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. 2013 Oct;41(18):8760-75.
doi: 10.1093/nar/gkt633. Epub 2013 Jul 26.

Architecturally Diverse Proteins Converge on an Analogous Mechanism to Inactivate Uracil-DNA Glycosylase

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Free PMC article

Architecturally Diverse Proteins Converge on an Analogous Mechanism to Inactivate Uracil-DNA Glycosylase

Ambrose R Cole et al. Nucleic Acids Res. .
Free PMC article

Abstract

Uracil-DNA glycosylase (UDG) compromises the replication strategies of diverse viruses from unrelated lineages. Virally encoded proteins therefore exist to limit, inhibit or target UDG activity for proteolysis. Viral proteins targeting UDG, such as the bacteriophage proteins ugi, and p56, and the HIV-1 protein Vpr, share no sequence similarity, and are not structurally homologous. Such diversity has hindered identification of known or expected UDG-inhibitory activities in other genomes. The structural basis for UDG inhibition by ugi is well characterized; yet, paradoxically, the structure of the unbound p56 protein is enigmatically unrevealing of its mechanism. To resolve this conundrum, we determined the structure of a p56 dimer bound to UDG. A helix from one of the subunits of p56 occupies the UDG DNA-binding cleft, whereas the dimer interface forms a hydrophobic box to trap a mechanistically important UDG residue. Surprisingly, these p56 inhibitory elements are unexpectedly analogous to features used by ugi despite profound architectural disparity. Contacts from B-DNA to UDG are mimicked by residues of the p56 helix, echoing the role of ugi's inhibitory beta strand. Using mutagenesis, we propose that DNA mimicry by p56 is a targeting and specificity mechanism supporting tight inhibition via hydrophobic sequestration.

Figures

Figure 1.
Figure 1.
HHV-1, human and E. coli UDG are among the most completely characterized UDGs, including structural data of their association with the PBS1 encoded stoichiometric inhibitor, ugi. Bacillus subtilis UDG is the natural target of the inhibitors ugi and p56. Sequence identity between these UDGs is highlighted in cyan. Positions marked with a gold triangle show important points of contact between HHV-1 UDG and p56 observed in this study, which are also seen in contacts between distorted B-DNA with human UDG-DNA in structure 1SSP (Figure 3, and Supplementary Table S1). MUG (E. coli), TDG (human) and SMUG (Xenopus) are other enzymes from the wider UDG superfamily, but important residues in ugi and p56 inhibition are not conserved, these are indicated by aligning only these residues; those residues that are conserved between these enzymes and family 1 UDGs shown, are indicated by a circle above. The orange highlighted region is the UDG minor groove intercalation loop apical hydrophobic residue (phenylalanine in B. subtilis, leucine in other UDGs shown) sequestered by ugi and p56. The presence of an arginine at this position in SMUG and TDG suggests lack of susceptibility to ugi or p56 (see main text). Residue substitutions across the much more highly conserved family 1 UDGs can affect attributes such as turnover and dissociation kinetics, and therefore might also modulate interactions with inhibitor proteins.
Figure 2.
Figure 2.
HHV-1 UDG (molecule A, in main text; shown here in light blue) in complex with PZA p56 [PDB accession 4L5N]. The subunits of p56 are coloured to support the descriptions in the text: molecule C (shown here in yellow and brown) makes the most extensive interactions via its docked helix (the brown segment), which includes forming two sides of a hydrophobic pit that traps UDG leucine 214 (see main text for details), whereas molecule D, as well as forming the remaining two sides of the hydrophobic pit, makes just one other notable interaction with UDG (see main text).
Figure 3.
Figure 3.
Comparative orientations of UDG bound to p56, DNA, and ugi, with detailed views of the UDG-binding pocket containing both substrate and inhibitor elements on a UDG surface. UDG residues are shown with blue carbon atoms as is the surface, whereas substrate and inhibitor residues are shown with yellow carbon atoms. The second dimer of p56 is shown in green. (A) The p56 inhibitory helix bound to the HHV-1 UDG. Only the interacting side chains of the inhibitory helix and Glu26 of p56 are shown alongside UDG interacting residues. (B) Backbone of the uracil-containing strand of DNA bound to human UDG (PDB: 1SSP). (C) Residues of the ugi inhibitory strand bound to human UDG (PDB: 1UGH).
Figure 4.
Figure 4.
Comparison of UDG-DNA backbone phosphate interactions versus UDG with the inhibitory helix of p56. The UDG carbon atoms are shown in blue, whereas DNA and inhibitor carbon atoms are in yellow. Numbering of UDG residues in left hand side panels (A,C,E,G) refer to human UDG, whereas numbering in right hand side panels (B,D,F,H) refer to HHV-1 UDG; these are equivalent views of the two enzymes. (A/B) Area 1 base upstream of the catalysed uracil base compared with the equivalent region with the p56 inhibitor bound. (C/D) Region containing the phosphate of the catalysed uracil compared with that with p56 bound. (E/F) The phosphate 1 base downstream of the catalysed uracil compared with that with p56 bound. (G/H) The final phosphate with significant interactions and the equivalent region with p56 bound.
Figure 5.
Figure 5.
The ‘box-shaped’ hydrophobic pit of the p56 inhibitor dimer with UDG (mol A) leucine 214 inserted. The surface of p56, as well as important residues, is shown in yellow, whereas the UDG carbon atoms are shown in light blue. UDG (mol A) serines 212, and 215, interact with both charged and polar elements of p56 (mol C) Glu37 at the upper rim of the pit, creating a ‘lid’ (see main text).

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