Structural Characterization of Novel Pseudomonas aeruginosa Type IV Pilins

Pseudomonas aeruginosa type IV pili, composed of PilA subunits, are used for attachment and twitching motility on surfaces. P. aeruginosa strains express one of five phylogenetically distinct PilA proteins, four of which are associated with accessory proteins that are involved either in pilin posttranslational modification or in modulation of pilus retraction dynamics. Full understanding of pilin diversity is crucial for the development of a broadly protective pilus-based vaccine. Here, we report the 1.6-Å X-ray crystal structure of an N-terminally truncated form of the novel PilA from strain Pa110594 (group V), which represents the first non-group II pilin structure solved. Although it maintains the typical T4a pilin fold, with a long N-terminal α-helix and four-stranded antiparallel β-sheet connected to the C-terminus by a disulfide-bonded loop, the presence of an extra helix in the αβ-loop and a disulfide-bonded loop with helical character gives the structure T4b pilin characteristics. Despite the presence of T4b features, the structure of PilA from strain Pa110594 is most similar to the Neisseria gonorrhoeae pilin and is also predicted to assemble into a fiber similar to the GC pilus, based on our comparative pilus modeling. Interactions between surface-exposed areas of the pilin are suggested to contribute to pilus fiber stability. The non-synonymous sequence changes between group III and V pilins are clustered in the same surface-exposed areas, possibly having an effect on accessory protein interactions. However, based on our high-confidence model of group III PilA_PA14, compensatory changes allow for maintenance of a similar shape.     

Structural Studies on the Pseudomonas aeruginosa Sialidase-Like Enzyme PA2794 Suggest Substrate and Mechanistic Variations

Pseudomonas aeruginosa encodes an enzyme (PA2794) that is annotated as a sialidase (or neuraminidase), as it possesses three bacterial neuraminidase repeats that are a signature of nonviral sialidases. A recent report showed that when the gene encoding this sialidase is knocked out, this led to a reduction in biofilm production in the lungs of mice, and it was suggested that the enzyme recognizes pseudaminic acid, a sialic acid analogue that decorates the flagella of Pseudomonas, Helicobacter, and Campylobacter species. Here, we present the crystal structure of the P. aeruginosa enzyme and show that it adopts a trimeric structure, partly held together by an immunoglobulin-like trimerization domain that is C-terminal to a classical β-propeller sialidase domain. The recombinant enzyme does not show any sialidase activity with the standard fluorogenic sialic-acid-based substrate. The proposed active site contains certain conserved features of a sialidase: a nucleophilic tyrosine with its associated glutamic acid, and two of the usual three arginines that interact with the carboxylic acid group of the substrate, but is missing the first arginine and the aspartic acid that acts as an acid/base in all sialidases studied to date. We show, by in silico docking, that the active site may accommodate pseudaminic acid but not sialic acid and that this is due, in part, to a phenylalanine in the hydrophobic pocket that selects for the alternative stereochemistry of pseudaminic acid at C5 compared to sialic acid. Mutation of this phenylalanine to an alanine converts the enzyme into a sialidase, albeit a poor one, which we confirm by kinetics and NMR, and this allowed us to probe the function of other amino acids. We propose that a histidine plays the role of the acid/base, whose state is altered through a charge-relay system involving a novel His-Tyr-Glu triad. The location of this relay system precludes the presence of one of the three arginines usually found in a sialidase active site.     

Discoidin I from Dictyostelium discoideum and Interactions with Oligosaccharides: Specificity, Affinity, Crystal Structures, and Comparison with Discoidin II

Discoidin I (DiscI) and discoidin II (DiscII) are N-acetylgalactosamine (GalNAc)-binding proteins from Dictyostelium discoideum. They consist of two domains: an N-terminal discoidin domain and a C-terminal H-type lectin domain. They were cloned and expressed in high yield in recombinant form in Escherichia coli. Although both lectins bind galactose (Gal) and GalNAc, glycan array experiments performed on the recombinant proteins displayed strong differences in their specificity for oligosaccharides. DiscI and DiscII bind preferentially to Gal/GalNAcβ1-3Gal/GalNAc-containing and Gal/GalNAcβ1-4GlcNAcβ1-6Gal/GalNAc-containing glycans, respectively. The affinity of the interaction of DiscI with monosaccharides and disaccharides was evaluated using isothermal titration calorimetry experiments. The three-dimensional structures of native DiscI and its complexes with GalNAc, GalNAcβ1-3Gal, and Galβ1-3GalNAc were solved by X-ray crystallography. DiscI forms trimers with involvement of calcium at the monomer interface. The N-terminal discoidin domain presents a structural similarity to F-type lectins such as the eel agglutinin, where an amphiphilic binding pocket suggests possible carbohydrate-binding activity. In the C-terminal H-type lectin domain, the GalNAc residue establishes specific hydrogen bonds that explain the observed affinity (K_d = 3 × 10^− 4 M). The different specificities of DiscI and DiscII for oligosaccharides were rationalized from the different structures obtained by either X-ray crystallography or molecular modeling.     

