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. 2011 Dec;39(22):9779-88.
doi: 10.1093/nar/gkr667. Epub 2011 Sep 2.

Novel dimeric structure of phage φ29-encoded protein p56: insights into uracil-DNA glycosylase inhibition

Juan Luis Asensio  1 , Laura Pérez-Lago, José M Lázaro, Carlos González, Gemma Serrano-Heras, Margarita Salas
Affiliations

Novel dimeric structure of phage φ29-encoded protein p56: insights into uracil-DNA glycosylase inhibition

Juan Luis Asensio et al. Nucleic Acids Res. 2011 Dec.

Abstract

Protein p56 encoded by the Bacillus subtilis phage φ29 inhibits the host uracil-DNA glycosylase (UDG) activity. To get insights into the structural basis for this inhibition, the NMR solution structure of p56 has been determined. The inhibitor defines a novel dimeric fold, stabilized by a combination of polar and extensive hydrophobic interactions. Each polypeptide chain contains three stretches of anti-parallel β-sheets and a helical region linked by three short loops. In addition, microcalorimetry titration experiments showed that it forms a tight 2:1 complex with UDG, strongly suggesting that the dimer represents the functional form of the inhibitor. This was further confirmed by the functional analysis of p56 mutants unable to assemble into dimers. We have also shown that the highly anionic region of the inhibitor plays a significant role in the inhibition of UDG. Thus, based on these findings and taking into account previous results that revealed similarities between the association mode of p56 and the phage PBS-1/PBS-2-encoded inhibitor Ugi with UDG, we propose that protein p56 might inhibit the enzyme by mimicking its DNA substrate.

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Figures

Figure 1.
Figure 1.
Stoichiometry of the p56–UDG interaction as deduced from isothermal titration calorimetry (ITC). UDG solutions were titrated with p56 inhibitor at 25°C in 10 mM sodium phosphate, pH 6.5 and 100 mM NaCl. The experimental curves were fitted assuming a single set of sites on the enzyme. The obtanied stoichiometry factor (n), binding constant (Kb), binding free energy (ΔG) and enthalpy (ΔH) values are shown.
Figure 2.
Figure 2.
(A) Backbone trace for the 20 lowest energy structures in the final NMR ensemble of the p56 dimer (Top) together with a ribbon representation of a single conformer (Bottom). Different monomeric units are labelled with subscripts a and b. Secondary structure elements are indicated by colour coding: α-helices (α1, red), β-strands (β1–β3, green), and loops (L1–L3, yellow). The disordered N-terminal tails (residues 1–7) are omitted for clarity. (B) Molecular surface representations of p56 showing the electrostatic potential (blue is positive and red is negative). The orientation of the p56 dimer in the top view is identical to that of the NMR ensemble shown in (A). Bottom view is related with the former by a 180° rotation around the x-axis. The position of the charged residues contributing to the inhibitor electrostatic potential is indicated. (C) Top: ribbon representation of the UDG-bound form of the inhibitor Ugi from phage PBS-1/PBS-2 (16) (pdb code 1EUI). Bottom: molecular surface representation showing the electrostatic potential at the UDG-binding surface of Ugi inhibitor.
Figure 3.
Figure 3.
(A) Double-filtered (left) and half-filtered (right) NOESY experiments acquired for p56 at 35°C (buffer conditions: 100 mM NaCl and 10 mM sodium phosphate, pH 5.0). Inter-protein NOE contacts involving the aromatic ring of Y40 and residues E37, L33 and L52 are clearly observable in the half-filtered spectrum (labelled in red). (B) Structural details of p56 dimerization interface. Protein side-chains involved in relevant protein–protein contacts are highlighted (aromatic, in green; aliphatic, in yellow; positively charged, in cyan; negatively charged, in red). The protein backbone is shown in grey. Different monomeric units are labelled with subscripts a and b. Top and bottom views are related by a 180o rotation around the x-axis. (C) Ensemble of 20 NMR structures showing the inhibitor side-chains involved in dimerization.
Figure 4.
Figure 4.
Influence of selected residues on the stability of the p56 dimer and its inhibition activity. (A) The oligomerization state of p56 mutants was analysed by glycerol gradient sedimentation. Wild-type p56 and mutants, Y40A, D53A and K49A/R51A (12 µg of each), were loaded on a 15–30% glycerol gradient and subjected to centrifugation for 49 h at 59 000 rpm in a Beckman SW.65 rotor. As markers, 15 µg of lysozyme (14.7 kDa) and 15 µg of aprotinine (6.5 kDa) were loaded in the same gradient. Sedimentation was from right to left. After fractionation, aliquots from each fraction were analysed by SDS–PAGE. Densitometry scanning of the gels stained with Coomassie Blue was used to determine the amount of the different proteins in each fraction (arbitrary units). The arrows indicate fractions at which the maximal amount of each marker was detected. (B) Inhibition activity of p56 dimerization mutants. Increasing amounts of the p56 mutants (from 0.2 to 5.4 ng) were incubated with 5 pg of UDG. Then, the DNA substrate was added and incubated for 20 min at 37°C and the product was analysed in 8 M urea–20% polyacrylamide gels. (C) Bands corresponding to DNA substrate and DNA product were quantified by densitometry and the percentage of inhibition was calculated taking the wild-type p56 values as 100% inhibition. Data are depicted in a bar chart and average values of three independent experiments with standard deviations are represented.
Figure 5.
Figure 5.
Influence of acidic residues on the p56 inhibition activity. The inhibition ability of two triple mutants (D5A/D8A/D11A and D18A/D19A/D20A) and two single mutants, E26A(A) and E37A(B) of p56 was assayed. Different doses of the mutant proteins (from 0.2 to 18.8 ng for the triple mutants and the E26A mutant and from 0.25 to 16 ng for the E37A mutant) were incubated with 5 pg of UDG. Then, the DNA substrate was added and incubated for 20 min at 37°C and the product was analysed in 8 M urea-20% polyacrylamide gels. (C) After quantification by densitometry, the percentage of inhibition for the mutants was calculated with respect to the wild-type protein. Data are depicted in a bar chart and average values of three independent experiments with standard deviations are represented.
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