Li, Huiguang
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Publication Structure And Metal Binding Properties Of A Poxvirus Resolvase(2016-01-01) Li, HuiguangPoxviruses, including smallpox virus, replicate their linear genomes by forming concatemers that must be resolved into monomeric units in order to produce new virions. Poxviruses encode a resolvase enzyme that cleaves 4-way junctions extruded at the concatemer junctions, a process that is essential for viral replication. Given its important role in the viral life cycle, the resolvase is an attractive target for identifying small molecule inhibitors of poxvirus infection. To provide a platform for interpreting and designing experiments and to establish a framework for inhibitor design, we have determined the crystal structure of the canarypox virus (CPV) resolvase. CPV resolvase is a RuvC-like dimer composed of RNaseH family folds, with an active site lined by highly conserved, acidic, metal-binding residues. However, there are a number of intriguing structural differences between resolvase and RuvC. A model of the CPV resolvase•HJ complex provides insights into the likely consequences of these changes, including a plausible explanation for the weak sequence specificity exhibited by the poxvirus enzymes. The model also explains why the poxvirus resolvases are more promiscuous than RuvC, cleaving variety of branched, bulge, and flap-containing substrates. Comparing to bacterial RuvC, poxvirus resolvase has one additional aspartic acid (Asp130) at the A site in the active site pocket, which locates at a well-structured α helix. Asp130 side chain carboxylates hydrogen bonds to a water molecule that coordinates the bound A site metal. Thus, Asp130 contributes indirectly to metal binding and could be important for resolvase activity. Based on the unique active site structure observed for CPV resolvase, we have carried out a series of experiments designed to test divalent ion usage and preferences. We find that the two resolvase metal binding sites have different preferences for Mg2+ vs. Mn2+ relative to their expected physiological concentrations. Optimal resolvase activity is maintained with 5 µM Mn2+ and 100 µM Mg2+, concentrations that are well below those required for either metal alone. The additional Asp130 increases the affinity for divalent ions by 10-15 folds and increases resolvase enzymatic activity. Biochemistry studies on the resolvase inhibitor demonstrated that it inhibits resolvase activity in uncompetitive manner. Together, our findings provide biochemical insights and structural models that will facilitate studying poxvirus replication and the search for efficient poxvirus inhibitors. We developed one novel method (Metal&EDTA) to purify RNaseH family members. This method takes full advantage that RNaseH family members have different binding affinities to cationic exchange chromatography column in the presence of EDTA and divalent ions. High purity of enzymes could be obtained in a two-step purification using one SP-sepharose chromatography column. Metal&EDTA method has the potential to purify all types of metal dependent nucleic acids enzymes and metalloproteinases.