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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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Plot of enthalpy, ΔH (kJ/mol), versus temperature (°C) for the interaction of either unmodified Ocr or chemically modified Ocr with M.EcoKI. The ΔH of interaction was determined from ITC experiments at temperatures ranging from 10 to 30 °C. “A” and “B” show unmodified Ocr in the absence and in the presence of 500 mM NaCl, respectively. “C” and “D” show the analysis of the interaction between M.EcoKI and the N60 and the D15 chemically modified Ocr samples in the absence of NaCl, respectively.
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fig9: Plot of enthalpy, ΔH (kJ/mol), versus temperature (°C) for the interaction of either unmodified Ocr or chemically modified Ocr with M.EcoKI. The ΔH of interaction was determined from ITC experiments at temperatures ranging from 10 to 30 °C. “A” and “B” show unmodified Ocr in the absence and in the presence of 500 mM NaCl, respectively. “C” and “D” show the analysis of the interaction between M.EcoKI and the N60 and the D15 chemically modified Ocr samples in the absence of NaCl, respectively.

Mentions: The ITC experiment was initially performed using unmodified Ocr in 20 mM Tris–HCl, pH 8.0, 6 mM MgCl2, 7 mM 2-mercaptoethanol and 100 μM S-adenosyl-l-methionine (SAM) with or without 500 mM NaCl at a range of temperatures from 10 to 30 °C. The experiment at 25 °C was repeated using 20 mM Hepes buffer (heat of ionization = 20.5 kJ/mol) in place of Tris–HCl (heat of ionization = 47.4 kJ/mol) in the absence of NaCl, which gave a very similar enthalpy of interaction (ΔH of − 90.0 kJ/mol in Hepes compared with − 86.2 kJ/mol in Tris). These experiments indicated that there was no major contribution to ΔH due to effects of buffer ionization arising from protonation changes during binding. The enthalpy change upon interaction was strongly exothermic in the absence of NaCl but showed significant temperature dependence characteristic of a heat capacity change, ΔCp, upon formation of the M.EcoKI–Ocr complex. This was quantified from the slope of the plot of enthalpy change versus temperature (assumed to be linear) using the standard thermodynamic relationship:ΔCp=dΔH/dTWe determined the ΔCp of unmodified Ocr in low salt buffer to be − 4221 ± 372 J/mol K (Fig. 9). In 500 mM NaCl, the transition was less sharp (i.e., sigmoidal and indicating a slightly weaker interaction) and endothermic at all temperatures, and the slope of the linear regression yielded a binding heat capacity of − 2071 ± 372 J/mol K.


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)

Plot of enthalpy, ΔH (kJ/mol), versus temperature (°C) for the interaction of either unmodified Ocr or chemically modified Ocr with M.EcoKI. The ΔH of interaction was determined from ITC experiments at temperatures ranging from 10 to 30 °C. “A” and “B” show unmodified Ocr in the absence and in the presence of 500 mM NaCl, respectively. “C” and “D” show the analysis of the interaction between M.EcoKI and the N60 and the D15 chemically modified Ocr samples in the absence of NaCl, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: Plot of enthalpy, ΔH (kJ/mol), versus temperature (°C) for the interaction of either unmodified Ocr or chemically modified Ocr with M.EcoKI. The ΔH of interaction was determined from ITC experiments at temperatures ranging from 10 to 30 °C. “A” and “B” show unmodified Ocr in the absence and in the presence of 500 mM NaCl, respectively. “C” and “D” show the analysis of the interaction between M.EcoKI and the N60 and the D15 chemically modified Ocr samples in the absence of NaCl, respectively.
Mentions: The ITC experiment was initially performed using unmodified Ocr in 20 mM Tris–HCl, pH 8.0, 6 mM MgCl2, 7 mM 2-mercaptoethanol and 100 μM S-adenosyl-l-methionine (SAM) with or without 500 mM NaCl at a range of temperatures from 10 to 30 °C. The experiment at 25 °C was repeated using 20 mM Hepes buffer (heat of ionization = 20.5 kJ/mol) in place of Tris–HCl (heat of ionization = 47.4 kJ/mol) in the absence of NaCl, which gave a very similar enthalpy of interaction (ΔH of − 90.0 kJ/mol in Hepes compared with − 86.2 kJ/mol in Tris). These experiments indicated that there was no major contribution to ΔH due to effects of buffer ionization arising from protonation changes during binding. The enthalpy change upon interaction was strongly exothermic in the absence of NaCl but showed significant temperature dependence characteristic of a heat capacity change, ΔCp, upon formation of the M.EcoKI–Ocr complex. This was quantified from the slope of the plot of enthalpy change versus temperature (assumed to be linear) using the standard thermodynamic relationship:ΔCp=dΔH/dTWe determined the ΔCp of unmodified Ocr in low salt buffer to be − 4221 ± 372 J/mol K (Fig. 9). In 500 mM NaCl, the transition was less sharp (i.e., sigmoidal and indicating a slightly weaker interaction) and endothermic at all temperatures, and the slope of the linear regression yielded a binding heat capacity of − 2071 ± 372 J/mol K.

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