Conserved Residues of the Putative L6 Loop of Escherichia coli BamA Play a Critical Role in the Assembly of β-Barrel Outer Membrane Proteins, Including That of BamA Itself

Many members of the Omp85 family of proteins form essential β-barrel outer membrane protein (OMP) biogenesis machinery in Gram-negative bacteria, chloroplasts, and mitochondria. In Escherichia coli, BamA, a member of the Omp85 family, folds into an outer membrane-embedded β-barrel domain and a soluble periplasmic polypeptide-transport-associated (POTRA) domain. Although the high-resolution structures of only the BamA POTRA domain of E. coli are available, the crystal structure of FhaC, an Omp85 family member and a component of the two-partner secretion system in Bordetella pertussis, suggests that the BamA β-barrel likely folds into a 16-stranded β-barrel. The FhaC β-barrel is occluded by an N-terminal α-helix and a large β-barrel loop, L6, which carries residues that are highly conserved among the Omp85 family members. Deletion of L6 in FhaC did not affect its biogenesis but abolished its secretion function. In this study, we tested the hypothesis that the conserved residues of the putative L6 loop, which presumably folds back into the lumen of the BamA β-barrel like the FhaC counterpart, play an important role in OMP and/or BamA biogenesis. The conserved (641)RGF(643) residues of L6 were either deleted or replaced with alanine in various permutations. Phenotypic and biochemical characterization of various BamA L6 mutants revealed that the conserved RGF residues are critical for OMP biogenesis. Moreover, three BamA L6 alterations, ΔRGF, AAA, and AGA, produced a conditional lethal phenotype, concomitant with severely reduced BamA levels and folding defects. Thus, the conserved (641)RGF(643) residues of the BamA L6 loop are important for BamA folding and biogenesis.     

Crystal Structure of the Dengue Virus Methyltransferase Bound to a 5′-Capped Octameric RNA

The N-terminal domain of the flavivirus NS5 protein functions as a methyltransferase (MTase). It sequentially methylates the N7 and 2′-O positions of the viral RNA cap structure (GpppA→^7meGpppA→^7meGpppA_2′-O-me). The same NS5 domain could also have a guanylyltransferase activity (GTP+ppA-RNA→GpppA). The mechanism by which this protein domain catalyzes these three distinct functions is currently unknown. Here we report the crystallographic structure of DENV-3 MTase in complex with a 5′-capped RNA octamer (G_pppAGAACCUG) at a resolution of 2.9 Å. Two RNA octamers arranged as kissing loops are encircled by four MTase monomers around a 2-fold non-crystallography symmetry axis. Only two of the four monomers make direct contact with the 5′ end of RNA. The RNA structure is stabilised by the formation of several intra and intermolecular base stacking and non-canonical base pairs. The structure may represent the product of guanylylation of the viral genome prior to the subsequent methylation events that require repositioning of the RNA substrate to reach to the methyl-donor sites. The crystal structure provides a structural explanation for the observed trans-complementation of MTases with different methylation defects.     

Crystal structure of Escherichia coli BamB, a lipoprotein component of the β-barrel assembly machinery complex

In Gram-negative bacteria, the BAM (β-barrel assembly machinery) complex catalyzes the essential process of assembling outer membrane proteins. The BAM complex in Escherichia coli consists of five proteins: one β-barrel membrane protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE. Despite their role in outer membrane protein biogenesis, there is currently a lack of functional and structural information on the lipoprotein components of the BAM complex. Here, we report the first crystal structure of BamB, the largest and most functionally characterized lipoprotein component of the BAM complex. The crystal structure shows that BamB has an eight-bladed β-propeller structure, with four β-strands making up each blade. Mapping onto the structure the residues previously shown to be important for BamA interaction reveals that these residues, despite being far apart in the amino acid sequence, are localized to form a continuous solvent-exposed surface on one side of the β-propeller. Found on the same side of the β-propeller is a cluster of residues conserved among BamB homologs. Interestingly, our structural comparison study suggests that other proteins with a BamB-like fold often participate in protein or ligand binding, and that the binding interface on these proteins is located on the surface that is topologically equivalent to where the conserved residues and the residues that are important for BamA interaction are found on BamB. Our structural and bioinformatic analyses, together with previous biochemical data, provide clues to where the BamA and possibly a substrate interaction interface may be located on BamB.     

