The three-dimensional structure of the extracellular adhesion domain of the sialic acid-binding adhesin SabA from Helicobacter pylori

Abstract

The gastric pathogen Helicobacter pylori is a major cause of acute chronic gastritis and the development of stomach and duodenal ulcers. Chronic infection furthermore predisposes to the development of gastric cancer. Crucial to H. pylori survival within the hostile environment of the digestive system are the adhesins SabA and BabA; these molecules belong to the same protein family and permit the bacteria to bind tightly to sugar moieties Lewis(B) and sialyl-Lewis(X), respectively, on the surface of epithelial cells lining the stomach and duodenum. To date, no representative SabA/BabA structure has been determined, hampering the development of strategies to eliminate persistent H. pylori infections that fail to respond to conventional therapy. Here, using x-ray crystallography, we show that the soluble extracellular adhesin domain of SabA shares distant similarity to the tetratricopeptide repeat fold family. The molecule broadly resembles a golf putter in shape, with the head region featuring a large cavity surrounded by loops that vary in sequence between different H. pylori strains. The N-terminal and C-terminal helices protrude at right angles from the head domain and together form a shaft that connects to a predicted outer membrane protein-like β-barrel trans-membrane domain. Using surface plasmon resonance, we were able to detect binding of the SabA adhesin domain to sialyl-Lewis(X) and Lewis(X) but not to Lewis(A), Lewis(B), or Lewis(Y). Substitution of the highly conserved glutamine residue 159 in the predicted ligand-binding pocket abrogates the binding of the SabA adhesin domain to sialyl-Lewis(X) and Lewis(X). Taken together, these data suggest that the adhesin domain of SabA is sufficient in isolation for specific ligand binding.

Keywords: Bacterial Adhesion; Carbohydrate Binding Protein; Helicobacter pylori; Membrane Proteins; Outer Membrane Protein; X-ray Crystallography.

FIGURE 1.

Domain organisation of SabA. A, schematic illustrating the predicted domain structure of SabA. B, crystal structure of SabA N-terminal adhesion region. The handle is in blue, the tetratricopeptide repeat region is colored in magenta and the remainder of the molecule is in orange. Dashed lines indicate disordered regions. The two conserved disulfide bonds are represented as cyan sticks. The short loop interruption in Helix-1 is marked with an asterisk. The C-terminal helix is connected to a trans-membrane β-barrel domain, which anchors the SabA adhesion region to the outer membrane of H. pylori.

FIGURE 2.

Structural superposition of SabA (magenta and gray) and pectin methylesterase inhibitor (green) around the tetratricopeptide repeat portion as calculated by DALI.

FIGURE 3.

Primary sequence alignment of SabA and BabA N-terminal adhesion regions. The alignment was generated with Clustal Omega using 33 SabA and 41 BabA sequences from various H. pylori strains (KEGG database). The figure shows a representative of the complete alignment (five sequences from each protein). Residues in yellow are identical in all SabA and BabA, red are identical surface residues in the predicted binding pocket, cyan are cysteine residues involved in disulfide bonds in SabA. The position of the alternative conserved cysteine residue in BabA is marked (#). Ser-80 and Pro-81 (@) map to the interruption in Helix-1, and Gly-357 (§) causes a slight kink in Helix-4. Tyr-148, Lys-152, Gln-159, and Gln-162 were mutated to alanine in this study are marked with an asterisk.

FIGURE 4.

Ligand-binding pockets calculated by an automatic ligand-binding-site prediction program (POCASA). A and B, the binding pockets (dark gray surface) are predicted mostly toward to the tip of the SabA head domain. C and D, view from the top toward the predicted ligand binding pockets (removed in D for clarity). A number of highly conserved residues (cyan sticks), in particular Ser-80, Pro-81, Trp-97, Tyr-148, Lys-152, Gln-159, and Gln-162, line the surface of these pockets. Tyr-148, Lys-152, Gln-159, and Gln-162 were mutated to alanine in this study are marked with an asterisk. A glycerol molecule (pink sticks) is observed in the crystal structure and forms hydrogen bond interactions with Lys-152 and Gln-159. E, electrostatic potential surface representation of the SabA head domain showing the predicted pockets (gray dots) generated using CCP4 mg (30). A glycerol molecule (pink sticks) binds in a positively charged cavity.