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Structural basis of Lewis(b) antigen binding by the Helicobacter pylori adhesin BabA.

Hage N, Howard T, Phillips C, Brassington C, Overman R, Debreczeni J, Gellert P, Stolnik S, Winkler GS, Falcone FH - Sci Adv (2015)

Bottom Line: No conformational change occurs in BabA upon binding of Le(b), which is characterized by low affinity under acidic [K D (dissociation constant) of ~227 μM] and neutral (K D of ~252 μM) conditions.Binding is mediated by a network of hydrogen bonds between Le(b) Fuc1, GlcNAc3, Fuc4, and Gal5 residues and a total of eight BabA amino acids (C189, G191, N194, N206, D233, S234, S244, and T246) through both carbonyl backbone and side-chain interactions.The structural model was validated through the generation of two BabA variants containing N206A and combined D233A/S244A substitutions, which result in a reduction and complete loss of binding affinity to Le(b), respectively.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

ABSTRACT
Helicobacter pylori is a leading cause of peptic ulceration and gastric cancer worldwide. To achieve colonization of the stomach, this Gram-negative bacterium adheres to Lewis(b) (Le(b)) antigens in the gastric mucosa using its outer membrane protein BabA. Structural information for BabA has been elusive, and thus, its molecular mechanism for recognizing Le(b) antigens remains unknown. We present the crystal structure of the extracellular domain of BabA, from H. pylori strain J99, in the absence and presence of Le(b) at 2.0- and 2.1-Å resolutions, respectively. BabA is a predominantly α-helical molecule with a markedly kinked tertiary structure containing a single, shallow Le(b) binding site at its tip within a β-strand motif. No conformational change occurs in BabA upon binding of Le(b), which is characterized by low affinity under acidic [K D (dissociation constant) of ~227 μM] and neutral (K D of ~252 μM) conditions. Binding is mediated by a network of hydrogen bonds between Le(b) Fuc1, GlcNAc3, Fuc4, and Gal5 residues and a total of eight BabA amino acids (C189, G191, N194, N206, D233, S234, S244, and T246) through both carbonyl backbone and side-chain interactions. The structural model was validated through the generation of two BabA variants containing N206A and combined D233A/S244A substitutions, which result in a reduction and complete loss of binding affinity to Le(b), respectively. Knowledge of the molecular basis of Le(b) recognition by BabA provides a platform for the development of therapeutics targeted at inhibiting H. pylori adherence to the gastric mucosa.

No MeSH data available.


Related in: MedlinePlus

The Leb binding site of BabA.(A) Leb binds to BabA at one edge of the crown. The electron density map around Leb (2Fo − Fc map, contoured at 2.0σ) in complex with BabA is shown. (B) An electrostatic surface representation shows Leb binding to a shallow, solvent-exposed pocket at the tip of BabA containing charged and neutral patches. Fucose, galactose, and N-acetylglucosamine residues are colored orange, yellow, and blue, respectively.
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Figure 2: The Leb binding site of BabA.(A) Leb binds to BabA at one edge of the crown. The electron density map around Leb (2Fo − Fc map, contoured at 2.0σ) in complex with BabA is shown. (B) An electrostatic surface representation shows Leb binding to a shallow, solvent-exposed pocket at the tip of BabA containing charged and neutral patches. Fucose, galactose, and N-acetylglucosamine residues are colored orange, yellow, and blue, respectively.

Mentions: To obtain structural insight into Leb binding, we solved the crystal structure of BabA in complex with a hexasaccharide form of the Leb antigen to 2.1-Å resolution (table S1). This oligosaccharide contains two fucose residues (Fuc1 and Fuc4), two galactose residues (Gal2 and Gal5), an N-acetylglucosamine residue (GlcNAc3), and a glucose residue (Glc6). Structure determination revealed a single, shallow binding site at the tip of the BabA crown (Fig. 2). All Leb residues were visible in the electron density map except the terminal Glc6 residue, which is consequently not modeled (Fig. 2A). A comparison of the BabA crystal structure in the absence or presence of Leb indicated that no conformational change occurred upon sugar binding [fig. S4; RMSD = 0.25Å for all Cα atoms (22)].