Crystal Structure and Molecular Imaging of the Nav Channel β3 Subunit Indicates a Trimeric Assembly

The vertebrate sodium (Na_v) channel is composed of an ion-conducting α subunit and associated β subunits. Here, we report the crystal structure of the human β3 subunit immunoglobulin (Ig) domain, a functionally important component of Na_v channels in neurons and cardiomyocytes. Surprisingly, we found that the β3 subunit Ig domain assembles as a trimer in the crystal asymmetric unit. Analytical ultracentrifugation confirmed the presence of Ig domain monomers, dimers, and trimers in free solution, and atomic force microscopy imaging also detected full-length β3 subunit monomers, dimers, and trimers. Mutation of a cysteine residue critical for maintaining the trimer interface destabilized both dimers and trimers. Using fluorescence photoactivated localization microscopy, we detected full-length β3 subunit trimers on the plasma membrane of transfected HEK293 cells. We further show that β3 subunits can bind to more than one site on the Na_v 1.5 α subunit and induce the formation of α subunit oligomers, including trimers. Our results suggest a new and unexpected role for the β3 subunits in Na_v channel cross-linking and provide new structural insights into some pathological Na_v channel mutations.     

Crystal Structure of the GalNAc/Gal-Specific Agglutinin from the Phytopathogenic Ascomycete Sclerotinia sclerotiorum Reveals Novel Adaptation of a β-Trefoil Domain

A lectin from the phytopathogenic ascomycete Sclerotinia sclerotiorum that shares only weak sequence similarity with characterized fungal lectins has recently been identified. S. sclerotiorum agglutinin (SSA) is a homodimeric protein consisting of two identical subunits of ∼ 17 kDa and displays specificity primarily towards Gal/GalNAc. Glycan array screening indicates that SSA readily interacts with Gal/GalNAc-bearing glycan chains. The crystal structures of SSA in the ligand-free form and in complex with the Gal-β1,3-GalNAc (T-antigen) disaccharide have been determined at 1.6 and 1.97 Å resolution, respectively. SSA adopts a β-trefoil domain as previously identified for other carbohydrate-binding proteins of the ricin B-like lectin superfamily and accommodates terminal non-reducing galactosyl and N-acetylgalactosaminyl glycans. Unlike other structurally related lectins, SSA contains a single carbohydrate-binding site at site α. SSA reveals a novel dimeric assembly markedly dissimilar to those described earlier for ricin-type lectins. The present structure exemplifies the adaptability of the β-trefoil domain in the evolution of fungal lectins.     

The Structure of CcmP,a Tandem Bacterial Microcompartment Domain Protein from the β-Carboxysome,Forms a Subcompartment Within a Microcompartment

The carboxysome is a bacterial organelle found in all cyanobacteria; it encapsulates CO₂ fixation enzymes within a protein shell. The most abundant carboxysome shell protein contains a single bacterial microcompartment (BMC) domain. We present in vivo evidence that a hypothetical protein (dubbed CcmP) encoded in all β-cyanobacterial genomes is part of the carboxysome. We show that CcmP is a tandem BMC domain protein, the first to be structurally characterized from a β-carboxysome. CcmP forms a dimer of tightly stacked trimers, resulting in a nanocompartment-containing shell protein that may weakly bind 3-phosphoglycerate, the product of CO₂ fixation. The trimers have a large central pore through which metabolites presumably pass into the carboxysome. Conserved residues surrounding the pore have alternate side-chain conformations suggesting that it can be open or closed. Furthermore, CcmP and its orthologs in α-cyanobacterial genomes form a distinct clade of shell proteins. Members of this subgroup are also found in numerous heterotrophic BMC-associated gene clusters encoding functionally diverse bacterial organelles, suggesting that the potential to form a nanocompartment within a microcompartment shell is widespread. Given that carboxysomes and architecturally related bacterial organelles are the subject of intense interest for applications in synthetic biology/metabolic engineering, our results describe a new type of building block with which to functionalize BMC shells.     

