Escherichia coli O127 group 4 capsule proteins assemble at the outer membrane.

Enteropathogenic Escherichia coli O127 is encapsulated by a protective layer of polysaccharide made of the same strain specific O-antigen as the serotype lipopolysaccharide. Seven genes encoding capsule export functions comprise the group 4 capsule (gfc) operon. Genes gfcE, etk and etp encode homologs of the group 1 capsule secretion system but the upstream gfcABCD genes encode unknown functions specific to group 4 capsule export. We have developed an expression system for the large-scale production of the outer membrane protein GfcD. Contrary to annotations, we find that GfcD is a non-acylated integral membrane protein. Circular dichroism spectroscopy, light-scattering data, and the HHomp server suggested that GfcD is a monomeric ß-barrel with 26 ß-strands and an internal globular domain. We identified a set of novel protein-protein interactions between GfcB, GfcC, and GfcD, both in vivo and in vitro, and quantified the binding properties with isothermal calorimetry and biolayer interferometry. GfcC and GfcB form a high-affinity heterodimer with a KD near 100 nM. This heterodimer binds to GfcD (KD = 28 µM) significantly better than either GfcB or GfcC alone. These gfc proteins may form a complex at the outer membrane for group 4 capsule secretion or for a yet unknown function.

Structural basis of peptidoglycan endopeptidase regulation.

Most bacteria surround themselves with a cell wall, a strong meshwork consisting primarily of the polymerized aminosugar peptidoglycan (PG). PG is essential for structural maintenance of bacterial cells, and thus for viability. PG is also constantly synthesized and turned over; the latter process is mediated by PG cleavage enzymes, for example, the endopeptidases (EPs). EPs themselves are essential for growth but also promote lethal cell wall degradation after exposure to antibiotics that inhibit PG synthases (e.g., ß-lactams). Thus, EPs are attractive targets for novel antibiotics and their adjuvants. However, we have a poor understanding of how these enzymes are regulated in vivo, depriving us of novel pathways for the development of such antibiotics. Here, we have solved crystal structures of the LysM/M23 family peptidase ShyA, the primary EP of the cholera pathogen Vibrio cholerae Our data suggest that ShyA assumes two drastically different conformations: a more open form that allows for substrate binding and a closed form, which we predicted to be catalytically inactive. Mutations expected to promote the open conformation caused enhanced activity in vitro and in vivo, and these results were recapitulated in EPs from the divergent pathogens Neisseria gonorrheae and Escherichia coli Our results suggest that LysM/M23 EPs are regulated via release of the inhibitory Domain 1 from the M23 active site, likely through conformational rearrangement in vivo.

Crystal Structure Of Photorespiratory Alanine:Glyoxylate Aminotransferase 1 (AGT1) From Arabidopsis thaliana.

Photorespiration is an energetically costly metabolic pathway for the recycling of phosphoglycolate produced by the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) to phosphoglycerate. Arabidopsis alanine:glyoxylate aminotransferase 1 (AGT1) is a peroxisomal aminotransferase with a central role in photorespiration. This enzyme catalyzes various aminotransferase reactions, including serine:glyoxylate, alanine:glyoxylate, and asparagine:glyoxylate transaminations. To better understand structural features that govern the specificity of this enzyme, its crystal structures in the native form (2.2-Å resolution) and in the presence of l-serine (2.1-Å resolution) were solved. The structures confirm that this enzyme is dimeric, in agreement with studies of the active enzyme in solution. In the crystal, another dimer related by noncrystallographic symmetry makes close interactions to form a tetramer mediated in part by an extra carboxyl-terminal helix conserved in plant homologs of AGT1. Pyridoxal 5'-phosphate (PLP) is bound at the active site but is not held in place by covalent interactions. Residues Tyr35' and Arg36', entering the active site from the other subunits in the dimer, mediate interactions between AGT and l-serine when used as a substrate. In comparison, AGT1 from humans and AGT1 from Anabaena lack these two residues and instead position a tyrosine ring into the binding site, which accounts for their preference for l-alanine instead of l-serine. The structure also rationalizes the phenotype of the sat mutant, Pro251 to Leu, which likely affects the dimer interface near the catalytic site. This structural model of AGT1 provides valuable new information about this protein that may enable improvements to the efficiency of photorespiration.

