Translocation of group 1 capsular polysaccharide in Escherichia coli serotype K30. Structural and functional analysis of the outer membrane lipoprotein Wza

The late steps in assembly of capsular polysaccharides (CPS) and their translocation to the bacterial cell surface are not well understood. The Wza protein was shown previously to be required for the formation of the prototype group 1 capsule structure on the surface of Escherichia coli serotype K30 (Drummelsmith, J., and Whitfield, C. (2000) EMBO J. 19, 57-66). Wza is a conserved outer membrane lipoprotein that forms multimers adopting a ringlike structure, and collective evidence suggests a role for these structures in the export of capsular polymer across the outer membrane. Wza was purified in the native form and with a C-terminal hexahistidine tag. WzaHis6 was acylated and functional in capsule assembly, although its efficiency was slightly reduced in comparison to the native Wza protein. Ordered two-dimensional crystals of WzaHis6 were obtained after reconstitution of purified multimers into lipids. Electron microscopy of negatively stained crystals and Fourier filtering revealed ringlike multimers with an average outer diameter of 8.84 nm and an average central cavity diameter of 2.28 nm. Single particle analysis yielded projection structures at an estimated resolution of 3 nm, favoring a structure for the WzaHis6 containing eight identical subunits. A derivative of Wza (Wza*) in which the original signal sequence was replaced with that from OmpF showed that the native acylated N terminus of Wza is critical for formation of normal multimeric structures and for their competence for CPS assembly, but not for targeting Wza to the outer membrane. In the presence of Wza*, CPS accumulated in the periplasm but was not detected on the cell surface. Chemical cross-linking of intact cells suggested formation of a transmembrane complex minimally containing Wza and the inner membrane tyrosine autokinase Wzc.     

Wza: a new structural paradigm for outer membrane secretory proteins?

Gram-negative bacteria need to be able to transport a large variety of macromolecules across their outer membranes. In Escherichia coli, the passage of the group 1 capsular polysaccharide is mediated by an integral outer membrane protein, Wza. The crystal structure of Wza, determined recently, reveals a novel transmembrane alpha-helical barrel and a large central cavity within the core of the vase-shaped protein complex. The structure has similarities with that of the secretin protein, PilQ, which mediates the transition of type IV pili across the outer membrane. We propose that the large internal chamber, which can accommodate the secreted assembled macromolecule, is likely to be a common feature found in other outer membrane proteins involved in secretion processes.     

Structure of the Ni/Fe-S protein subcomponent of the acetyl-CoA decarbonylase/synthase complex from Methanosarcina thermophila at 26-A resolution

The acetyl-CoA decarbonylase/synthase (ACDS) complex is responsible for synthesis and cleavage of acetyl-CoA in methanogens. The complex is composed of five different subunits, with a probable stoichiometry of alpha(8)beta(8)gamma(8)delta(8)epsilon(8). The native molecular mass of a subcomponent of the ACDS complex from Methanosarcina thermophila, the Ni/Fe-S protein containing the 90-kDa alpha and 19-kDa epsilon subunits, was determined by scanning transmission electron microscopy. A value of 218.6 +/- 19.6 kDa (n = 566) was obtained, thus establishing that the oligomeric structure of this subcomponent is alpha(2)epsilon(2). The three-dimensional structure of alpha(2)epsilon(2) was determined at 26-A resolution by analysis of a large number of electron microscopic images of negatively stained, randomly oriented particles. The alpha(2)epsilon(2) subcomponent has a globular appearance, 110 A in diameter, and consists of two large, hemisphere-like masses that surround a hollow internal cavity. The two large masses are connected along one face by a bridge-like structure and have relatively less protein density joining them at other positions. The internal cavity has four main openings to the outside, one of which is directly adjacent to the bridge. The results are consistent with a structure in which the large hemispheric masses are assigned to the two alpha subunits, with epsilon(2) as the bridge forming a structural link between them. The structure of the alpha(2)epsilon(2) subcomponent is discussed in connection with biochemical data from gel filtration, crosslinking, and dissociation experiments and in the context of its function as a major component of the ACDS complex.     

