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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

Conservation of the Leb binding site of BabA.(A) Sequence alignment of the crown (residues 183 to 253) from BabA J99 with 21 H. pylori strains shown to bind Leb glycoconjugates. Amino acids involved in hydrogen bond formation to each Leb residue are indicated. (B) The crown β-strand unit is well conserved (gold) across the analyzed strains with the exception of the hypervariable crown loop (residues 198 to 207; gray). Fucose, galactose, and N-acetylglucosamine residues are colored orange, yellow, and blue, respectively. Hydrogen bonds are represented by dotted black lines.
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Figure 4: Conservation of the Leb binding site of BabA.(A) Sequence alignment of the crown (residues 183 to 253) from BabA J99 with 21 H. pylori strains shown to bind Leb glycoconjugates. Amino acids involved in hydrogen bond formation to each Leb residue are indicated. (B) The crown β-strand unit is well conserved (gold) across the analyzed strains with the exception of the hypervariable crown loop (residues 198 to 207; gray). Fucose, galactose, and N-acetylglucosamine residues are colored orange, yellow, and blue, respectively. Hydrogen bonds are represented by dotted black lines.

Mentions: Alignment of BabA J99, used in this study, with BabA from 21 H. pylori strains reported to bind Leb glycoconjugates (23–25) revealed that the amino acids identified in mediating Leb binding in our structural model are highly conserved with the exception of N206 (Fig. 4A). N206 is found within a region (residues 198 to 207 in BabA J99) with low amino acid conservation across the Leb binding strains. We refer to this segment, which connects two antiparallel β-strands in the BabA J99 crown, as the hypervariable crown loop (Fig. 4B).


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)

Conservation of the Leb binding site of BabA.(A) Sequence alignment of the crown (residues 183 to 253) from BabA J99 with 21 H. pylori strains shown to bind Leb glycoconjugates. Amino acids involved in hydrogen bond formation to each Leb residue are indicated. (B) The crown β-strand unit is well conserved (gold) across the analyzed strains with the exception of the hypervariable crown loop (residues 198 to 207; gray). Fucose, galactose, and N-acetylglucosamine residues are colored orange, yellow, and blue, respectively. Hydrogen bonds are represented by dotted black lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Conservation of the Leb binding site of BabA.(A) Sequence alignment of the crown (residues 183 to 253) from BabA J99 with 21 H. pylori strains shown to bind Leb glycoconjugates. Amino acids involved in hydrogen bond formation to each Leb residue are indicated. (B) The crown β-strand unit is well conserved (gold) across the analyzed strains with the exception of the hypervariable crown loop (residues 198 to 207; gray). Fucose, galactose, and N-acetylglucosamine residues are colored orange, yellow, and blue, respectively. Hydrogen bonds are represented by dotted black lines.
Mentions: Alignment of BabA J99, used in this study, with BabA from 21 H. pylori strains reported to bind Leb glycoconjugates (23–25) revealed that the amino acids identified in mediating Leb binding in our structural model are highly conserved with the exception of N206 (Fig. 4A). N206 is found within a region (residues 198 to 207 in BabA J99) with low amino acid conservation across the Leb binding strains. We refer to this segment, which connects two antiparallel β-strands in the BabA J99 crown, as the hypervariable crown loop (Fig. 4B).

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