Structure of the capsule and lipopolysaccharide O-antigen from the channel catfish pathogen, Aeromonas hydrophila.

A hypervirulent A. hydrophila (vAh) pathotype has been identified as the etiologic agent responsible for disease outbreaks in farmed carp species and channel catfish (Ictalurus punctatus) in China and the Southeastern United States, respectively. The possible route of infection has previously been unknown; however, virulence is believed to be multifactorial, involving the production/secretion of several virulence factors, including a high molecular weight group 4 capsular polysaccharide. Here we present chemical structural evidence of a novel capsule- and LPS-associated O-antigen found present in vAh isolated during these disease outbreaks. In this study, the chemical structure of the vAh O-antigen was determined by chemical analysis, Smith degradation, mass spectrometry, and 2D proton and carbon nuclear magnetic resonance (NMR) spectroscopy and found to be unique among described bacterial O-antigens. The O-antigen consists of hexasaccharide repeating units featuring a 4)-α-l-Fucp-(1-3)-ß-d-GlcpNAc-(1-4)-α-l-Fucp-(1-4)-ß-d-Glcp-(1- backbone, substituted with single residue side chains of α-d-Glcp and α-d-Quip3NAc linked to O-3 of the two fucose residues. The polysaccharide is partially O-acetylated on O-6 of the 4-substituted ß-Glcp residue.

Crystal structures of the amino-terminal domain of LpoA from Escherichia coli and Haemophilus influenzae.

The bacterial periplasmic protein LpoA is an outer membrane lipoprotein and an activator for the cross-linking activity of PBP1A, a bifunctional peptidoglycan synthase. Previous structures of the amino-terminal (N) domain of LpoA showed it to consist entirely of helices and loops, with at least four tetratricopeptide-like repeats. Although the previously determined orthorhombic crystal structure of the N domain of Haemophilus influenzae LpoA showed a typical curved structure with a concave groove, an NMR structure of the same domain from Escherichia coli was relatively flat. Here, a crystal structure of the N domain of E. coli LpoA was determined to a resolution of 2.1 Šand was found to be more similar to the H. influenzae crystal structure than to the E. coli NMR structure. To provide a quantitative description for these comparisons, the various structures were superimposed pairwise by fitting the first half of each structure to its pairwise partner and then calculating the rotation axis that would optimally superimpose the second half. Differences in both the magnitude of the rotation and the direction of the rotation axis were observed between different pairs of structures. A 1.35 Šresolution structure of a monoclinic crystal form of the N domain of H. influenzae LpoA was also determined. In this structure, the subdomains rotate 10° relative to those in the original orthorhombic H. influenzae crystal structure to further narrow the groove between the subdomains. To accommodate this, a bound chloride ion (in place of sulfate) allowed the closer approach of a helix that forms one side of the groove.

Structural analyses of the Haemophilus influenzae peptidoglycan synthase activator LpoA suggest multiple conformations in solution.

In many Gram-negative bacteria, the peptidoglycan synthase PBP1A requires the outer membrane lipoprotein LpoA for constructing a functional peptidoglycan required for bacterial viability. Previously, we have shown that the C-terminal domain of Haemophilus influenzae LpoA (HiLpoA) has a highly conserved, putative substrate-binding cleft between two α/ß lobes. Here, we report a 2.0 Å resolution crystal structure of the HiLpoA N-terminal domain. Two subdomains contain tetratricopeptide-like motifs that form a concave groove, but their relative orientation differs by ∼45° from that observed in an NMR structure of the Escherichia coli LpoA N domain. We also determined three 2.0-2.8 Å resolution crystal structures containing four independent full-length HiLpoA molecules. In contrast to an elongated model previously suggested for E. coli LpoA, each HiLpoA formed a U-shaped structure with a different C-domain orientation. This resulted from both N-domain twisting and rotation of the C domain (up to 30°) at the end of the relatively immobile interdomain linker. Moreover, a previously predicted hinge between the lobes of the LpoA C domain exhibited variations of up to 12°. Small-angle X-ray scattering data revealed excellent agreement with a model calculated by normal mode analysis from one of the full-length HiLpoA molecules but even better agreement with an ensemble of this molecule and two of the partially extended normal mode analysis-predicted models. The different LpoA structures helped explain how an outer membrane-anchored LpoA can either withdraw from or extend toward the inner membrane-bound PBP1A through peptidoglycan gaps and hence regulate the synthesis of peptidoglycan necessary for bacterial viability.

