Figure 1.
Oligomeric state of D4 and His-D4/A201–50.
(A) 15% SDS-PAGE analysis of purified D4 and His-D4/A201–50 complex. Lane 1: Molecular weight standards. Lane 2: D4 (∼10 µg). Lane 3: His-D4/A201–50 (∼10 µg). Proteins were stained with InstantBlue (Expedeon). (B) Analysis of D4 and His-D4/A201–50 by SEC-MALLS. D4 elutes at 11.5 mL (black dashed line). The molecular mass of 43±2 kDa at the maximum peak height decreases down to ∼28 kDa suggesting that a monomer/dimer equilibrium of D4 (calculated molecular mass of 25.4 kDa) exists in solution (grey dashed line). His-D4/A201–50 (calculated molecular mass of 32.4 kDa) elutes as a symmetrical peak at 12.2 mL (black line) with a constant molecular mass of 32±2 kDa (grey line) suggesting a 1∶1 stoichiometry for the complex.
Table 1.
Data collection and refinement statistics.
Figure 2.
Crystal structure of His-D4/A201–50 complex.
D4 is shown with yellow carbon atoms, A201–50 in violet. Oxygen atoms are colored in red, nitrogen atoms in blue. Panels (A), (B), (C) and (D) use the same orientation. (A) View of the complex in cartoon representation. (B) SigmaA-weighted 2Fo-Fc electron density map of the refined model of His-D4/A201–50-T2A at 1.85 Å resolution contoured at 1σ in the vicinity of Trp43 of A20 and Arg167 and Pro173 of D4. A cyan dotted line indicates a hydrogen bond. (C) Surface representation of D4, where only atoms within 4.5 Å of A20 are colored, with the principal side chains of A201–50 forming the interface (see panel E) shown in stick representation. When contacts involve main chain atoms, the main chain of the corresponding residues is shown. Hydrogen bond donors and acceptors are highlighted with cyan dots. (D) The principal residues of D4 which are involved in the contact with A201–50 are shown in light green; residues involved in hydrophobic contacts are printed in brown. Cyan dots mark hydrogen bond acceptors and donors. The arrow indicates the position of the Gly179Arg mutation described in [10]. (E) The D4/A20 contacts. Residues involved in α-helices are printed in red, the ones involved in β-sheets in blue. Residues with some of their surface buried in the contact are underlined. Residues providing hydrogen bond donors or acceptors for inter-subunit hydrogen bonds are marked «H». H-bonds in capitals involve main chain atoms; in small letters side chain atoms; H-bonds in bold indicate residues forming H-bonds with both side chain and main chain atoms. H-bonds are numbered in blue. Stars indicate the residues which contribute with more than 0.2 kcal⋅mol−1 to the interaction according to the the PISA server [31] used for analysis of the interface. (F) The D4/D4 contacts. Residues which are involved in the dimer contact on both monomers are underlined in bold; residues interacting asymmetrically are simply underlined. The analysis was performed with the pdb entry 4DOF, chain A/B.
Figure 3.
Point mutations at the D4/A201–50 interface affect complex formation.
D4, His-D4/A201–50WT and His-D4/A201–50 mutants were purified as described in the Materials and Methods section. Protein elution profiles after the last purification step (i.e. size exclusion chromatography) are presented. 15% SDS-PAGE analysis of the peak fractions (11 to 18) is aligned with each chromatogram. Proteins were stained with InstantBlue (Expedeon). Migration of D4, His-D4 and A201–50 is indicated. Peak 1 and peak 2 are labelled.
Figure 4.
Point mutations at the D4/A201–50 interface affect complex stability.
(A) Fractions from Peak 1 after purification of WT and His-D4/A201–50 mutants (see Figure 3) were pooled and loaded again onto a gel filtration column. Chromatograms of WT and His-D4/A201–50 mutants are superimposed with 0.05 OD offset. A typical 15% SDS-PAGE analysis of the peak fractions (12 to 18) is shown below (in this case His-D4/A201–50W43A). Proteins were stained with InstantBlue (Expedeon). Migration of His-D4 and A201–50 is indicated. (B) A representative thermal shift experiment is shown. For D4 and each His-D4/A201–50 complex calculated Tm values obtained from three independent experiments are given together with their standard deviation.
Figure 5.
Comparison of D4/A201–50 and D4/D4 interfaces.
(A) Cartoon representation of the D4 (yellow)/A201–50 (magenta) complex. (B) Cartoon representation of the D4 dimer (pdb entry 4DOF chain A in light green and chain B in brown). (C) Surface representation of D4 in the His-D4/A201–50 complex structure. The atoms in contact with A201–50 (meaning closer than 4.5 Å to atoms of A201–50) are colored in grey. (D) Surface representation of D4 (pdb entry 4DOF, chain A) in the context of the D4 dimer structure. Atoms of D4 involved in the dimer contact are shown in brown (distance <4.5 Å). (E) Surface representation of A201–50. The atoms in contact with D4 (distance <4.5 Å) are colored in grey. A201–50 has been turned by 180° around a horizontal axis. (F) Three representative D4/D4 dimer structures present in the pdb differing in the relative orientation of the two subunits are shown. The A chains of the dimers have been superposed and are shown in light colors. Chain B from pdb entry 4DOF represents one class and is shown in green; the chain of the crystallographic dimer from pdb entry 4DOG which represents another class of orientations is shown in red, chain B from the dimer in pdb entry 2OWR with an intermediate orientation is shown in turquoise.
Figure 6.
Model of D4/A201–50 heterodimer bound to DNA.
D4 from the D4/A201–50 complex was superimposed onto the human UDG from the hUDG/DNA complex structure (pdb entry 1SSP). (A) The surface of D4 from the D4/A201–50 complex is shown in yellow, atoms within 4.5 Å from A20 in grey, A20 as cartoon in violet. The DNA is shown in cartoon representation. (B) Basic residues of D4 (in yellow) mutated in the study of Druck Shudowsky et al. [28] and affecting the processivity of the vaccinia virus polymerase holoenzyme are shown in light green and labeled in black. The superposed structure of human UDG is shown in orange-red. Structurally equivalent basic residues are shown in red with red labels. An uracil molecule bound in the uracil binding site of the human enzyme is shown as space filling representation with white carbon atoms. (C) Residues of vaccinia virus and human UDG forming the uracil recognition pocket (Tyr70, Phe79 and Asn120) and required for glycosylase activity (Asp68 and His181) are shown in stick representation. The structure of the human enzyme in complex with dsDNA and uracil (white carbon atoms) is shown with orange-red carbon atoms; corresponding residues of D4 are labeled and shown with yellow carbon atoms. The superposition is based on the shown active site residues. The hydrogen bonds involving the uracil molecule are shown as dotted lines.
Table 2.
Primers used for cloning.