The synthesis of pABA: Coupling between the glutamine amidotransferase and aminodeoxychorismate synthase domains of the bifunctional aminodeoxychorismate synthase from Arabidopsis thaliana.

International audienceAminodeoxychorismate (ADC) synthase in plants is a bifunctional enzyme containing glutamine amidotransferase (GAT) and ADC synthase (ADCS) domains. The GAT domain releases NH(3) from glutamine and the ADCS domain uses NH(3) to aminate chorismate. This enzyme is involved in folate (vitamin B9) biosynthesis. We produced a stable recombinant GAT-ADCS from Arabidopsis. Its kinetic properties were characterized, and activities and coupling of the two domains assessed. Both domains could operate independently, but not at their optimal capacities. When coupled, the activity of one domain modified the catalytic properties of the other. The GAT activity increased in the presence of chorismate, an activation process that probably involved conformational changes. The ADCS catalytic efficiency was 10(4) fold higher with glutamine than with NH(4)Cl, indicating that NH(3) released from glutamine and used for ADC synthesis did not equilibrate with the external medium. We observed that the GAT activity was always higher than that of ADCS, the excess of NH(3) being released in the external medium. In addition, we observed that ADC accumulation retro-inhibited ADCS activity. Altogether, these results indicate that channeling of NH(3) between the two domains and/or amination of chorismate are the limiting step of the whole process, and that ADC cannot accumulate

Structural analysis of point mutations at the Vaccinia virus A20/D4 interface

International audienceThe Vaccinia virus polymerase holoenzyme is composed of three subunits: E9, the catalytic DNA polymerase subunit; D4, a uracil-DNA glycosylase; and A20, a protein with no known enzymatic activity. The D4/A20 heterodimer is the DNA polymerase cofactor, the function of which is essential for processive DNA synthesis. The recent crystal structure of D4 bound to the first 50 amino acids of A20 (D4/A201-50) revealed the importance of three residues, forming a cation-π interaction at the dimerization interface, for complex formation. These are Arg167 and Pro173 of D4 and Trp43 of A20. Here, the crystal structures of the three mutants D4-R167A/A201-50, D4-P173G/A201-50 and D4/A201-50-W43A are presented. The D4/A20 interface of the three structures has been analysed for atomic solvation parameters and cation-π interactions. This study confirms previous biochemical data and also points out the importance for stability of the restrained conformational space of Pro173. Moreover, these new structures will be useful for the design and rational improvement of known molecules targeting the D4/A20 interface

Crystal Structure of the Vaccinia Virus Uracil-DNA Glycosylase in Complex with DNA.

International audienceVaccinia virus polymerase holoenzyme is composed of the DNA polymerase catalytic subunit E9 associated with its heterodimeric co-factor A20·D4 required for processive genome synthesis. Although A20 has no known enzymatic activity, D4 is an active uracil-DNA glycosylase (UNG). The presence of a repair enzyme as a component of the viral replication machinery suggests that, for poxviruses, DNA synthesis and base excision repair is coupled. We present the 2.7 Å crystal structure of the complex formed by D4 and the first 50 amino acids of A20 (D4·A201-50) bound to a 10-mer DNA duplex containing an abasic site resulting from the cleavage of a uracil base. Comparison of the viral complex with its human counterpart revealed major divergences in the contacts between protein and DNA and in the enzyme orientation on the DNA. However, the conformation of the dsDNA within both structures is very similar, suggesting a dominant role of the DNA conformation for UNG function. In contrast to human UNG, D4 appears rigid, and we do not observe a conformational change upon DNA binding. We also studied the interaction of D4·A201-50 with different DNA oligomers by surface plasmon resonance. D4 binds weakly to nonspecific DNA and to uracil-containing substrates but binds abasic sites with a Kd of <1.4 μm. This second DNA complex structure of a family I UNG gives new insight into the role of D4 as a co-factor of vaccinia virus DNA polymerase and allows a better understanding of the structural determinants required for UNG action

