Biophysical study of the DNA charge mimicry displayed by the T7 Ocr protein
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Date
2010Author
Stephanou, Augoustinos S.
Metadata
Abstract
The homodimeric Ocr protein of bacteriophage T7 is a molecular mimic of a
bent double-stranded DNA molecule ~24 bp in length. As such, Ocr is a highly
effective competitive inhibitor of the bacterial Type I restriction modification (R/M)
system. Thus, Ocr facilitates phage infection of the bacterial cell to proceed
unhindered by the action of the R/M defense system. The main aim of this work was
to understand the basis of the DNA mimicry displayed by Ocr. The surface of the
protein is replete with acidic residues, most or all of which mimic the phosphate
backbone of DNA. Aspartate and glutamate residues on the surface of Ocr were
either mutated or chemically modified in order to investigate their contribution to the
tight binding between Ocr and the EcoKI Type I R/M enzyme. Single or double
mutations of Ocr had no discernable effect on binding to EcoKI or its
methyltransferase component (M.EcoKI). Chemical modification was then used to
specifically modify the carboxyl moieties of Ocr, thereby neutralizing the negative
charges on the protein surface. Ocr samples modified to varying degrees were
analysed to establish the extent of derivatisation prior to extensive biophysical
characterisation to assess the impact of these changes in terms of binding to the
EcoKI R/M system. The results of this analysis revealed that the electrostatic
mimicry of Ocr increases the binding affinity for its target enzyme by at least
~800-fold. In addition, based on the known 3-D structure of the protein, a set of
multiple mutations were introduced into Ocr aimed at eliminating patches of negative
charge from the protein surface. Specifically, between 5 and 17 acidic residues were
targeted for mutation (Asp and Glu to Asn and Gln, respectively). Analysis of the in
vivo activity of the mutant Ocr along with biophysical characterisation of the purified
proteins was then performed. Results from these studies identified regions of the Ocr
protein that were critical in forming a tight association with the EcoKI R/M system.
Furthermore by comparing the relative contribution of different groups of acidic
residues to the free energy of binding, the actual mechanism by which Ocr mimics
the charge distribution of DNA has been delineated.