Translocation of group 1 capsular polysaccharide to the surface of Escherichia coli requires a multimeric complex in the outer membrane

Surface expression of the group 1 K30 capsular polysaccharide of Escherichia coli strain E69 (O9a:K30) requires Wza(K30), a member of the outer membrane auxiliary (OMA) protein family. A mutation in wza(K30) severely restricts the formation of the K30 capsular structure on the cell surface, but does not interfere with the biosynthesis or polymerization of the K30 repeat unit. Here we show that Wza(K30) is a surface-exposed outer membrane lipoprotein. Wza(K30) multimers form ring-like structures in the outer membrane that are reminiscent of the secretins of type II and III protein translocation systems. We propose that Wza(K30) forms an outer membrane pore through which the K30-capsular antigen is translocated. This is the first evidence of a potential mechanism for translocation of high molecular weight polysaccharide across the outer membrane. The broad distribution of the OMA protein family suggests a similar process for polysaccharide export in diverse Gram-negative bacteria.     

Structure of the vacuolar ATPase by electron microscopy.

The structure of the vacuolar ATPase from bovine brain clathrin-coated vesicles has been determined by electron microscopy of negatively stained, detergent-solubilized enzyme molecules. Preparations of both lipid-containing and delipidated enzyme have been analyzed. The complex is organized in two major domains, a V(1) and V(0), with overall dimensions of 28 x 14 x 14 nm. The V(1) is a more or less spherical molecule with a central cavity. The V(0) has the shape of a flattened sphere or doughnut with a radius of about 100 A. The V(1) and V(0) are joined by a 60-A long and 40-A wide central stalk, consisting of several individual protein densities. Two kinds of smaller densities are visible at the top periphery of the V(1), and one of these seems to extend all the way down to the stalk domain in some averages. Images of both the lipid-containing and the delipidated complex show a 30-50-kDa protein density on the lumenal side of the complex, opposite the central stalk, centered in the ring of c subunits. A large trans-membrane mass, probably the C-terminal domain of the 100-kDa subunit a, is seen at the periphery of the c subunit ring in some projections. This large mass has both a lumenal and a cytosolic domain, and it is the cytosolic domain that interacts with the central stalk. Two to three additional protein densities can be seen in the V(1)-V(0) interface, all connected to the central stalk. Overall, the structure of the V-ATPase is similar to the structure of the related F(1)F(0)-ATP synthase, confirming their common origin.     

Characterization of the translocon of the outer envelope of chloroplasts

The protein translocon of the outer envelope of chloroplasts (Toc) consists of the core subunits Toc159, Toc75, and Toc34. To investigate the molecular structure, the core complex was purified. This core complex has an apparent molecular mass of approximately 500 kD and a molecular stoichiometry of 1:4:4-5 between Toc159, Toc75, and Toc34. The isolated translocon recognizes both transit sequences and precursor proteins in a GTP-dependent manner, suggesting its functional integrity. The complex is embedded by the lipids phosphatidylcholine and digalactosyldiacylglyceride. Two-dimensional structural analysis by EM revealed roughly circular particles consistent with the formation of a stable core complex. The particles show a diameter of approximately 130 A with a solid ring and a less dense interior structure. A three-dimensional map obtained by random conical tilt reconstruction of electron micrographs suggests that a "finger"-like central region separates four curved translocation channels within one complex.     

Structure of Ala24/Asp61 --> Asp24/Asn61 substituted subunit c of Escherichia coli ATP synthase: implications for the mechanism of proton transport and rotary movement in the F0 complex

The structure of the A24D/D61N substituted subunit c of Escherichia coli ATP synthase, in which the essential carboxylate has been switched from residue 61 of the second transmembrane helix (TMH) to residue 24 of the first TMH, has been determined by heteronuclear multidimensional NMR in a monophasic chloroform/methanol/water (4:4:1) solvent mixture. As in the case of the wild-type protein, A24D/D61N substituted subunit c forms a hairpin of two extended alpha-helices (residues 5-39 and 46-78), with residues 40-45 forming a connecting loop at the center of the protein. The structure was determined at pH 5, where Asp24 is fully protonated. The relative orientation of the two extended helices in the A24D/D61N structure is different from that in the protonated form of the wild-type protein, also determined at pH 5. The C-terminal helix is rotated by 150 degrees relative to the wild-type structure, and the N-terminal helix is rotated such that the essential Asp24 carboxyl group packs on the same side of the molecule as Asp61 in the wild-type protein. The changes in helix-helix orientation lead to a structure that is quite similar to that of the deprotonated form of wild-type subunit c, determined at pH 8. When a decameric ring of c subunits was modeled from the new structure, the Asp24 carboxyl group was found to pack in a cavity at the interface between two subunits that is similar to the cavity in which Asp61 of the wild-type protein is predicted to pack. The interacting faces of the packed subunits in this model are also similar to those in the wild-type model. The results provide further evidence that subunit c is likely to fold in at least two conformational states differing most notably in the orientation of the C-terminal helix. Based upon the structure, a mechanistic model is discussed that indicates how the wild-type and A24D/D61N subunits could utilize similar helical movements during H(+) transport-coupled rotation of the decameric c ring.     

