Florian D. Schubot

Selection and characterization of Yersinia pestis YopN mutants that constitutively block Yop secretion

Secretion of Yop effector proteins by the Yersinia pestis plasmid pCD1‐encoded type III secretion system (T3SS) is regulated in response to specific environmental signals. Yop secretion is activated by contact with a eukaryotic cell or by growth at 37°C in the absence of calcium. The secreted YopN protein, the SycN/YscB chaperone and TyeA form a cytosolic YopN/SycN/YscB/TyeA complex that is required to prevent Yop secretion in the presence of calcium and prior to contact with a eukaryotic cell. The mechanism by which these proteins prevent secretion and the subcellular location where the block in secretion occurs are not known. To further investigate both the mechanism and location of the YopN‐dependent block, we isolated and characterized several YopN mutants that constitutively block Yop secretion. All the identified amino‐acid substitutions that resulted in a constitutive block in Yop secretion mapped to a central domain of YopN that is not directly involved in the interaction with the SycN/YscB chaperone or TyeA. The YopN mutants required an intact TyeA‐binding domain and TyeA to block secretion, but did not require an N‐terminal secretion signal, an intact chaperone‐binding domain or the SycN/YscB chaperone. These results suggest that a C‐terminal domain of YopN complexed with TyeA blocks Yop secretion from a cytosolic, not an extracellular, location. A hypothetical model for how the YopN/SycN/YscB/TyeA complex regulates Yop secretion is presented.

A pivotal role for reductive methylation in the de novo crystallization of a ternary complex composed of Yersinia pestis virulence factors YopN, SycN and YscB.

Structural studies of a ternary complex composed of the Yersina pestis virulence factors YopN, SycN and YscB were initially hampered by poor solubility of the individual proteins. Co-expression of all three proteins in Escherichia coli yielded a well behaved complex, but this sample proved to be recalcitrant to crystallization. As crystallization efforts remained fruitless, even after the proteolysis-guided engineering of a truncated YopN polypeptide, reductive methylation of lysine residues was employed to alter the surface properties of the complex. The methylated complex yielded crystals that diffracted X-rays to a maximal resolution of 1.8 A. The potential utility of reductive methylation as a remedial strategy for high-throughput structural biology was further underscored by the successful modification of a selenomethionine-substituted sample.

The First Agmatine/Cadaverine Aminopropyl Transferase: Biochemical and Structural Characterization of an Enzyme Involved in Polyamine Biosynthesis in the Hyperthermophilic Archaeon Pyrococcus furiosus

We report here the characterization of the first agmatine/cadaverine aminopropyl transferase (ACAPT), the enzyme responsible for polyamine biosynthesis from an archaeon. The gene PF0127 encoding ACAPT in the hyperthermophile Pyrococcus furiosus was cloned and expressed in Escherichia coli, and the recombinant protein was purified to homogeneity. P. furiosus ACAPT is a homodimer of 65 kDa. The broad substrate specificity of the enzyme toward the amine acceptors is unique, as agmatine, 1,3-diaminopropane, putrescine, cadaverine, and sym-nor-spermidine all serve as substrates. While maximal catalytic activity was observed with cadaverine, agmatine was the preferred substrate on the basis of the k(cat)/K(m) value. P. furiosus ACAPT is thermoactive and thermostable with an apparent melting temperature of 108 degrees C that increases to 112 degrees C in the presence of cadaverine. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. The crystal structure of the enzyme determined to 1.8-A resolution confirmed its dimeric nature and provided insight into the proteolytic analyses as well as into mechanisms of thermal stability. Analysis of the polyamine content of P. furiosus showed that spermidine, cadaverine, and sym-nor-spermidine are the major components, with small amounts of sym-nor-spermine and N-(3-aminopropyl)cadaverine (APC). This is the first report in Archaea of an unusual polyamine APC that is proposed to play a role in stress adaptation.

