaperone and peptidyl-prolyl isomerase (PPIase) SurA is a major factor in the biogenesis of β-barrel outer membrane proteins (OMPs) and as such plays an integral role in cell envelope homeostasis and cell envelope functions. Recently, the biological importance of SurA was further substantiated by the finding that SurA also affects pathogenicity, being required for full virulence of uropathogenic Escherichia coli, Salmonella, and Shigella spp. Moreover, given the conservation of the protein, SurA likely plays similar roles in other Gram-negative bacteria and may hence prove a valuable drug target against Gram-negative pathogens. While our understanding on how SurA promotes transport and folding of β-barrel OMPs, how it provides support to virulence, and how it functions at a molecular level is still limited, major contributions have recently been made on our way to find answers to these questions. This review is a compilation of our current state of knowledge on E. coli SurA function and a discussion of recent findings with a particular emphasis on the pleiotropic contributions of SurA to pathogenicity.

AlgK Is a TPR-Containing Protein and the Periplasmic Component of a Novel Exopolysaccharide Secretin

The opportunistic pathogen Pseudomonas aeruginosa causes chronic biofilm infections in cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by overproduction of the exopolysaccharide alginate. Here we show that AlgK, a protein essential for production of high molecular weight alginate, is an outer membrane lipoprotein that contributes to the correct localization of the porin AlgE. Our 2.5 A structure shows AlgK is composed of 9.5 tetratricopeptide-like repeats, and three putative sites of protein-protein interaction have been identified. Bioinformatics analysis suggests that BcsA, PgaA, and PelB, involved in the production and export of cellulose, poly-beta-1,6-N-Acetyl-D-glucosamine, and Pel exopolysaccharide, respectively, share the same topology as AlgK/E. Together, our data suggest that AlgK plays a role in the assembly of the alginate biosynthetic complex and represents the periplasmic component of a new type of outer membrane secretin that differs from canonical bacterial capsular polysaccharide secretion systems.

3P305 Development of High Performance Predictor of Bacterial and Mitochondrial Outer Membrane β-barrel Proteins.(Bioinformatics: Functional genomics,The 48th Annual Meeting of the Biophysical Society of Japan)

3P305 Development of High Performance Predictor of Bacterial and Mitochondrial Outer Membrane β-barrel Proteins.(Bioinformatics: Functional genomics,The 48th Annual Meeting of the Biophysical Society of Japan)

Ferrous iron-binding protein Omb of Salmonella enterica serovar Choleraesuis promotes resistance to hydrophobic antibiotics and contributes to its virulence

Salmonella enterica serovar Choleraesuis (SC) is an important enteric pathogen that causes serious systemic infections in swine and humans. To identify the genes required for resistance to antimicrobial peptides, we constructed a bank of SC transposon mutants and screened them for hypersensitivity to the cationic peptide polymyxin B. Here we report one isolated polymyxin B-susceptible mutant that also exhibited increased sensitivity toward human neutrophil peptide alpha-defensin 1 (HNP-1) and hydrophobic antibiotics including erythromycin and novobiocin. The mutant had a mutation in an ORF identified as outer membrane beta-barrel protein gene omb. The purified recombinant Omb protein was characterized as a ferrous iron-binding protein. The constructed omb isogenic mutant grew more slowly in iron-limiting conditions than the wild-type (WT) parent strain. In addition, compared with the WT strain, the omb mutant exhibited an increase in net negative charge upon the cell surface and was more easily killed by polymyxin B, HNP-1 and hydrophobic antibiotics. The omb gene was transcribed, regardless of the iron content within the growth medium, and the Omb protein appeared exclusively in the outer membrane fraction. Infection experiments demonstrated virulence attenuation when the mutant was administered orally or intraperitoneally to mice. This study indicates that Omb is a previously unrecognized ferrous iron-binding protein. In vivo, Omb may be involved in the acquisition of ferrous iron during the initial stages of SC infection and appears to be an important virulence factor for SC in mice.

