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Architecturally diverse proteins converge on an analogous mechanism to inactivate Uracil-DNA glycosylase.

Cole AR, Ofer S, Ryzhenkova K, Baltulionis G, Hornyak P, Savva R - Nucleic Acids Res. (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck College, University of London, Malet Street, London WC1E 7HX, UK and Research Department of Structural and Molecular Biology, Institute of Structural and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK.

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.

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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).
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gkt633-F3: 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).

Mentions: 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.


Architecturally diverse proteins converge on an analogous mechanism to inactivate Uracil-DNA glycosylase.

Cole AR, Ofer S, Ryzhenkova K, Baltulionis G, Hornyak P, Savva R - Nucleic Acids Res. (2013)

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).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3794593&req=5

gkt633-F3: 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).
Mentions: 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.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck College, University of London, Malet Street, London WC1E 7HX, UK and Research Department of Structural and Molecular Biology, Institute of Structural and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK.

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.

Show MeSH
Related in: MedlinePlus