The Role of the Amino-Terminal β-Barrel Domain of the α and β Subunits in the Yeast F1-ATPase

The crystal structure of mitochondrial F₁-ATPase indicatesthat the and subunits fold into a structure defined by threedomains: the top -barrel domain, the middle nucleotide-binding domain,and the C-terminal -helix bundle domain (Abraham et al.1994); Bianchet et al., 1998). The -barrel domains of the and subunits form a crown structure at the top ofF₁, which was suggested to stabilize it (Abraham et al.1994). In this study. the role of the -barrel domain in the and subunits of the yeast Saccharomyces cerevisiae F₁,with regard to its folding and assembly, was investigated. The -barreldomains of yeast F₁ and subunits were expressedindividually and together in Escherichia coli. When expressedseperately, the -barrel domain of the subunit formed a largeaggregate structure, while the domain of the subunit waspredominately a monomer or dimer. However, coexpression of the -barreldomain of subunit domain. Furthermore, the two domains copurified incomplexes with the major portion of the complex found in a small molecularweight form. These results indicate that the -barrel domain of the and subunits interact specifically with each other and thatthese interactions prevent the aggregation of the -barrel domain of the subunit. These results mimic in vivo results and suggest thatthe interactions of the -barrel domains may be critical during thefolding and assembly of F₁.     

Remote Coupled Drastic β-Barrel to β-Sheet Transition of the Protein Translocation Motor

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The β1 Integrin Subunit is Not a Specific Component of the Costamere Domain in Human Myocardial Cells

Studies on altered integrin receptor expression during cardiac hypertrophy and heart failure requires accurate knowledge of the distributional pattern of integrins in myocardial cells. At present the general consensus is that in cardiac muscle the β₁ integrin receptor is mainly localized to the same sarcolemmal domain as vinculin at Z-band levels (‘costamere’). Since most previous studies have been focusing on myocardial integrin distribution in lower mammals, the myocardial localization of the β₁ integrin subunit was investigated in biopsies collected from the auricle of patients undergoing a coronary bypass operation. Non-invasive serial optical sectioning was carried out by immuno-laser scanning confocal microscopy. Double-labelling for vinculin/α-actinin, and the cytoplasmic domain for the β₁ integrin subunit, showed that β₁ integrin is deposited throughout both the vinculin/α-actinin domains and the non-vinculin/α-actinin domains. These results were supported by a semi-quantitative analysis in extended focus images of the latter preparations. Higher magnification views at the electron microscopical levels of the large, extracellular domain of the β₁ integrin subunit disclosed a pronounced labelling in the form of a dense, irregular punctuate pattern that was distributed at Z-disc domains as well as along the entire sarcolemmal area between Z-discs. Our findings show that in human, myocardial cells, the β₁ integrin receptor does not only localize to the surface membrane at the Z-disc level (‘costamere’ in cardiac muscle), but has a widespread distribution along the sarcolemma.     

Crystal structure of BamD: an essential component of the β-Barrel assembly machinery of gram-negative bacteria

Folding and insertion of integral β-barrel proteins in the outer membrane (OM) is an essential process for Gram-negative bacteria that requires the β-barrel assembly machinery (BAM). Efficient OM protein (OMP) folding and insertion appears to require a consensus C-terminal signal in OMPs characterized by terminal F or W residues. The BAM complex is embedded in the OM and, in Escherichia coli, consists of the β-barrel BamA and four lipoproteins BamBCDE. BamA and BamD are broadly distributed across all species of Gram-negative bacteria, whereas the other components are present in only a subset of species. BamA and BamD are also essential for viability, suggesting that these two proteins constitute the functional core of the bacterial BAM complex. Here, we present the crystal structure of BamD from the thermophilic bacteria Rhodothermus marinus refined to 2.15 Å resolution. The protein contains five tetratricopeptide repeats (TPRs) organized into two offset tandems, each capped by a terminal helix. The N-terminal domain contains three TPRs and displays remarkable structural similarity with proteins that recognize targeting signals in extended conformations. The C-terminal domain harbors the remaining two TPRs and previously described mutations that impair binding to other BAM components map to this domain. Therefore, the structure suggests a model where the C-terminal domain provides a scaffold for interaction with BAM components, while the N-terminal domain participates in interaction with the substrates, either recognizing the C-terminal consensus sequence or binding unfolded OMP intermediates.     

A New Crystal Form of the Fab Fragment of a Monoclonal Antibody to Human Interleukin-2: The Three-Dimensional Structure at 2.7 Å Resolution

The three-dimensional structure of the antigen-binding fragment of a monoclonal antibody to human interleukin-2 in a new crystal form (space group P2₁2₁2₁; unit cell parameters: a = 42.82 Å, b = 90.68 Å, and c = 139.82 Å) was determined by the X-ray molecular replacement method at the resolution of 2.7 Å. The protein folding and the stereochemistry of its antigen-binding site were comparatively analyzed.     