Integral and peripheral association of proteins and protein complexes with Yersinia pestis inner and outer membranes

Yersinia pestis proteins were sequentially extracted from crude membranes with a high salt buffer (2.5 M NaBr), an alkaline solution (180 mM Na2CO3, pH 11.3) and membrane denaturants (8 M urea, 2 M thiourea and 1% amidosulfobetaine-14). Separation of proteins by 2D gel electrophoresis was followed by identification of more than 600 gene products by MS. Data from differential 2D gel display experiments, comparing protein abundances in cytoplasmic, periplasmic and all three membrane fractions, were used to assign proteins found in the membrane fractions to three protein categories: (i) integral membrane proteins and peripheral membrane proteins with low solubility in aqueous solutions (220 entries); (ii) peripheral membrane proteins with moderate to high solubility in aqueous solutions (127 entries); (iii) cytoplasmic or ribosomal membrane-contaminating proteins (80 entries). Thirty-one proteins were experimentally associated with the outer membrane (OM). Circa 50 proteins thought to be part of membrane-localized, multi-subunit complexes were identified in high Mr fractions of membrane extracts via size exclusion chromatography. This data supported biologically meaningful assignments of many proteins to the membrane periphery. Since only 32 inner membrane (IM) proteins with two or more predicted transmembrane domains (TMDs) were profiled in 2D gels, we resorted to a proteomic analysis by 2D-LC-MS/MS. Ninety-four additional IM proteins with two or more TMDs were identified. The total number of proteins associated with Y. pestis membranes increased to 456 and included representatives of all six β-barrel OM protein families and 25 distinct IM transporter families.

Crystal Structure of a Major Outer Membrane Protein from Thermus thermophilus HB27

The thermophilic eubacterium Thermus thermophilus belongs to one of the oldest branches of evolution and has a multilayered cell envelope that differs from that of modern Gram-negative bacteria. Its outer membrane contains integral outer membrane proteins (OMPs), of which only a few are characterized. TtoA, a new β-barrel OMP, was identified by searching the genome sequence of strain HB27 for the presence of a C-terminal signature sequence. The structure of TtoA was determined to a resolution of 2.8 Å, representing the first crystal structure of an OMP from a thermophilic bacterium. TtoA consists of an eight-stranded β-barrel with a large extracellular part to which a divalent cation is bound. A five-stranded extracellular β-sheet protrudes out of the membrane-embedded transmembrane barrel and is stabilized by a disulfide bridge. The edge of this β-sheet forms crystal contacts that could mimic interactions with other proteins. In modern Gram-negative bacteria, the C-terminal signature sequence of OMPs is required for binding to an Omp85 family protein as a prerequisite for its assembly. We present hints that a similar assembly pathway exists in T. thermophilus by an in vitro binding assay, where unfolded TtoA binds to the Thermus Omp85 family protein TtOmp85, while a mutant without the signature sequence does not.

β-Barrel Proteins That Reside in the Escherichia coli Outer Membrane in Vivo Demonstrate Varied Folding Behavior in Vitro

Little is known about the dynamic process of membrane protein folding, and few models exist to explore it. In this study we doubled the number of Escherichia coli outer membrane proteins (OMPs) for which folding into lipid bilayers has been systematically investigated. We cloned, expressed, and folded nine OMPs: outer membrane protein X (OmpX), OmpW, OmpA, the crcA gene product (PagP), OmpT, outer membrane phospholipase A (OmpLa), the fadl gene product (FadL), the yaet gene product (Omp85), and OmpF. These proteins fold into the same bilayer in vivo and share a transmembrane beta-barrel motif but vary in sequence and barrel size. We quantified the ability of these OMPs to fold into a matrix of bilayer environments. Several trends emerged from these experiments: higher pH values, thinner bilayers, and increased bilayer curvature promote folding of all OMPs. Increasing the incubation temperature promoted folding of several OMPs but inhibited folding of others. We discovered that OMPs do not have the same ability to fold into any single bilayer environment. This suggests that although environmental factors influence folding, OMPs also have intrinsic qualities that profoundly modulate their folding. To rationalize the differences in folding efficiency, we performed kinetic and thermal denaturation experiments, the results of which demonstrated that OMPs employ different strategies to achieve the observed folding efficiency.