Crystal structure of the vaccinia virus DNA polymerase holoenzyme subunit D4 in complex with the A20 N-terminal domain

International audienceVaccinia virus polymerase holoenzyme is composed of the DNA polymerase E9, the uracil-DNA glycosylase D4 and A20, a protein with no known enzymatic activity. The D4/A20 heterodimer is the DNA polymerase co-factor whose function is essential for processive DNA synthesis. Genetic and biochemical data have established that residues located in the N-terminus of A20 are critical for binding to D4. However, no information regarding the residues of D4 involved in A20 binding is yet available. We expressed and purified the complex formed by D4 and the first 50 amino acids of A20 (D4/A20₁₋₅₀). We showed that whereas D4 forms homodimers in solution when expressed alone, D4/A20₁₋₅₀ clearly behaves as a heterodimer. The crystal structure of D4/A20₁₋₅₀ solved at 1.85 Å resolution reveals that the D4/A20 interface (including residues 167 to 180 and 191 to 206 of D4) partially overlaps the previously described D4/D4 dimer interface. A20₁₋₅₀ binding to D4 is mediated by an α-helical domain with important leucine residues located at the very N-terminal end of A20 and a second stretch of residues containing Trp43 involved in stacking interactions with Arg167 and Pro173 of D4. Point mutations of the latter residues disturb D4/A20₁₋₅₀ formation and reduce significantly thermal stability of the complex. Interestingly, small molecule docking with anti-poxvirus inhibitors selected to interfere with D4/A20 binding could reproduce several key features of the D4/A20₁₋₅₀ interaction. Finally, we propose a model of D4/A20₁₋₅₀ in complex with DNA and discuss a number of mutants described in the literature, which affect DNA synthesis. Overall, our data give new insights into the assembly of the poxvirus DNA polymerase cofactor and may be useful for the design and rational improvement of antivirals targeting the D4/A20 interface

Primers used for cloning.

<p>Primers used for cloning.</p

Oligomeric state of D4 and His-D4/A20_1–50.

<p>(A) 15% SDS-PAGE analysis of purified D4 and His-D4/A20_1–50 complex. Lane 1: Molecular weight standards. Lane 2: D4 (∼10 µg). Lane 3: His-D4/A20_1–50 (∼10 µg). Proteins were stained with InstantBlue (Expedeon). (B) Analysis of D4 and His-D4/A20_1–50 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/A20_1–50 (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.</p

Comparison of D4/A20_1–50 and D4/D4 interfaces.

<p>(A) Cartoon representation of the D4 (yellow)/A20_1–50 (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/A20_1–50 complex structure. The atoms in contact with A20_1–50 (meaning closer than 4.5 Å to atoms of A20_1–50) 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 A20_1–50. The atoms in contact with D4 (distance <4.5 Å) are colored in grey. A20_1–50 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.</p

Model of D4/A20_1–50 heterodimer bound to DNA.

<p>D4 from the D4/A20_1–50 complex was superimposed onto the human UDG from the hUDG/DNA complex structure (pdb entry 1SSP). (A) The surface of D4 from the D4/A20_1–50 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 <i>et al</i>. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003978#ppat.1003978-DruckShudofsky1" target="_blank">[28]</a> 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.</p

Point mutations at the D4/A20_1–50 interface affect complex stability.

<p>(A) Fractions from Peak 1 after purification of WT and His-D4/A20_1–50 mutants (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003978#ppat-1003978-g003" target="_blank">Figure 3</a>) were pooled and loaded again onto a gel filtration column. Chromatograms of WT and His-D4/A20_1–50 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/A20_1–50W43A). Proteins were stained with InstantBlue (Expedeon). Migration of His-D4 and A20_1–50 is indicated. (B) A representative thermal shift experiment is shown. For D4 and each His-D4/A20_1–50 complex calculated <i>T_m</i> values obtained from three independent experiments are given together with their standard deviation.</p