Crystal structure of the Vibrio cholerae colonization factor TcpF and identification of a functional immunogenic site

Vibrio cholerae relies on two main virulence factors—toxin-coregulated pilus (TCP) and cholera toxin—to cause the gastrointestinal disease cholera. TCP is a type IV pilus that mediates bacterial autoagglutination and colonization of the intestine. TCP is encoded by the tcp operon, which also encodes TcpF, a protein of unknown function that is secreted by V. cholerae in a TCP-dependent manner. Although TcpF is not required for TCP biogenesis, a tcpF mutant has a colonization defect in the infant mouse cholera model that is as severe as a pilus mutant. Furthermore, TcpF antisera protect against V. cholerae infection. TcpF has no apparent sequence homology to any known protein. Here, we report the de novo X-ray crystal structure of TcpF and the identification of an epitope that is critical for its function as a colonization factor. A monoclonal antibody recognizing this epitope is protective against V. cholerae challenge and adds to the protection provided by an anti-TcpA antibody. These data suggest that TcpF has a novel function in V. cholerae colonization and define a region crucial for this function.     

Crystal Structures of A. acidocaldarius Endoglucanase Cel9A in Complex with Cello-Oligosaccharides: Strong − 1 and − 2 Subsites Mimic Cellobiohydrolase Activity

Alicyclobacillus acidocaldarius endoglucanase Cel9A (AaCel9A) is an inverting glycoside hydrolase with β-1,4-glucanase activity on soluble polymeric substrates. Here, we report three X-ray structures of AaCel9A: a ligand-free structure at 1.8 Å resolution and two complexes at 2.66 and 2.1 Å resolution, respectively, with cellobiose obtained by co-crystallization and with cellotetraose obtained by the soaking method. AaCel9A forms an (α/α)₆-barrel like other glycoside hydrolase family 9 enzymes. When cellobiose is used as a ligand, three glucosyl binding subsites are occupied, including two on the glycone side, while with cellotetraose as a ligand, five subsites, including four on the glycone side, are occupied. A structural comparison showed no conformational rearrangement of AaCel9A upon ligand binding. The structural analysis demonstrates that of the four minus subsites identified, subsites − 1 and − 2 show the strongest interaction with bound glucose. In conjunction with the open active-site cleft of AaCel9A, this is able to reconcile the previously observed cleavage of short-chain oligosaccharides with cellobiose as main product with the endo mode of action on larger polysaccharides.     

Crystal Structures of the CBS and DRTGG Domains of the Regulatory Region of Clostridiumperfringens Pyrophosphatase Complexed with the Inhibitor, AMP, and Activator, Diadenosine Tetraphosphate

Nucleotide-binding cystathionine β-synthase (CBS) domains serve as regulatory units in numerous proteins distributed in all kingdoms of life. However, the underlying regulatory mechanisms remain to be established. Recently, we described a subfamily of CBS domain-containing pyrophosphatases (PPases) within family II PPases. Here, we express a novel CBS-PPase from Clostridium perfringens (CPE2055) and show that the enzyme is inhibited by AMP and activated by a novel effector, diadenosine 5′,5-P1,P4-tetraphosphate (AP₄A). The structures of the AMP and AP₄A complexes of the regulatory region of C. perfringens PPase (cpCBS), comprising a pair of CBS domains interlinked by a DRTGG domain, were determined at 2.3 Å resolution using X-ray crystallography. The structures obtained are the first structures of a DRTGG domain as part of a larger protein structure. The AMP complex contains two AMP molecules per cpCBS dimer, each bound to a single monomer, whereas in the activator-bound complex, one AP₄A molecule bridges two monomers. In the nucleotide-bound structures, activator binding induces significant opening of the CBS domain interface, compared with the inhibitor complex. These results provide structural insight into the mechanism of CBS-PPase regulation by nucleotides.     

