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Dissection of the DNA mimicry of the bacteriophage T7 Ocr protein using chemical modification.

Stephanou AS, Roberts GA, Cooper LP, Clarke DJ, Thomson AR, MacKay CL, Nutley M, Cooper A, Dryden DT - J. Mol. Biol. (2009)

Bottom Line: Our analysis reveals that removal of about 46% of the carboxylate groups per Ocr monomer results in an approximately 50-fold reduction in binding affinity for a methyltransferase from a model type I restriction/modification system.The reduced affinity between Ocr with this degree of modification and the methyltransferase is comparable with the affinity of DNA for the methyltransferase.Our results show that the electrostatic mimicry of Ocr increases the binding affinity for its target enzyme by up to approximately 800-fold.

View Article: PubMed Central - PubMed

Affiliation: EastChem School of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ, UK.

ABSTRACT
The homodimeric Ocr (overcome classical restriction) protein of bacteriophage T7 is a molecular mimic of double-stranded DNA and a highly effective competitive inhibitor of the bacterial type I restriction/modification system. The surface of Ocr is replete with acidic residues that mimic the phosphate backbone of DNA. In addition, Ocr also mimics the overall dimensions of a bent 24-bp DNA molecule. In this study, we attempted to delineate these two mechanisms of DNA mimicry by chemically modifying the negative charges on the Ocr surface. Our analysis reveals that removal of about 46% of the carboxylate groups per Ocr monomer results in an approximately 50-fold reduction in binding affinity for a methyltransferase from a model type I restriction/modification system. The reduced affinity between Ocr with this degree of modification and the methyltransferase is comparable with the affinity of DNA for the methyltransferase. Additional modification to remove approximately 86% of the carboxylate groups further reduces its binding affinity, although the modified Ocr still binds to the methyltransferase via a mechanism attributable to the shape mimicry of a bent DNA molecule. Our results show that the electrostatic mimicry of Ocr increases the binding affinity for its target enzyme by up to approximately 800-fold.

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Derivatisation of carboxylate side chains of Ocr (Asp or Glu residues) using either dimethylamine (D-series) or ammonium hydroxide (N-series) as a nucleophile.
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fig2: Derivatisation of carboxylate side chains of Ocr (Asp or Glu residues) using either dimethylamine (D-series) or ammonium hydroxide (N-series) as a nucleophile.

Mentions: We used 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), a water-soluble carbodiimide, to specifically modify the carboxyl groups of Asp and Glu residues and the C-terminus of Ocr (Fig. 2). The physical and chemical properties of EDC have been extensively studied.17–20 In aqueous solution under acidic conditions, EDC is anticipated to react with Asp, Glu, Cys and Tyr residues. The absence of a Cys residue in Ocr rules out any unwanted side reactions with sulfhydryl groups. Regeneration of unsubstituted tyrosyl residues, the phenolic hydroxyl group of which reacts with EDC to form a relatively stable O-arylisourea, is achieved by treating the protein with hydroxylamine.21,22 EDC reacts with surface-exposed carboxyl groups to form highly reactive O-acylisourea intermediates (Fig. 3, reaction 1) that are susceptible to nucleophilic attack. The reactions were done in the presence of a large molar excess of dimethylamine (i.e., D-series) or ammonium hydroxide (i.e., N-series) as a nucleophile. The end result is the formation of a stable amide bond with concomitant loss of one negative charge for each residue modified (Fig. 3). The O-acylisourea intermediate is however unstable and can either react with water, regenerating the initial carboxylate and hydrolysing EDC to its urea derivative, or rearrange to an N-acylurea, thus forming a stable adduct on the protein. This undesirable N-acylurea rearrangement is largely avoided by carrying out the reaction in the presence of (i) a high concentration of nucleophile and (ii) N-hydroxybenzotriazole (HOBt). HOBt reacts with the O-acylisourea to form a more stable activated ester (Fig. 3, reaction 2), greatly increasing the overall coupling efficiency (Fig. 3, reaction 3) and preferentially mediating the reaction with an amine.23 This can however include the ɛ- and α-amino groups of Lys residues and the protein N-terminus, respectively, leading to intramolecular cross-linking and possibly protein polymerisation. Nevertheless, HOBt minimises the adventitious formation of ester-bond cross-links because the HOBt ester is less susceptible than the O-acylisourea to nucleophilic attack by OH groups (i.e., Tyr, Ser and Thr residues). An additional mechanism has been reported by Nakajima and Ikada, whereby the O-acylisourea intermediate may react with a neighbouring free carboxylate to form an acid anhydride.20 This labile anhydride, which is highly susceptible to hydrolysis, can react with an amine and a hydroxyl to form an amide bond and an ester bond, respectively.24 It is probable that the modification reaction of surface-exposed carboxyl groups on Ocr occurs through a combination of the aforementioned species (O-acylisourea, HOBt ester and acid anhydride). Considering the close proximity of side chains within a protein and the variability of their pKa values, depending on their specific microenvironment, it is clear that a limited number of side reactions are unavoidable.