Omp85 Tt from Thermus thermophilus HB27: an Ancestral Type of the Omp85 Protein Family

Proteins belonging to the Omp85 family are involved in the assembly of beta-barrel outer membrane proteins or in the translocation of proteins across the outer membrane in bacteria, mitochondria, and chloroplasts. The cell envelope of the thermophilic bacterium Thermus thermophilus HB27 is multilayered, including an outer membrane that is not well characterized. Neither the precise lipid composition nor much about integral membrane proteins is known. The genome of HB27 encodes one Omp85-like protein, Omp85(Tt), representing an ancestral type of this family. We overexpressed Omp85(Tt) in T. thermophilus and purified it from the native outer membranes. In the presence of detergent, purified Omp85(Tt) existed mainly as a monomer, composed of two stable protease-resistant modules. Circular dichroism spectroscopy indicated predominantly beta-sheet secondary structure. Electron microscopy of negatively stained lipid-embedded Omp85(Tt) revealed ring-like structures with a central cavity of approximately 1.5 nm in diameter. Single-channel conductance recordings indicated that Omp85(Tt) forms ion channels with two different conducting states, characterized by conductances of approximately 0.4 nS and approximately 0.65 nS, respectively.

Colicin N Binds to the Periphery of Its Receptor and Translocator, Outer Membrane Protein F

SummaryColicins kill Escherichia coli after translocation across the outer membrane. Colicin N displays an unusually simple translocation pathway, using the outer membrane protein F (OmpF) as both receptor and translocator. Studies of this binary complex may therefore reveal a significant component of the translocation pathway. Here we show that, in 2D crystals, colicin is found outside the porin trimer, suggesting that translocation may occur at the protein-lipid interface. The major lipid of the outer leaflet interface is lipopolysaccharide (LPS). It is further shown that colicin N binding displaces OmpF-bound LPS. The N-terminal helix of the pore-forming domain, which is not required for pore formation, rearranges and binds to OmpF. Colicin N also binds artificial OmpF dimers, indicating that trimeric symmetry plays no part in the interaction. The data indicate that colicin is closely associated with the OmpF-lipid interface, providing evidence that this peripheral pathway may play a role in colicin transmembrane transport.

NMR of Membrane-Associated Peptides and Proteins

In living cells, membrane proteins are essential to signal transduction, nutrient use, and energy exchange between the cell and environment. Due to challenges in protein expression, purification and crystallization, deposition of membrane protein structures in the Protein Data Bank lags far behind existing structures for soluble proteins. This review describes recent advances in solution NMR allowing the study of a select set of peripheral and integral membrane proteins. Surface-binding proteins discussed include amphitropic proteins, antimicrobial and anticancer peptides, the HIV-1 gp41 peptides, human alpha-synuclein and apolipoproteins. Also discussed are transmembrane proteins including bacterial outer membrane beta-barrel proteins and oligomeric alpha-helical proteins. These structural studies are possible due to solubilization of the proteins in membrane-mimetic constructs such as detergent micelles and bicelles. In addition to protein dynamics, protein-lipid interactions such as those between arginines and phosphatidylglycerols have been detected directly by NMR. These examples illustrate the unique role solution NMR spectroscopy plays in structural biology of membrane proteins.

2P-286 βシグナルの特徴解析とそれを用いた新規ミトコンドリアβバレル型外膜タンパク質の探索(生命情報科学・構造ゲノミクス,第46回日本生物物理学会年会)

2P-286 βシグナルの特徴解析とそれを用いた新規ミトコンドリアβバレル型外膜タンパク質の探索(生命情報科学・構造ゲノミクス,第46回日本生物物理学会年会)

A TolC-Like Protein Is Required for Heterocyst Development inAnabaenasp. Strain PCC 7120

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 forms heterocysts in a semiregular pattern when it is grown on N2 as the sole nitrogen source. The transition from vegetative cells to heterocysts requires marked metabolic and morphological changes. We show that a trimeric pore-forming outer membrane beta-barrel protein belonging to the TolC family, Alr2887, is up-regulated in developing heterocysts and is essential for diazotrophic growth. Mutants defective in Alr2887 did not form the specific glycolipid layer of the heterocyst cell wall, which is necessary to protect nitrogenase from external oxygen. Comparison of the glycolipid contents of wild-type and mutant cells indicated that the protein is not involved in the synthesis of glycolipids but might instead serve as an exporter for the glycolipid moieties or enzymes involved in glycolipid attachment. We propose that Alr2887, together with an ABC transporter like DevBCA, is part of a protein export system essential for assembly of the heterocyst glycolipid layer. We designate the alr2887 gene hgdD (heterocyst glycolipid deposition protein).

