Acharya, Narottam and Roy, Sudipta and Varshney, Umesh (2002) Mutational Analysis of the Uracil DNA Glycosylase Inhibitor Protein and Its Interaction with Escherichia coli Uracil DNA Glycosylase. In: Journal of Molecular Biology, 321 (4). pp. 579-590.
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Uracil DNA glycosylase inhibitor (Ugi), a protein of 9.4 kDa consists of a five-stranded antiparallel $\beta$ sheet flanked on either side by single $\alpha$ helices, forms an exclusive complex with uracil DNA glycosylases (UDGs) that is stable in 8 M urea. We report on the mutational analysis of various structural elements in Ugi, two of which (hydrophobic pocket and the $\beta 1$ edge)establish key interactions with Escherichia coli UDG. The point mutations in helix $\alpha 1$ (amino acid residues 3–14) do not affect the stability of the UDG–Ugi complexes in urea. And, while the complex of the $\Delta N13$ mutant with UDG is stable in only $\sim 4$ M urea, its overall structure and thermostability are maintained. The identity of P37, stacked between P26 and W68, was not important for the maintenance of the hydrophobic pocket or for the stability of the complex. However, the M24K mutation at the rim of the hydrophobic pocket lowered the stability of the complex in 6 M urea. On the other hand, non-conservative mutations E49G, D61G (cancels the only ionic interaction with UDG) and N76K, in three of the loops connecting the $\beta$ strands, conferred no such phenotype. The L23R and S21P mutations ($\beta 1$ edge) at the UDG–Ugi interface, and the N35D mutation far from the interface resulted in poor stability of the complex. However, the stability of the complexes was restored in the L23A, S21T and N35A mutations. These analyses and the studies on the exchange of Ugi mutants in preformed complexes with the substrate or the native Ugi have provided insights into the two-step mechanism of UDG–Ugi complex formation. Finally, we discuss the application of the Ugi isolates in overproduction of UDG mutants, toxic to cells.
|Item Type:||Journal Article|
|Additional Information:||Copyright of this article belongs to Elsevier Science.|
|Keywords:||UDG;Ugi;Ung;Bacteriophage PBS-1 and -2;Protein mimicry of DNA|
|Department/Centre:||Division of Biological Sciences > Microbiology & Cell Biology|
|Date Deposited:||15 Oct 2007|
|Last Modified:||23 Feb 2012 10:26|
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