Refined solution structure of a liganded type 2 wheat nonspecific lipid transfer protein

The refined structure of a wheat type 2 nonspecific lipid transfer protein (ns-LTP2) liganded with l-alpha-palmitoylphosphatidylglycerol has been determined by NMR. The (15)N-labeled protein was produced in Pichia pastoris. Physicochemical conditions and ligandation were intensively screened to obtain the best NMR spectra quality. This ns-LTP2 is a 67-residue globular protein with a diameter of about 30 A. The structure is composed of five helices forming a right superhelix. The protein presents an inner cavity, which has been measured at 341 A(3). All of the helices display hydrophobic side chains oriented toward the cavity. The phospholipid is found in this cavity. Its fatty acid chain is completely inserted in the protein, the l-alpha-palmitoylphosphatidylglycerol glycerol moiety being located on a positively charged pocket on the surface of the protein. The superhelix structure of the protein is coiled around the fatty acid chain. The overall structure shows similarities with ns-LTP1. Nevertheless, large three-dimensional structural discrepancies are observed for the H3 and H4 alpha-helices, the C-terminal region, and the last turn of the H2 helix. The lipid is orthogonal to the orientation observed in ns-LTP1. The volume of the hydrophobic cavity appears to be in the same range as the one of ns-LTP1, despite the fact that ns-LTP2 is shorter by 24 residues.     

Three-dimensional structure of the Neisseria meningitidis secretin PilQ determined from negative-stain transmission electron microscopy

The PilQ secretin from the pathogenic bacterium Neisseria meningitidis is an integral outer membrane protein complex which plays a crucial role in the biogenesis of type IV pili. We present here the first three-dimensional structure of this type of secretin at 2.5-nm resolution, obtained by single-particle averaging methods applied to the purified protein complex visualized in a negative stain. In projection, the PilQ complex is circular, with a donut-like appearance. When viewed from the side it has a rounded, conical profile. The complex was demonstrated to have 12-fold rotational symmetry, and this property was used to improve the quality of the density map by symmetry averaging. The dominant feature of the structure is a cavity, 10 nm deep, within the center of the molecule. The cavity is funnel-shaped in cross section, measures 6.5 nm in diameter at the top of the complex, and tapers to a closed point, effectively blocking formation of a continuous pore through the PilQ complex. These results suggest that the complex would have to undergo a conformational change in order to accommodate an assembled pilus fiber of diameter 6.5 nm running through the outer membrane.     

Three-dimensional structure of the anaphase-promoting complex

The anaphase-promoting complex (APC) is a cell cycle-regulated ubiquitin-protein ligase, composed of at least 11 subunits, that controls progression through mitosis and G1. Using cryo-electron microscopy and angular reconstitution, we have obtained a three-dimensional model of the human APC at a resolution of 24 A. The APC has a complex asymmetric structure 140 A x 140 A x 135 A in size, in which an outer protein wall surrounds a large inner cavity. We discuss the possibility that this cavity represents a reaction chamber in which ubiquitination reactions take place, analogous to the inner cavities formed by other protein machines such as the 26S proteasome and chaperone complexes. This cage hypothesis could help to explain the great subunit complexity of the APC.     

Periplasmic protein-protein contacts in the inner membrane protein Wzc form a tetrameric complex required for the assembly of Escherichia coli group 1 capsules

The K antigenic capsular polysaccharide forms a structural layer, the capsule, on the surfaces of Escherichia coli cells. The capsule provides an important protective covering that helps protect encapsulated bacteria from host immune defenses. The assembly and translocation of the capsule requires proteins in the inner and outer membranes. The inner membrane protein Wzc is a tyrosine autokinase that plays an essential role in what is believed to be a coordinated biosynthesis and secretion process. Mutants lacking Wzc can form K antigen oligosaccharides but are unable to polymerize high molecular weight capsular polymers. Wzc homologs have been identified in exopolymer biosynthesis systems in many different Gram-negative and -positive bacteria. Using single particle averaging on cryo-negatively stained samples, we have produced the first three-dimensional structure of this type of membrane protein in its phosphorylated state at approximately 14 A resolution. Perfluoro-octanoate-PAGE analysis of detergent-solubilized oligomeric Wzc and symmetry analysis of the transmission electron microscopy data clearly demonstrated that Wzc forms a tetrameric complex with C4 rotational symmetry. Viewed from the top of the complex, the oligomer is square with a diameter of approximately 100 A and can be divided into four separate densities. From the side, Wzc is approximately 110 A high and has a distinctive appearance similar to an extracted molar tooth. The upper "crown" region is approximately 55 A high and forms a continuous ring of density. Four unconnected "roots" ( approximately 65 A high) emerge from the underside of the crown. We propose that the crown is formed by protein-protein contacts from the four Wzc periplasmic domains, while each root represents an individual cytoplasmic tyrosine autokinase domain.     