Role of Aromatic Side Chains in the Folding and Thermodynamic Stability of Integral Membrane Proteins

Aromatic residues are frequently found in helical and beta-barrel integral membrane proteins enriched at the membrane-water interface. Although the importance of these residues in membrane protein folding has been rationalized by thermodynamic partition measurements using peptide model systems, their contribution to the stability of bona fide membrane proteins has never been demonstrated. Here, we have investigated the contribution of interfacial aromatic residues to the thermodynamic stability of the beta-barrel outer membrane protein OmpA from Escherichia coli in lipid bilayers by performing extensive mutagenesis and equilibrium folding experiments. Isolated interfacial tryptophanes contribute -2.0 kcal/mol, isolated interfacial tyrosines contribute -2.6 kcal/mol, and isolated interfacial phenylalanines contribute -1.0 kcal/mol to the stability of this protein. These values agree well with the prediction from the Wimley-White interfacial hydrophobicity scale, except for tyrosine residues, which contribute more than has been expected from the peptide models. Double mutant cycle analysis reveals that interactions between aromatic side chains become significant when their centroids are separated by less than 6 A but are nearly insignificant above 7 A. Aromatic-aromatic side chain interactions are on the order of -1.0 to -1.4 kcal/mol and do not appear to depend on the type of aromatic residue. These results suggest that the clustering of aromatic side chains at membrane interfaces provides an additional heretofore not yet recognized driving force for the folding and stability of integral membrane proteins.

Lipoprotein SmpA is a component of the YaeT complex that assembles outer membrane proteins in Escherichia coli

A major role of the outer membrane (OM) of Gram-negative bacteria is to provide a protective permeability barrier for the cell, and proper maintenance of the OM is required for cellular viability. OM biogenesis requires the coordinated assembly of constituent lipids and proteins via dedicated OM assembly machineries. We have previously shown that, in Escherichia coli, the multicomponent YaeT complex is responsible for the assembly of OM beta-barrel proteins (OMPs). This complex contains the OMP YaeT and three OM lipoproteins. Here, we report another component of the YaeT complex, the OM lipoprotein small protein A (SmpA). Strains carrying loss-of-function mutations in smpA are viable but exhibit defects in OMP assembly. Biochemical experiments show that SmpA is involved in maintaining complex stability. Taken together, these experiments establish an important role for SmpA in both the structure and function of the YaeT complex.

The morphology proteins Mdm12/Mmm1 function in the major β-barrel assembly pathway of mitochondria

The beta-barrel proteins of mitochondria are synthesized on cytosolic ribosomes. The proteins are imported by the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It has been assumed that the SAM(core) complex with the subunits Sam35, Sam37 and Sam50 represents the last import stage common to all beta-barrel proteins, followed by splitting in a Tom40-specific route and a route for other beta-barrel proteins. We have identified new components of the beta-barrel assembly machinery and show that the major beta-barrel pathway extends beyond SAM(core). Mdm12/Mmm1 function after SAM(core) yet before splitting of the major pathway. Mdm12/Mmm1 have been known for their role in maintenance of mitochondrial morphology but we reveal assembly of beta-barrel proteins as their primary function. Moreover, Mdm10, which functions in the Tom40-specific route, can associate with SAM(core) as well as Mdm12/Mmm1 to form distinct assembly complexes, indicating a dynamic exchange between the machineries governing mitochondrial beta-barrel assembly. We conclude that assembly of mitochondrial beta-barrel proteins represents a major function of the morphology proteins Mdm12/Mmm1.

