Research Article: Crystal Structure of the Vaccinia Virus DNA Polymerase Holoenzyme Subunit D4 in Complex with the A20 N-Terminal Domain

Date Published: March 6, 2014

Publisher: Public Library of Science

Author(s): Céline Contesto-Richefeu, Nicolas Tarbouriech, Xavier Brazzolotto, Stéphane Betzi, Xavier Morelli, Wim P. Burmeister, Frédéric Iseni, Bernard Moss.

http://doi.org/10.1371/journal.ppat.1003978

Abstract

Vaccinia 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.

Partial Text

The well-studied vaccinia virus (VACV) belongs to the orthopoxvirus genus of the family poxviridae. The Orthopoxvirus genus also comprises well-known pathogens such as monkeypox virus and cowpox virus (which can be transmitted to humans) as well as the most virulent member variola virus. Unlike other DNA viruses, orthopoxviruses replicate entirely in the cytoplasm of the infected host-cell. Viral genome synthesis takes place in perinuclear foci called viral factories and is thought to depend almost exclusively on virally encoded-proteins. Four of these proteins, presumably positioned at the replication fork, were shown to be essential for DNA synthesis [1]. For VACV these are: E9, the catalytic subunit of the DNA polymerase; D5, a DNA-independent nucleoside triphosphatase which contains a putative helicase domain [2] and primase activity [3]; D4, a uracil-DNA glycosylase (UDG) [4] and A20, a central component linking E9 and D4 [5], [6] and interacting with D5 [7], [8].

It has been demonstrated that the complex formed by the VACV D4 and A20 proteins is essential to convert the distributive DNA polymerase E9 into a processive mode [5], [10]. Yet, much work remains to be done to understand the molecular mechanisms driving D4/A20 assembly and how it stimulates long-chain DNA synthesis. A first glimpse into the complex structure was obtained from the study of Schormann, et al. which has shown that bacterially expressed VACV D4 was found to be dimeric in solution and crystallized as a dimer [29]. The dimerization of D4 was intriguing since UDGs from different organisms are structurally well conserved and known to be small monomeric enzymes that do not require co-factors or even divalent cations for activity [33]. Additional biochemical and structural studies of the D4/A20 complex did not favor the model in which D4 functions as a dimer but rather suggested that within the DNA polymerase holoenzyme D4 is in a monomeric state [5], [6]. The data presented in this report strengthen this last model and explain at the molecular level how A20 prevents D4/D4 dimerization by binding to D4. The molecular mass ranging from 43 to 28 kDa obtained for D4 in the SEC-MALLS experiment is consistent with the protein existing as a mixture of monomer/dimer in solution, with a relatively large dissociation constant and fast kinetics. In contrast, when co-expressed with its partner A201–50, a D4/A201–50 complex is formed with a 1∶1 stoichiometry (molecular mass of 32 kDa, Figure 1).

 

Source:

http://doi.org/10.1371/journal.ppat.1003978

 

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