Phosphorylation and Dephosphorylation among Dif Chemosensory Proteins Essential for Exopolysaccharide Regulation in Myxococcus xanthus

Myxococcus xanthus social gliding motility, which is powered by type IV pili, requires the presence of exopolysaccharides (EPS) on the cell surface. The Dif chemosensory system is essential for the regulation of EPS production. It was demonstrated previously that DifA (methyl-accepting chemotaxis protein [MCP]-like), DifC (CheW-like), and DifE (CheA-like) stimulate whereas DifD (CheY-like) and DifG (CheC-like) inhibit EPS production. DifD was found not to function downstream of DifE in EPS regulation, as a difD difE double mutant phenocopied the difE single mutant. It has been proposed that DifA, DifC, and DifE form a ternary signaling complex that positively regulates EPS production through the kinase activity of DifE. DifD was proposed as a phosphate sink of phosphorylated DifE (DifE approximately P), while DifG would augment the function of DifD as a phosphatase of phosphorylated DifD (DifD approximately P). Here we report in vitro phosphorylation studies with all the Dif chemosensory proteins that were expressed and purified from Escherichia coli. DifE was demonstrated to be an autokinase. Consistent with the formation of a DifA-DifC-DifE complex, DifA and DifC together, but not individually, were found to influence DifE autophosphorylation. DifD, which did not inhibit DifE autophosphorylation directly, was found to accept phosphate from autophosphorylated DifE. While DifD approximately P has an unusually long half-life for dephosphorylation in vitro, DifG efficiently dephosphorylated DifD approximately P as a phosphatase. These results support a model where DifE complexes with DifA and DifC to regulate EPS production through phosphorylation of a downstream target, while DifD and DifG function synergistically to divert phosphates away from DifE approximately P.

Analysis of the crystal structure of the ExsC.ExsE complex reveals distinctive binding interactions of the Pseudomonas aeruginosa type III secretion chaperone ExsC with ExsE and ExsD.

Pseudomonas aeruginosa, like many Gram-negative bacterial pathogens, requires its type III secretion system (T3SS) to facilitate acute infections. In P. aeruginosa, the expression of all T3SS-related genes is regulated by the transcriptional activator ExsA. A signaling cascade involving ExsA and three additional proteins, ExsC, ExsD, and ExsE, directly ties the upregulation of ExsA-mediated transcription to the activation of the type III secretion apparatus. In order to characterize the events underlying the signaling process, the crystal structure of the T3SS chaperone ExsC in complex with its cognate effector ExsE has been determined. The structure reveals critical contacts that mediate the interactions between these two proteins. Particularly striking is the presence of two Arg-X-Val-X-Arg motifs in ExsE that form identical interactions along opposite sides of an ExsC dimer. The structure also provides insights into the interactions of ExsC with the antiactivator protein ExsD. It was shown that the amino-terminal 46 residues of ExsD are sufficient for ExsC binding. On the basis of these findings, a new model for the ExsC.ExsD complex is proposed to explain its distinctive 2:2 stoichiometry and why ExsC displays a weaker affinity for ExsD than for ExsE.

Crystal Structure of a Type IV Pilus Assembly ATPase: Insights into the Molecular Mechanism of PilB from Thermus thermophilus

Type IV pili (T4P) mediate bacterial motility and virulence. The PilB/GspE family ATPases power the assembly of T4P and type 2 secretion systems. We determined the structure of the ATPase region of PilB (PilBATP) in complex with ATPγS to provide a model of a T4P assembly ATPase and a view of a PilB/GspE family hexamer at better than 3-Å resolution. Spatial positioning and conformations of the protomers suggest a mechanism of force generation. All six PilBATP protomers contain bound ATPγS. Two protomers form a closed conformation poised for ATP hydrolysis. The other four molecules assume an open conformation but separate into two pairs with distinct active-site accessibilities. We propose that one pair represents the post-hydrolysis phase while the other pair appears poised for ADP/ATP exchange. Collectively, the data suggest that T4P assembly is powered by coordinating concurrent substrate binding with ATP hydrolysis across the PilB hexamer.