Cycling of Etk and Etp phosphorylation states is involved in formation of group 4 capsule by Escherichia coli.

Capsules frequently play a key role in bacterial interactions with their environment. Escherichia coli capsules were categorized as groups 1 through 4, each produced by a distinct mechanism. Etk and Etp are members of protein families required for the production of group 1 and group 4 capsules. These members function as a protein tyrosine kinase and protein tyrosine phosphatase, respectively. We show that Etp dephosphorylates Etk in vivo, and mutations rendering Etk or Etp catalytically inactive result in loss of group 4 capsule production, supporting the notion that cyclic phosphorylation and dephosphorylation of Etk is required for capsule formation. Notably, Etp also becomes tyrosine phosphorylated in vivo and catalyzes rapid auto-dephosphorylation. Further analysis identified Tyr121 as the phosphorylated residue of Etp. Etp containing Phe, Glu or Ala in place of Tyr121 retained phosphatase activity and catalyzed dephosphorylation of Etp and Etk. Although EtpY121E and EtpY121A still supported capsule formation, EtpY121F failed to do so. These results suggest that cycles of phosphorylation and dephosphorylation of Etp, as well as Etk, are involved in the formation of group 4 capsule, providing an additional regulatory layer to the complex control of capsule production.

Improved X-ray diffraction from Bacillus megaterium penicillin G acylase crystals through long cryosoaking dehydration.

Penicillin G acylase from Bacillus megaterium (BmPGA) is currently used in the pharmaceutical industry as an alternative to PGA from Escherichia coli (EcPGA) for the hydrolysis of penicillin G to produce 6-aminopenicillanic acid (6-APA), a penam nucleus for semisynthetic penicillins. Despite the significant differences in amino-acid sequence between PGAs from Gram-positive and Gram-negative bacteria, a representative PGA structure of Gram-positive origin has never been reported. In this study, crystallization and diffraction studies of BmPGA are described. Poor diffraction patterns with blurred spots at higher resolution were typical for BmPGA crystals cryocooled after a brief immersion in cryoprotectant solution. Overnight soaking in the same cryo-solution substantially improved both the mosaicity and resolution limit through the establishment of a new crystal-packing equilibrium. A crystal of BmPGA diffracted X-rays to 2.20 Å resolution and belonged to the monoclinic space group P2(1) with one molecule of BmPGA in the asymmetric unit.

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

We report the 1.9 Å 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 ß-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 ß-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 ß-barrel pore in some species and fusion proteins necessary for secreting biofilm-forming exopolysaccharides.

Structure of YraM, a protein essential for growth of Haemophilus influenzae.

Nontypeable Haemophilus influenzae is an obligate human parasite that often causes middle ear infections in children and exacerbates chronic obstructive pulmonary disorder, the fourth leading cause of death in the United States. There are no effective vaccines available for this strain. The lipoprotein YraM (gene HI1655) was identified as essential for the growth and viability of H. influenzae but its function is unknown. Sequence comparisons showed that YraM is a fusion of two protein modules. We grew crystals of the carboxyl-terminal module of YraM comprising residues 257-573 (YraM-C), phased the diffraction data by the multiwavelength anomalous diffraction technique, and refined the model to a crystallographic R-factor of 0.16 (R(free) = 0.19) with data to 1.35 A resolution. The two-domain structure of YraM-C adopts a fold similar to that observed for the open, unliganded forms of several periplasmic binding proteins (PBPs) involved in bacterial active transport. Sequence alignments of YraM homologues from other Gram-negative species showed that the most conserved residues of YraM-C cluster between the two domains in the location where other PBPs bind their cognate ligand. Modeling of YraM-C into a closed conformation similar to the leucine-bound form of the Leu/Ile/Val-binding protein (LIVBP) shows a putative binding pocket larger than the leucine-binding site in LIVBP. The pocket has both polar and nonpolar surfaces, with the latter located in the same area where a leucine side chain binds to LIVBP. We discuss possible biological functions of YraM considering its predicted location in the outer membrane, a novel place for such a binding protein.