The Crystal Structure of the Catalytic Domain of the NF-κB Inducing Kinase Reveals a Narrow but Flexible Active Site

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Highlights? Production of soluble NIK kinase domain ? Structures of apo murine and human NIK possess active conformation ? Structure of mNIK bound to inhibitors reveals conformational flexibility ? Inhibitor potency varies against mNIK and hNIK due to substitution in the active site     

The Protein-disulfide Isomerase DsbC Cooperates with SurA and DsbA in the Assembly of the Essential β-Barrel Protein LptD

The assembly of the β-barrel proteins present in the outer membrane (OM) of Gram-negative bacteria is poorly characterized. After translocation across the inner membrane, unfolded β-barrel proteins are escorted across the periplasm by chaperones that reside within this compartment. Two partially redundant chaperones, SurA and Skp, are considered to transport the bulk mass of β-barrel proteins. We found that the periplasmic disulfide isomerase DsbC cooperates with SurA and the thiol oxidase DsbA in the folding of the essential β-barrel protein LptD. LptD inserts lipopolysaccharides in the OM. It is also the only β-barrel protein with more than two cysteine residues. We found that surAdsbC mutants, but not skpdsbC mutants, exhibit a synthetic phenotype. They have a decreased OM integrity, which is due to the lack of the isomerase activity of DsbC. We also isolated DsbC in a mixed disulfide complex with LptD. As such, LptD is identified as the first substrate of DsbC that is localized in the OM. Thus, electrons flowing from the cytoplasmic thioredoxin system maintain the integrity of the OM by assisting the folding of one of the most important β-barrel proteins.     

Crystal Structure of the Human Pol α B Subunit in Complex with the C-terminal Domain of the Catalytic Subunit

In eukaryotic DNA replication, short RNA-DNA hybrid primers synthesized by primase-DNA polymerase α (Prim-Pol α) are needed to start DNA replication by the replicative DNA polymerases, Pol δ and Pol ϵ. The C terminus of the Pol α catalytic subunit (p180_C) in complex with the B subunit (p70) regulates the RNA priming and DNA polymerizing activities of Prim-Pol α. It tethers Pol α and primase, facilitating RNA primer handover from primase to Pol α. To understand these regulatory mechanisms and to reveal the details of human Pol α organization, we determined the crystal structure of p70 in complex with p180_C. The structured portion of p70 includes a phosphodiesterase (PDE) domain and an oligonucleotide/oligosaccharide binding (OB) domain. The N-terminal domain and the linker connecting it to the PDE domain are disordered in the reported crystal structure. The p180_C adopts an elongated asymmetric saddle shape, with a three-helix bundle in the middle and zinc-binding modules (Zn1 and Zn2) on each side. The extensive p180_C-p70 interactions involve 20 hydrogen bonds and a number of hydrophobic interactions resulting in an extended buried surface of 4080 Ų. Importantly, in the structure of the p180_C-p70 complex with full-length p70, the residues from the N-terminal to the OB domain contribute to interactions with p180_C. The comparative structural analysis revealed both the conserved features and the differences between the human and yeast Pol α complexes.     

The Crystal Structure of the Adenylation Enzyme VinN Reveals a Unique β-Amino Acid Recognition Mechanism

Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp²³⁰ residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes.     

Truncation of a β-Barrel Scaffold Dissociates Intrinsic Stability from Its Propensity to Aggregation

Δ98Δ is a functional all-β sheet variant of intestinal fatty acid binding protein (IFABP) that was generated by controlled proteolysis. This framework is useful to study the molecular determinants related to aggregation of β-barrel proteins. Albeit displaying increased conformational plasticity, Δ98Δ exhibits a nativelike β-barrel topology and is able to support a cooperative folding behavior. Here we present a comparative study of IFABP and Δ98Δ regarding their conformational perturbation and aggregation propensity triggered by trifluoroethanol. Both proteins share a common nucleation-elongation mechanism, whereby the rate-limiting step is the formation of stable dimeric nuclei followed by the association of monomers to the growing aggregates. Despite leading to a less stable structure, the extensive truncation of IFABP yields a form exhibiting a somewhat lower tendency to aggregate. This finding appears at odds with the established notion that a perturbation of the native compact fold should necessarily favor the population of aggregation-prone species. In addition to the aggregation propensity dictated by a given amino-acid sequence, our contention holds that long-range interactions might also play a major role in determining the overall aggregation propensity.