Structure and Specificity of a Binary Tandem Domain F-Lectin from Striped Bass (Morone saxatilis)

The plasma of the striped bass Morone saxatilis contains a fucose-specific lectin (MsaFBP32) that consists of two F-type carbohydrate recognition domains (CRDs) in tandem. The crystal structure of the complex of MsaFBP32 with l-fucose reported here shows a cylindrical  81-Å-long and  60-Å-wide trimer divided into two globular halves: one containing N-terminal CRDs (N-CRDs) and the other containing C-terminal CRDs (C-CRDs). The resulting binding surfaces at the opposite ends of the cylindrical trimer have the potential to cross-link cell surface or humoral carbohydrate ligands. The N-CRDs and C-CRDs of MsaFBP32 exhibit significant structural differences, suggesting that they recognize different glycans. Analysis of the carbohydrate binding sites provides the structural basis for the observed specificity of MsaFBP32 for simple carbohydrates and suggests that the N-CRD recognizes more complex fucosylated oligosaccharides and with a relatively higher avidity than the C-CRD. Modeling of MsaFBP32 complexed with fucosylated glycans that are widely distributed in prokaryotes and eukaryotes rationalizes the observation that binary tandem CRD F-type lectins function as opsonins by cross-linking “non-self” carbohydrate ligands and “self” carbohydrate ligands, such as sugar structures displayed by microbial pathogens and glycans on the surface of phagocytic cells from the host.     

Crystal structure of the polyextremophilic alpha-amylase AmyB from Halothermothrix orenii: details of a productive enzyme-substrate complex and an N domain with a role in binding raw starch

The gene for a membrane-bound, halophilic, and thermostable α-amylase, AmyB, from Halothermothrix orenii was cloned and sequenced. The crystal structure shows that, in addition to the typical domain organization of family 13 glycoside hydrolases, AmyB carries an additional N-terminal domain (N domain) that forms a large groove—the N-C groove—some 30 Å away from the active site. The structure of AmyB with the inhibitor acarbose at 1.35 Å resolution shows that a nonasaccharide has been synthesized through successive transglycosylation reactions of acarbose. Unexpectedly, in a complex of wild-type AmyB with α-cyclodextrin and maltoheptaose at 2.2 Å resolution, a maltotetraose molecule is bound in subsites − 1 to + 3, spanning the cleavage point at − 1/+ 1, with the − 1 glucosyl residue present as a ²S_o skew boat. This wild-type AmyB complex was obtained in the presence of a large excess of substrate, a condition under which it is possible to capture Michaelis complexes, which may explain the observed binding across − 1/+ 1 and ring distortion. We observe three methionine side chains that serve as “binding platforms” for glucosyl rings in AmyB, a seemingly rare occurrence in carbohydrate-binding proteins. The structures and results from the biochemical characterization of AmyB and AmyB lacking the N domain show that the N domain increases binding of the enzyme to raw starch. Furthermore, theoretical modeling suggests that the N-C groove can accommodate, spatially and chemically, large substrates such as A-starch.     

The Indispensable N-Terminal Half of eIF3j/HCR1 Cooperates with its Structurally Conserved Binding Partner eIF3b/PRT1-RRM and with eIF1A in Stringent AUG Selection

Despite recent progress in our understanding of the numerous functions of individual subunits of eukaryotic translation initiation factor (eIF) 3, little is known on the molecular level. Using NMR spectroscopy, we determined the first solution structure of an interaction between eIF3 subunits. We revealed that a conserved tryptophan residue in the human eIF3j N-terminal acidic motif (NTA) is held in the helix α1 and loop 5 hydrophobic pocket of the human eIF3b RNA recognition motif (RRM). Mutating the corresponding “pocket” residues in its yeast orthologue reduces cellular growth rate, eliminates eIF3j/HCR1 association with eIF3b/PRT1 in vitro and in vivo, affects 40S occupancy of eIF3, and produces a leaky scanning defect indicative of a deregulation of the AUG selection process. Unexpectedly, we found that the N-terminal half of eIF3j/HCR1 containing the NTA is indispensable and sufficient for wild-type growth of yeast cells. Furthermore, we demonstrate that deletion of either j/HCR1 or its N-terminal half only, or mutation of the key tryptophan residues results in the severe leaky scanning phenotype partially suppressible by overexpressed eIF1A, which is thought to stabilize properly formed preinitiation complexes at the correct start codon. These findings indicate that eIF3j/HCR1 remains associated with the scanning preinitiation complexes and does not dissociate from the small ribosomal subunit upon mRNA recruitment, as previously believed. Finally, we provide further support for earlier mapping of the ribosomal binding site for human eIF3j by identifying specific interactions of eIF3j/HCR1 with small ribosomal proteins RPS2 and RPS23 located in the vicinity of the mRNA entry channel. Taken together, we propose that eIF3j/HCR1 closely cooperates with the eIF3b/PRT1 RRM and eIF1A on the ribosome to ensure proper formation of the scanning-arrested conformation required for stringent AUG recognition.