Dissection of the DNA mimicry of the bacteriophage T7 Ocr protein using chemical modification.

Stephanou AS, Roberts GA, Cooper LP, Clarke DJ, Thomson AR, MacKay CL, Nutley M, Cooper A, Dryden DT - J. Mol. Biol. (2009)

Derivatisation of carboxylate side chains of Ocr (Asp or Glu residues) using either dimethylamine (D-series) or ammonium hydroxide (N-series) as a nucleophile.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2806950&req=5

fig2: Derivatisation of carboxylate side chains of Ocr (Asp or Glu residues) using either dimethylamine (D-series) or ammonium hydroxide (N-series) as a nucleophile.
Mentions: We used 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), a water-soluble carbodiimide, to specifically modify the carboxyl groups of Asp and Glu residues and the C-terminus of Ocr (Fig. 2). The physical and chemical properties of EDC have been extensively studied.17–20 In aqueous solution under acidic conditions, EDC is anticipated to react with Asp, Glu, Cys and Tyr residues. The absence of a Cys residue in Ocr rules out any unwanted side reactions with sulfhydryl groups. Regeneration of unsubstituted tyrosyl residues, the phenolic hydroxyl group of which reacts with EDC to form a relatively stable O-arylisourea, is achieved by treating the protein with hydroxylamine.21,22 EDC reacts with surface-exposed carboxyl groups to form highly reactive O-acylisourea intermediates (Fig. 3, reaction 1) that are susceptible to nucleophilic attack. The reactions were done in the presence of a large molar excess of dimethylamine (i.e., D-series) or ammonium hydroxide (i.e., N-series) as a nucleophile. The end result is the formation of a stable amide bond with concomitant loss of one negative charge for each residue modified (Fig. 3). The O-acylisourea intermediate is however unstable and can either react with water, regenerating the initial carboxylate and hydrolysing EDC to its urea derivative, or rearrange to an N-acylurea, thus forming a stable adduct on the protein. This undesirable N-acylurea rearrangement is largely avoided by carrying out the reaction in the presence of (i) a high concentration of nucleophile and (ii) N-hydroxybenzotriazole (HOBt). HOBt reacts with the O-acylisourea to form a more stable activated ester (Fig. 3, reaction 2), greatly increasing the overall coupling efficiency (Fig. 3, reaction 3) and preferentially mediating the reaction with an amine.23 This can however include the ɛ- and α-amino groups of Lys residues and the protein N-terminus, respectively, leading to intramolecular cross-linking and possibly protein polymerisation. Nevertheless, HOBt minimises the adventitious formation of ester-bond cross-links because the HOBt ester is less susceptible than the O-acylisourea to nucleophilic attack by OH groups (i.e., Tyr, Ser and Thr residues). An additional mechanism has been reported by Nakajima and Ikada, whereby the O-acylisourea intermediate may react with a neighbouring free carboxylate to form an acid anhydride.20 This labile anhydride, which is highly susceptible to hydrolysis, can react with an amine and a hydroxyl to form an amide bond and an ester bond, respectively.24 It is probable that the modification reaction of surface-exposed carboxyl groups on Ocr occurs through a combination of the aforementioned species (O-acylisourea, HOBt ester and acid anhydride). Considering the close proximity of side chains within a protein and the variability of their pKa values, depending on their specific microenvironment, it is clear that a limited number of side reactions are unavoidable.

Bottom Line: Our analysis reveals that removal of about 46% of the carboxylate groups per Ocr monomer results in an approximately 50-fold reduction in binding affinity for a methyltransferase from a model type I restriction/modification system.The reduced affinity between Ocr with this degree of modification and the methyltransferase is comparable with the affinity of DNA for the methyltransferase.Our results show that the electrostatic mimicry of Ocr increases the binding affinity for its target enzyme by up to approximately 800-fold.

View Article: PubMed Central - PubMed

Affiliation: EastChem School of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ, UK.

ABSTRACT
The homodimeric Ocr (overcome classical restriction) protein of bacteriophage T7 is a molecular mimic of double-stranded DNA and a highly effective competitive inhibitor of the bacterial type I restriction/modification system. The surface of Ocr is replete with acidic residues that mimic the phosphate backbone of DNA. In addition, Ocr also mimics the overall dimensions of a bent 24-bp DNA molecule. In this study, we attempted to delineate these two mechanisms of DNA mimicry by chemically modifying the negative charges on the Ocr surface. Our analysis reveals that removal of about 46% of the carboxylate groups per Ocr monomer results in an approximately 50-fold reduction in binding affinity for a methyltransferase from a model type I restriction/modification system. The reduced affinity between Ocr with this degree of modification and the methyltransferase is comparable with the affinity of DNA for the methyltransferase. Additional modification to remove approximately 86% of the carboxylate groups further reduces its binding affinity, although the modified Ocr still binds to the methyltransferase via a mechanism attributable to the shape mimicry of a bent DNA molecule. Our results show that the electrostatic mimicry of Ocr increases the binding affinity for its target enzyme by up to approximately 800-fold.

Show MeSH
Related in: MedlinePlus