Structural basis of Lewis(b) antigen binding by the Helicobacter pylori adhesin BabA.

Hage N, Howard T, Phillips C, Brassington C, Overman R, Debreczeni J, Gellert P, Stolnik S, Winkler GS, Falcone FH - Sci Adv (2015)

The Leb binding site of BabA.(A) Leb binds to BabA at one edge of the crown. The electron density map around Leb (2Fo − Fc map, contoured at 2.0σ) in complex with BabA is shown. (B) An electrostatic surface representation shows Leb binding to a shallow, solvent-exposed pocket at the tip of BabA containing charged and neutral patches. Fucose, galactose, and N-acetylglucosamine residues are colored orange, yellow, and blue, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643811&req=5

Figure 2: The Leb binding site of BabA.(A) Leb binds to BabA at one edge of the crown. The electron density map around Leb (2Fo − Fc map, contoured at 2.0σ) in complex with BabA is shown. (B) An electrostatic surface representation shows Leb binding to a shallow, solvent-exposed pocket at the tip of BabA containing charged and neutral patches. Fucose, galactose, and N-acetylglucosamine residues are colored orange, yellow, and blue, respectively.
Mentions: To obtain structural insight into Leb binding, we solved the crystal structure of BabA in complex with a hexasaccharide form of the Leb antigen to 2.1-Å resolution (table S1). This oligosaccharide contains two fucose residues (Fuc1 and Fuc4), two galactose residues (Gal2 and Gal5), an N-acetylglucosamine residue (GlcNAc3), and a glucose residue (Glc6). Structure determination revealed a single, shallow binding site at the tip of the BabA crown (Fig. 2). All Leb residues were visible in the electron density map except the terminal Glc6 residue, which is consequently not modeled (Fig. 2A). A comparison of the BabA crystal structure in the absence or presence of Leb indicated that no conformational change occurred upon sugar binding [fig. S4; RMSD = 0.25Å for all Cα atoms (22)].

Bottom Line: No conformational change occurs in BabA upon binding of Le(b), which is characterized by low affinity under acidic [K D (dissociation constant) of ~227 μM] and neutral (K D of ~252 μM) conditions.Binding is mediated by a network of hydrogen bonds between Le(b) Fuc1, GlcNAc3, Fuc4, and Gal5 residues and a total of eight BabA amino acids (C189, G191, N194, N206, D233, S234, S244, and T246) through both carbonyl backbone and side-chain interactions.The structural model was validated through the generation of two BabA variants containing N206A and combined D233A/S244A substitutions, which result in a reduction and complete loss of binding affinity to Le(b), respectively.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

ABSTRACT
Helicobacter pylori is a leading cause of peptic ulceration and gastric cancer worldwide. To achieve colonization of the stomach, this Gram-negative bacterium adheres to Lewis(b) (Le(b)) antigens in the gastric mucosa using its outer membrane protein BabA. Structural information for BabA has been elusive, and thus, its molecular mechanism for recognizing Le(b) antigens remains unknown. We present the crystal structure of the extracellular domain of BabA, from H. pylori strain J99, in the absence and presence of Le(b) at 2.0- and 2.1-Å resolutions, respectively. BabA is a predominantly α-helical molecule with a markedly kinked tertiary structure containing a single, shallow Le(b) binding site at its tip within a β-strand motif. No conformational change occurs in BabA upon binding of Le(b), which is characterized by low affinity under acidic [K D (dissociation constant) of ~227 μM] and neutral (K D of ~252 μM) conditions. Binding is mediated by a network of hydrogen bonds between Le(b) Fuc1, GlcNAc3, Fuc4, and Gal5 residues and a total of eight BabA amino acids (C189, G191, N194, N206, D233, S234, S244, and T246) through both carbonyl backbone and side-chain interactions. The structural model was validated through the generation of two BabA variants containing N206A and combined D233A/S244A substitutions, which result in a reduction and complete loss of binding affinity to Le(b), respectively. Knowledge of the molecular basis of Le(b) recognition by BabA provides a platform for the development of therapeutics targeted at inhibiting H. pylori adherence to the gastric mucosa.

No MeSH data available.


Related in: MedlinePlus