Electron microscopic studies of the translin octameric ring

Translin is thought to participate in a variety of cellular activities including chromosomal translocations, translational regulation of mRNA expression, and mRNA transport. It forms an octameric ring structure capable of sequence-specific binding of both DNA and RNA substrates. We have used electron microscopy and single-particle image analysis to generate a three-dimensional reconstruction of the Translin ring. The subunits appear to have two distinct domains that assemble to form an open channel with diameter of approximately 30 A at one end and approximately 50 A at the opposite end. In the presence of either DNA or RNA containing consensus binding sequences, the largest opening into the central cavity is filled with density. Strikingly, although Translin shows significant sequence homology to only one other protein, Translin-associated factor X, the quaternary organization and the dimerization of subunits in the ring are very similar to those observed for hexameric ring helicases. This suggests that many of the structures in DNA and RNA metabolism may have similar quaternary organization.     

Three-dimensional electron microscopy of the clamp loader small subunit from Pyrococcus furiosus

An archaeal clamp loader, replication factor C (RFC), consists of two proteins, the small subunit (RFCS) and large subunit (RFCL), whose sequences are both highly homologous to those of the eukaryotic RFC components. We have investigated the oligomeric structure of RFCS from Pyrococcus furiosus by electron microscopy using single-particle analysis. RFCS forms mostly ring-shaped hexamers at pH 9.0, although it tends to form C-shaped tetramers or pentamers at a lower pH (pH 5.5). The three-dimensional (3D) structure of the RFCS hexamer was obtained by random conical tilt reconstruction at 24.0-A resolution. RFCS forms a hexameric ring with outer and inner diameters of 117 and 27 A, respectively, and with a height of about 55 A. The six subunits are arranged in a twisted manner with a sixfold symmetry around the channel. The 3D map revealed that the six subunits are arranged in a head-to-tail configuration. Although the RFC complex consists of RFCS and RFCL in vivo, RFCS alone, together with PCNA, substantially enhanced the DNA synthesizing activity of P. furiosus DNA polymerase I in vitro. The 3D reconstruction of RFCS with catalytic activity provides important insights into the organization mechanism and the functional state of the RFC complex.     

Crystal-structure determination of reduced nicotinamide adenine dinucleotide complex with horse liver alcohol dehydrogenase maintained in its apo conformation by zinc-bound imidazole

A crystallographic study to 2.4-A resolution of the ternary complex between horse liver alcohol dehydrogenase (LADH), NADH, and the effector molecule imidazole (Im) (LADH-NADH-Im) is presented. The ligand binding and the changes in the protein structure due to ligand interactions were interpreted from difference electron density maps calculated with phase angles derived from the refined native enzyme model. The complex crystallizes in the orthorhombic space group C2221, and the enzyme structure remains in the apo conformation in which the active-site cleft is not entirely shielded from the solvent. NADH binds in an extended conformation, and the protein-coenzyme interactions are weaker compared to other complexes. The B-stereospecific side of the nicotinamide ring faces the catalytic center (LADH is known to be an A-side-specific enzyme). However, the reactive carbon atom C4 of the ring has a similar position in relation to active-center groups in this structure compared to LADH complexes where the A side of the ring faces the substrate site. The carboxamide group is situated within hydrogen-bonding distance to the sulfur of Cys-46, which is one of the three protein ligands to the active-site zinc atom. The imidazole molecule is directly ligated to the metal ion, which has a roughly tetrahedral geometry in the complex.     