The catalytic domain of the germination-specific lytic transglycosylase SleB from Bacillus anthracis displays a unique active site topology.

Bacillus anthracis produces metabolically inactive spores. Germination of these spores requires germination‐specific lytic enzymes (GSLEs) that degrade the unique cortex peptidoglycan to permit resumption of metabolic activity and outgrowth. We report the first crystal structure of the catalytic domain of a GSLE, SleB. The structure revealed a transglycosylase fold with unique active site topology and permitted identification of the catalytic glutamate residue. Moreover, the structure provided insights into the molecular basis for the specificity of the enzyme for muramic‐δ‐lactam‐containing cortex peptidoglycan. The protein also contains a metal‐binding site that is positioned directly at the entrance of the substrate‐binding cleft. Proteins 2012;.

Crystal Structure and Oligomeric State of the RetS Signaling Kinase Sensory Domain

The opportunistic pathogen Pseudomonas aeruginosa may cause both acute and chronic‐persistent infections in predisposed individuals. Acute infections require the presence of a functional type III secretion system (T3SS), whereas chronic P. aeruginosa infections are characterized by the formation of drug‐resistant biofilms. The T3SS and biofilm formation are reciprocally regulated by the signaling kinases LadS, RetS, and GacS. RetS downregulates biofilm formation and upregulates expression of the T3SS through a unique mechanism. RetS forms a heterodimeric complex with GacS and thus prevents GacS autophosphorylation and downstream signaling. The signals that regulate RetS are not known but RetS possesses a distinctive periplasmic sensor domain that is believed to serve as receptor for the regulatory ligand. We have determined the crystal structure of the RetS sensory domain at 2.0 Å resolution. The structure closely resembles those of carbohydrate binding modules of other proteins, suggesting that the elusive ligands are likely carbohydrate moieties. In addition to the conserved beta‐sandwich structure, the sensory domain features two alpha helices which create a unique surface topology. Protein–protein crosslinking and fluorescence energy transfer experiments also revealed that the sensory domain dimerizes with a dissociation constant of Kd = 580 ± 50 nM, a result with interesting implications for our understanding of the underlying signaling mechanism. Proteins 2010.

Structural genomics of Pyrococcus furiosus: X-ray crystallography reveals 3D domain swapping in rubrerythrin

Wolfram Tempel, Zhi-Jie (James) Liu, Florian D. Schubot, Ashit Shah, Michael V. Weinberg, Francis E. Jenney, Jr., W. Bryan Arendall, III, Michael W. W. Adams, Jane S. Richardson, David C. Richardson, John P. Rose,* and Bi-Cheng Wang Southeast Collaboratory for Structural Genomics, Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia Department of Biochemistry, Duke University, Durham, North Carolina

Structural evidence suggests that antiactivator ExsD from Pseudomonas aeruginosa is a DNA binding protein

The opportunistic pathogen P. aeruginosa utilizes a type III secretion system (T3SS) to support acute infections in predisposed individuals. In this bacterium, expression of all T3SS‐related genes is dependent on the AraC‐type transcriptional activator ExsA. Before host contact, the T3SS is inactive and ExsA is repressed by the antiactivator protein ExsD. The repression, thought to occur through direct interactions between the two proteins, is relieved upon opening of the type III secretion (T3S) channel when secretion chaperone ExsC sequesters ExsD. We have solved the crystal structure of Δ20ExsD, a protease‐resistant fragment of ExsD that lacks only the 20 amino terminal residues of the wild‐type protein at 2.6 Å. Surprisingly the structure revealed similarities between ExsD and the DNA binding domain of transcriptional repressor KorB. A model of an ExsD‐DNA complex constructed on the basis of this homology produced a realistic complex that is supported by the prevalence of conserved residues in the putative DNA binding site and the results of differential scanning fluorimetry studies. Our findings challenge the currently held model that ExsD solely acts through interactions with ExsA and raise new questions with respect to the underlying mechanism of ExsA regulation.