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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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Related in: MedlinePlus

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).
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gkt633-F2: 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).

Mentions: Further rounds of refinement used the BUSTER suite of refinement tools (39), and building used the program COOT (35,40). Refinement of the structure yielded high-quality maps for the majority of observed molecules, with the exception of molecule B. Residues 17–60 of molecule B are located adjacent to a large solvent channel in the crystal and poorly ordered in the structure. Omission of the region resulted in clear positive density in fo-fc difference maps but the 2fo-fc maps with the region replaced were consistently poor, especially for side chains. Refinement improved with NCS dropped from this region and TLS refinement introduced with separate operators for the region as well as for each separate chain. Some side chains were still not visible, and these were cropped back to Cβ atoms. The final refinement statistics are shown in Table 1, the overall structure is shown in Figure 2, and the structure is deposited in the PDB with the accession number 4L5N.


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)

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

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

gkt633-F2: 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).
Mentions: Further rounds of refinement used the BUSTER suite of refinement tools (39), and building used the program COOT (35,40). Refinement of the structure yielded high-quality maps for the majority of observed molecules, with the exception of molecule B. Residues 17–60 of molecule B are located adjacent to a large solvent channel in the crystal and poorly ordered in the structure. Omission of the region resulted in clear positive density in fo-fc difference maps but the 2fo-fc maps with the region replaced were consistently poor, especially for side chains. Refinement improved with NCS dropped from this region and TLS refinement introduced with separate operators for the region as well as for each separate chain. Some side chains were still not visible, and these were cropped back to Cβ atoms. The final refinement statistics are shown in Table 1, the overall structure is shown in Figure 2, and the structure is deposited in the PDB with the accession number 4L5N.

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