Refolding a beta-barrel membrane protein

The field of membrane protein folding is relatively new compared to soluble protein folding. This thesis describes spectroscopy investigations of the refolding and dynamics of a β-barrel membrane protein. The amphiphilic, β-barrel outer membrane protein A (OmpA) refolds and inserts directly into a lipid vesicle or micelle from a denatured state in aqueous urea solution. Spectroscopic probes used to study this system are native tryptophans located at positions 7, 15, 57, 102, and 143. Steady-state and time-resolved fluorescence measurements were performed using single tryptophan mutants of full-length OmpA (325 residues) and the truncated, transmembrane domain (176 residues). Both full-length and truncated mutants exhibit similar tryptophan emission lifetimes, suggesting that the transmembrane microenvironment is not greatly perturbed by the presence of the C-terminus. While the microenvironments of folded full-length and truncated OmpA appear similar, the dynamics of refolding at each tryptophan position exhibit subtle differences when the C-terminus is present. Specifically, we observe that tryptophan-102, which faces the pore interior, inserts and folds the fastest while tryptophan-7, which does not cross the bilayer, is the slowest. Fluorescence anisotropy decays also indicate that tryptophan-7 is the most flexible residue compared to the other tryptophans. Temperature studies below the lipid gel-liquid transition temperature have also been performed. In the lipid gel phase, OmpA adsorbs to the surface of the vesicles but contains immediate β-sheet structure upon folding as well as very hydrophobic tryptophan environments. It is still uncertain from ensemble measurements whether this species is a true intermediate. Fluorescence energy transfer kinetics have successfully determined the intramolecular distance between tryptophan-7 and cysteine-175 labeled with a dansyl fluorophore. These results reveal that the barrel ends of OmpA come into contact early in the refolding process and remain close together up to the final assembly of the barrel. We also have evidence that the adsorbed species at low temperatures is not an intermediate in the folding pathway since no energy transfer is observed for this species. These spectroscopic investigations have provided the foundation for further fundamental studies to dissect the molecular mechanism of the folding pathway of OmpA as well as other integral membrane proteins.

In silico and microarray-based genomic approaches to identifying potential vaccine candidates against Leptospira interrogans

BackgroundCurrently available vaccines against leptospirosis are of low efficacy, have an unacceptable side-effect profile, do not induce long-term protection, and provide no cross-protection against the different serovars of pathogenic leptospira. The current major focus in leptospirosis research is to discover conserved protective antigens that may elicit longer-term protection against a broad range of Leptospira. There is a need to screen vaccine candidate genes in the genome of Leptospira interrogans.ResultsBioinformatics, comparative genomic hybridization (CGH) analysis and transcriptional analysis were used to identify vaccine candidates in the genome of L. interrogans serovar Lai strain #56601. Of a total of 4727 open reading frames (ORFs), 616 genes were predicted to encode surface-exposed proteins by P-CLASSIFIER combined with signal peptide prediction, α-helix transmembrane topology prediction, integral β-barrel outer membrane protein and lipoprotein prediction, as well as by retaining the genes shared by the two sequenced L. interrogans genomes and by subtracting genes with human homologues. A DNA microarray of L. interrogans strain #56601 was constructed for CGH analysis and transcriptome analysis in vitro. Three hundred and seven differential genes were identified in ten pathogenic serovars by CGH; 1427 genes had high transcriptional levels (Cy3 signal ≥ 342 and Cy5 signal ≥ 363.5, respectively). There were 565 genes in the intersection between the set encoding surface-exposed proteins and the set of 307 differential genes. The number of genes in the intersection between this set of 565 and the set of 1427 highly transcriptionally active genes was 226. These 226 genes were thus identified as putative vaccine candidates. The proteins encoded by these genes are not only potentially surface-exposed in the bacterium, but also conserved in two sequenced L. interrogans. Moreover, these genes are conserved among ten epidemic serovars in China and have high transcriptional levels in vitro.ConclusionOf the 4727 ORFs in the genome of L. interrogans, 226 genes were identified as vaccine candidates by bioinformatics, CGH and transcriptional analysis on the basis of the theory of reverse vaccinology. The proteins encoded by these genes might be useful as vaccine candidates as well as for diagnosis of leptospirosis.

Structure of the monomeric outer-membrane porin OmpG in the open and closed conformation

OmpG, a monomeric pore-forming protein from Escherichia coli outer membranes, was refolded from inclusion bodies and crystallized in two different conformations. The OmpG channel is a 14-stranded beta-barrel, with short periplasmic turns and seven extracellular loops. Crystals grown at neutral pH show the channel in the open state at 2.3 A resolution. In the 2.7 A structure of crystals grown at pH 5.6, the pore is blocked by loop 6, which folds across the channel. The rearrangement of loop 6 appears to be triggered by a pair of histidine residues, which repel one another at acidic pH, resulting in the breakage of neighbouring H-bonds and a lengthening of loop 6 from 10 to 17 residues. A total of 151 ordered LDAO detergent molecules were found in the 2.3 A structure, mostly on the hydrophobic outer surface of OmpG, mimicking the outer membrane lipid bilayer, with three LDAO molecules in the open pore. In the 2.7 A structure, OmpG binds one OG and one glucose molecule as sugar substrates in the closed pore.