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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.

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Binding affinity of wild-type and variant BabA proteins to Leb.(A and B) Calorimetric response (top) and binding isotherm (bottom) of (A) wild-type BabA titrated with Leb and (B) the BabA N206A variant titrated with Leb. The continuous line in both lower panels represents the least-squares fit of the data to a single-site binding model. The reported thermodynamic parameters are the average (±SEM) of three independent experiments. (C) No calorimetric response (top) or binding isotherm (bottom) was obtained by titrating BabA containing D233A/S244A substitutions with Leb. All calorimetric titrations were performed at pH 7.4.
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Figure 5: Binding affinity of wild-type and variant BabA proteins to Leb.(A and B) Calorimetric response (top) and binding isotherm (bottom) of (A) wild-type BabA titrated with Leb and (B) the BabA N206A variant titrated with Leb. The continuous line in both lower panels represents the least-squares fit of the data to a single-site binding model. The reported thermodynamic parameters are the average (±SEM) of three independent experiments. (C) No calorimetric response (top) or binding isotherm (bottom) was obtained by titrating BabA containing D233A/S244A substitutions with Leb. All calorimetric titrations were performed at pH 7.4.

Mentions: To further characterize the molecular interactions between BabA and Leb, we used isothermal titration calorimetry (ITC) to study binding at acidic and neutral pH (Fig. 5A and table S2). In agreement with the proposed model, binding was found to be a single-site interaction (N of ~0.91 and ~1.07 at pH 4.5 and 7.4, respectively). The thermodynamic parameters of the interaction indicate that binding is driven by noncovalent, that is, enthalpic contributions (ΔH of ~−12.2 and ~−10.9 kcal/mol at pH 4.5 and 7.4, respectively) rather than hydrophobic, that is, entropic contributions (−TΔS of ~7.2 and ~6.0 kcal/mol at pH 4.5 and 7.4, respectively). Binding between BabA and Leb molecules was observed to be a low-affinity interaction [KD (dissociation constant) of ~227 and ~252 μM at pH 4.5 and 7.4, respectively]. There are no significant differences (P > 0.05) between the thermodynamic parameters and dissociation constants of BabA:Leb binding at acidic and neutral pH.


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)

Binding affinity of wild-type and variant BabA proteins to Leb.(A and B) Calorimetric response (top) and binding isotherm (bottom) of (A) wild-type BabA titrated with Leb and (B) the BabA N206A variant titrated with Leb. The continuous line in both lower panels represents the least-squares fit of the data to a single-site binding model. The reported thermodynamic parameters are the average (±SEM) of three independent experiments. (C) No calorimetric response (top) or binding isotherm (bottom) was obtained by titrating BabA containing D233A/S244A substitutions with Leb. All calorimetric titrations were performed at pH 7.4.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Binding affinity of wild-type and variant BabA proteins to Leb.(A and B) Calorimetric response (top) and binding isotherm (bottom) of (A) wild-type BabA titrated with Leb and (B) the BabA N206A variant titrated with Leb. The continuous line in both lower panels represents the least-squares fit of the data to a single-site binding model. The reported thermodynamic parameters are the average (±SEM) of three independent experiments. (C) No calorimetric response (top) or binding isotherm (bottom) was obtained by titrating BabA containing D233A/S244A substitutions with Leb. All calorimetric titrations were performed at pH 7.4.
Mentions: To further characterize the molecular interactions between BabA and Leb, we used isothermal titration calorimetry (ITC) to study binding at acidic and neutral pH (Fig. 5A and table S2). In agreement with the proposed model, binding was found to be a single-site interaction (N of ~0.91 and ~1.07 at pH 4.5 and 7.4, respectively). The thermodynamic parameters of the interaction indicate that binding is driven by noncovalent, that is, enthalpic contributions (ΔH of ~−12.2 and ~−10.9 kcal/mol at pH 4.5 and 7.4, respectively) rather than hydrophobic, that is, entropic contributions (−TΔS of ~7.2 and ~6.0 kcal/mol at pH 4.5 and 7.4, respectively). Binding between BabA and Leb molecules was observed to be a low-affinity interaction [KD (dissociation constant) of ~227 and ~252 μM at pH 4.5 and 7.4, respectively]. There are no significant differences (P > 0.05) between the thermodynamic parameters and dissociation constants of BabA:Leb binding at acidic and neutral pH.

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