Identification of an Escherichia coli operon required for formation of the O-antigen capsule.

Escherichia coli produces polysaccharide capsules that, based on their mechanisms of synthesis and assembly, have been classified into four groups. The group 4 capsule (G4C) polysaccharide is frequently identical to that of the cognate lipopolysaccharide O side chain and has, therefore, also been termed the O-antigen capsule. The genes involved in the assembly of the group 1, 2, and 3 capsules have been described, but those required for G4C assembly remained obscure. We found that enteropathogenic E. coli (EPEC) produces G4C, and we identified an operon containing seven genes, ymcD, ymcC, ymcB, ymcA, yccZ, etp, and etk, which are required for formation of the capsule. The encoded proteins appear to constitute a polysaccharide secretion system. The G4C operon is absent from the genomes of enteroaggregative E. coli and uropathogenic E. coli. E. coli K-12 contains the G4C operon but does not express it, because of the presence of IS1 at its promoter region. In contrast, EPEC, enterohemorrhagic E. coli, and Shigella species possess an intact G4C operon.

Two substrate-targeting sites in the Yersinia protein tyrosine phosphatase co-operate to promote bacterial virulence.

YopH is a protein tyrosine phosphatase and an essential virulence determinant of the pathogenic bacterium Yersinia. Yersinia delivers YopH into infected host cells using a type III secretion mechanism. YopH dephosphorylates several focal adhesion proteins including p130Cas in human epithelial cells, resulting in disruption of focal adhesions and cell detachment from the extracellular matrix. How the C-terminal protein tyrosine phosphatase domain of YopH targets specific substrates such as p130Cas in the complex milieu of the host cell has not been fully elucidated. An N-terminal non-catalytic domain of YopH binds p130Cas in a phosphotyrosine-dependent manner and functions as a novel substrate-targeting site. The structure of the YopH protein tyrosine phosphatase domain bound to a model phosphopeptide substrate was solved and the resulting structure revealed a second substrate-targeting site ('site 2') within the catalytic domain. Site 2 binds to p130Cas in a phosphotyrosine-dependent manner, and co-operates with the N-terminal domain ('site 1') to promote efficient recognition of p130Cas by YopH in epithelial cells. The identification of two substrate-targeting sites in YopH that co-operate to promote epithelial cell detachment and bacterial virulence reinforces the importance of protein-protein interactions for determining protein tyrosine phosphatase specificity in vivo, and highlights the sophisticated nature of microbial pathogenicity factors.

Solution structure and phosphopeptide binding to the N-terminal domain of Yersinia YopH: comparison with a crystal structure.

Virulence of pathogenic bacteria of the genus Yersinia requires the injection of six effector proteins into the cytoplasm of host cells. The amino-terminal domain of one of these effectors, the tyrosine phosphatase YopH, is essential for translocation of YopH, as well as for targeting it to phosphotyrosine-containing substrates of the type pYxxP. We report the high-resolution solution structure of the N-terminal domain (residues 1-129) from the Yersinia pseudotuberculosis YopH (YopH-NT) in complex with N-acetyl-DEpYDDPF-NH(2), a peptide derived from an in vivo protein substrate. In contrast to the domain-swapped dimer observed in a crystal structure of the same protein (Smith, C. L., Khandelwal, P., Keliikuli, K., Zuiderweg, E. R. P., and Saper, M. A. (2001) Mol. Microbiol. 42, 967-979), YopH-NT is monomeric in solution. The peptide binding site is located on a beta-hairpin that becomes the crossover point in the dimer structure. The binding site has several characteristics that are reminiscent of SH2 domains, which also bind to pYxxP sequences.