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1.  Structural Analysis of the Oligomeric States of Helicobacter pylori VacA Toxin 
Journal of molecular biology  2012;425(3):524-535.
Helicobacter pylori is a Gram-negative bacterium that colonizes the human stomach and contributes to peptic ulceration and gastric adenocarcinoma. H. pylori secretes a pore-forming exotoxin known as vacuolating toxin (VacA). VacA contains two distinct domains, designated p33 and p55, and assembles into large “snowflake”-shaped oligomers. Thus far, no structural data are available for the p33 domain, which is essential for membrane channel formation. Using single-particle electron microscopy and the random conical tilt approach, we have determined the three-dimensional structures of six VacA oligomeric conformations at ~15-Å resolution. The p55 domain, composed primarily of β-helical structures, localizes to the peripheral arms, while the p33 domain consists of two globular densities that localize within the center of the complexes. By fitting the VacA p55 crystal structure into the electron microscopy densities, we have mapped inter-VacA interactions that support oligomerization. In addition, we have examined VacA variants/mutants that differ from wild-type (WT) VacA in toxin activity and/or oligomeric structural features. Oligomers formed by VacAΔ6–27, a mutant that fails to form membrane channels, lack an organized p33 central core. Mixed oligomers containing both WT and VacAΔ6–27 subunits also lack an organized core. Oligomers formed by a VacA s2m1 chimera (which lacks cell-vacuolating activity) and VacAΔ301–328 (which retains vacuolating activity) each contain p33 central cores similar to those of WT oligomers. By providing the most detailed view of the VacA structure to date, these data offer new insights into the toxin's channel-forming component and the intermolecular interactions that underlie oligomeric assembly.
doi:10.1016/j.jmb.2012.11.020
PMCID: PMC3612943  PMID: 23178866
pore-forming toxins; single-particle electron microscopy; oligomerization; pathogenesis
3.  Purification, crystallization and preliminary X-ray diffraction analysis of the carbohydrate-binding region of the Streptococcus gordonii adhesin GspB 
The carbohydrate-binding region of GspB from S. gordonii strain M99 was crystallized in space group P212121 and data were collected to 1.3 Å resolution.
The carbohydrate-binding region of the bacterial adhesin GspB from Strepto­coccus gordonii strain M99 (GspBBR) was expressed in Escherichia coli and purified using affinity and size-exclusion chromatography. Separate sparse-matrix screening of GspBBR buffered in either 20 mM Tris pH 7.4 or 20 mM HEPES pH 7.5 resulted in different crystallographic behavior such that different precipitants, salts and additives supported crystallization of GspBBR in each buffer. While both sets of conditions supported crystal growth in space group P212121, the crystals had distinct unit-cell parameters of a = 33.3, b = 86.7, c = 117.9 Å for crystal form 1 and a = 34.6, b = 98.3, c = 99.0 Å for crystal form 2. Additive screening improved the crystals grown in both conditions such that diffraction extended to beyond 2 Å resolution. A complete data set has been collected to 1.3 Å resolution with an overall R merge value of 0.04 and an R merge value of 0.33 in the highest resolution shell.
doi:10.1107/S1744309110036535
PMCID: PMC3001660  PMID: 21045307
GspB; glycoproteins; Streptococcus gordonii; sialic acid; adhesins; endocarditis; lectins
4.  Purification, crystallization, and preliminary x-ray diffraction analysis of the carbohydrate-binding region from the Streptococcus gordonii adhesin GspB 
The carbohydrate binding region of the bacterial adhesin GspB from Streptococcus gordonii strain M99 (GspBBR) was expressed in Escherichia coli and purified using affinity and size exclusion chromatography. Separate sparse-matrix screening of GspBBR buffered in either 20 mM Tris pH 7.4 or 20 mM HEPES pH 7.5 resulted in different crystallographic behavior such that different precipitants, salts, and additives supported crystallization of GspBBR in each buffer. While both sets of conditions supported crystal growth in space group P212121, these had distinct unit cell dimensions of a=33.3 Å, b=86.6 Å, c=117.9 Å for crystal form one and a=34.6 Å, b=98.3 Å, c=99.0 Å for crystal form two. Additive screening improved the crystals grown in both conditions such that diffraction extended beyond 2 Å resolution. A complete data set has been collected to 1.3 Å resolution with an overall Rsym value of 0.04 and an Rsym value of 0.33 in the highest resolution shell.
doi:10.1107/S1744309110036535
PMCID: PMC3001660  PMID: 21045307
GspB; glycoprotein; Streptococcus gordonii; sialic acid; adhesin; endocarditis; lectin
5.  A Structural Model for Binding of the Serine-Rich Repeat Adhesin GspB to Host Carbohydrate Receptors 
PLoS Pathogens  2011;7(7):e1002112.
GspB is a serine-rich repeat (SRR) adhesin of Streptococcus gordonii that mediates binding of this organism to human platelets via its interaction with sialyl-T antigen on the receptor GPIbα. This interaction appears to be a major virulence determinant in the pathogenesis of infective endocarditis. To address the mechanism by which GspB recognizes its carbohydrate ligand, we determined the high-resolution x-ray crystal structure of the GspB binding region (GspBBR), both alone and in complex with a disaccharide precursor to sialyl-T antigen. Analysis of the GspBBR structure revealed that it is comprised of three independently folded subdomains or modules: 1) an Ig-fold resembling a CnaA domain from prokaryotic pathogens; 2) a second Ig-fold resembling the binding region of mammalian Siglecs; 3) a subdomain of unique fold. The disaccharide was found to bind in a pocket within the Siglec subdomain, but at a site distinct from that observed in mammalian Siglecs. Confirming the biological relevance of this binding pocket, we produced three isogenic variants of S. gordonii, each containing a single point mutation of a residue lining this binding pocket. These variants have reduced binding to carbohydrates of GPIbα. Further examination of purified GspBBR-R484E showed reduced binding to sialyl-T antigen while S. gordonii harboring this mutation did not efficiently bind platelets and showed a significant reduction in virulence, as measured by an animal model of endocarditis. Analysis of other SRR proteins revealed that the predicted binding regions of these adhesins also had a modular organization, with those known to bind carbohydrate receptors having modules homologous to the Siglec and Unique subdomains of GspBBR. This suggests that the binding specificity of the SRR family of adhesins is determined by the type and organization of discrete modules within the binding domains, which may affect the tropism of organisms for different tissues.
Author Summary
The binding of bacteria to human platelets is thought to be important for development of infective endocarditis, a life-threatening infection of the cardiovascular system. Streptococcus gordonii is a leading cause of endocarditis. This pathogen uses a protein called GspB to attach to carbohydrates on human platelets. While this binding interaction appears to be mediated by a specific, contiguous domain within GspB, little is known about the molecular details of the interaction between GspB and the carbohydrate receptors on its human host. We therefore determined the crystal structure of the region of GspB that binds to platelet carbohydrates, both alone and in complex with a synthetic carbohydrate receptor. Using this structure as a guide, we were able to produce three strains of S. gordonii that lacked the ability to bind to platelet carbohydrates. One of these isogenic variants was studied more in-depth and lacked the ability to bind to human platelets in vitro and was reduced in virulence when tested in vivo. These studies provide the first structural information detailing the molecular interactions between any serine-rich repeat adhesin and its host receptor, and identify how different, related adhesins may have evolved different specificities for host receptors.
doi:10.1371/journal.ppat.1002112
PMCID: PMC3131266  PMID: 21765814

Results 1-5 (5)