Ultrastructure of C4b-binding protein fragments formed by limited proteolysis using chymotrypsin

C4b-binding protein is a regulator of the classical pathway of the complement system, acting as a cofactor to the serine protease factor I in the degradation of C4b. Its molecular weight is approximately 570,000 and it is composed of multiple, disulfide-linked 70-kDa subunits. Visualized by electron microscopy (Dahlb?ck, B., Smith, C. A., and Muller-Eberhard, H. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 3641-3645), it has an unusual spider-like structure with multiple thin (30 A), elongated (330 A) tentacles. The number of tentacles was estimated to be seven. Limited proteolysis by chymotrypsin produces fragments of approximately 50- and 160-kDa, the latter composed of multiple, disulfide-linked, 25-kDa polypeptides. We now have isolated the undenatured C4b-binding protein fragments formed by treatment of the protein with chymotrypsin and have visualized them by electron microscopy. The 160-kDa fragment comprises the central portion of the C4b-binding protein, which appears as a ringlike structure with an inner diameter of 13 A and an outer diameter of 60 A and having attached an approximately 40-A long piece of each tentacle. The liberated 50-kDa fragment constitutes the major part (290-A long) of the tentacles. Chymotrypsin digestion of C4b-binding protein was also monitored as a function of time by polyacrylamide gel electrophoresis and the number of subunits cleaved was found to be seven, supporting our previous ultrastructural data which suggested that C4b-binding protein contains seven identical tentacle-like subunits.     

Assembly of the secretion pores GspD,Wza and CsgG into bacterial outer membranes does not require the Omp85 proteins BamA or TamA

In Gram‐negative bacteria, β‐barrel proteins are integrated into the outer membrane by the β‐barrel assembly machinery, with key components of the machinery being the Omp85 family members BamA and TamA. Recent crystal structures and cryo‐electron microscopy show a diverse set of secretion pores in Gram‐negative bacteria, with α‐helix (Wza and GspD) or β‐strand (CsgG) transmembrane segments in the outer membrane. We developed assays to measure the assembly of three distinct secretion pores that mediate protein (GspD), curli fibre (CsgG) and capsular polysaccharide (Wza) secretion by bacteria and show that depletion of BamA and TamA does not diminish the assembly of Wza, GspD or CsgG. Like the well characterised pilotins for GspD and other secretins, small periplasmic proteins enhance the assembly of the CsgG β‐barrel. We discuss a model for integral protein assembly into the bacterial outer membrane, focusing on the commonalities and differences in the assembly of Wza, GspD and CsgG.     

The crystal structure of Escherichia coli group 4 capsule protein GfcC reveals a domain organization resembling that of Wza

We report the 1.9 ? resolution crystal structure of enteropathogenic Escherichia coli GfcC, a periplasmic protein encoded by the gfc operon, which is essential for assembly of group 4 polysaccharide capsule (O-antigen capsule). Presumed gene orthologs of gfcC are present in capsule-encoding regions of at least 29 genera of Gram-negative bacteria. GfcC, a member of the DUF1017 family, is comprised of tandem β-grasp (ubiquitin-like) domains (D2 and D3) and a carboxyl-terminal amphipathic helix, a domain arrangement reminiscent of that of Wza that forms an exit pore for group 1 capsule export. Unlike the membrane-spanning C-terminal helix from Wza, the GfcC C-terminal helix packs against D3. Previously unobserved in a β-grasp domain structure is a 48-residue helical hairpin insert in D2 that binds to D3, constraining its position and sequestering the carboxyl-terminal amphipathic helix. A centrally located and invariant Arg115 not only is essential for proper localization but also forms one of two mostly conserved pockets. Finally, we draw analogies between a GfcC protein fused to an outer membrane β-barrel pore in some species and fusion proteins necessary for secreting biofilm-forming exopolysaccharides.     

Crystal structure of a heptameric Sm-like protein complex from archaea: implications for the structure and evolution of snRNPs

The Sm/Lsm proteins associate with small nuclear RNA to form the core of small nuclear ribonucleoproteins, required for processes as diverse as pre-mRNA splicing, mRNA degradation and telomere formation. The Lsm proteins from archaea are likely to represent the ancestral Sm/Lsm domain. Here, we present the crystal structure of the Lsm alpha protein from the thermophilic archaeon Methanobacterium thermoautotrophicum at 2.0 A resolution. The Lsm alpha protein crystallizes as a heptameric ring comprised of seven identical subunits interacting via beta-strand pairing and hydrophobic interactions. The heptamer can be viewed as a propeller-like structure in which each blade consists of a seven-stranded antiparallel beta-sheet formed from neighbouring subunits. There are seven slots on the inner surface of the heptamer ring, each of which is lined by Asp, Asn and Arg residues that are highly conserved in the Sm/Lsm sequences. These conserved slots are likely to form the RNA-binding site. In archaea, the gene encoding Lsm alpha is located next to the L37e ribosomal protein gene in a putative operon, suggesting a role for the Lsm alpha complex in ribosome function or biogenesis.