DNA Cleavage by EcoRV Endonuclease:  Two Metal Ions in Three Metal Ion Binding Sites

Abstract

Four crystal structures of EcoRV endonuclease mutants K92A and K38A provide new insight into the mechanism of DNA bending and the structural basis for metal-dependent phosphodiester bond cleavage. The removal of a key active site positive charge in the uncleaved K92A-DNA-M(2+) substrate complex results in binding of a sodium ion in the position of the amine nitrogen, suggesting a key role for a positive charge at this position in stabilizing the sharp DNA bend prior to cleavage. By contrast, two structures of K38A cocrystallized with DNA and Mn(2+) ions in different lattice environments reveal cleaved product complexes featuring a common, novel conformation of the scissile phosphate group as compared to all previous EcoRV structures. In these structures, the released 5'-phosphate and 3'-OH groups remain in close juxtaposition with each other and with two Mn(2+) ions that bridge the conserved active site carboxylates. The scissile phosphates are found midway between their positions in the prereactive substrate and postreactive product complexes of the wild-type enzyme. Mn(2+) ions occupy two of the three sites previously described in the prereactive complexes and are plausibly positioned to generate the nucleophilic hydroxide ion, to compensate for the incipient additional negative charge in the transition state, and to ionize a second water for protonation of the 3'-oxyanion. Reconciliation of these findings with earlier X-ray and fluorescence studies suggests a novel mechanism in which a single initially bound metal ion in a third distinct site undergoes a shift in position together with movement of the scissile phosphate deeper into the active site cleft. This reconfigures the local environment to permit binding of the second metal ion followed by movement toward the pentacovalent transition state. The new mechanism suggested here embodies key features of previously proposed two- and three-metal catalytic models, and offers a view of the stereochemical pathway that integrates much of the copious structural and functional data that are available from exhaustive studies in many laboratories.

An Integrated Biological Approach to Guide the Development of Metal-Chelating Inhibitors of Influenza Virus PA Endonuclease

“…In thermodynamic models using Mg 21 as the cofactor, the Mg 21 to Mg 21 distance was predicted to decrease from 4.91 to 3.85 Å upon RNA binding (Xiao et al, 2014). These changes in the intermetal distance along the catalytic pathway were already demonstrated for Bacillus halodurans RNase H (Yang et al, 2006) and EcoRV (Horton and Perona, 2004).…”

Mutational and Metal Binding Analysis of the Endonuclease Domain of the Influenza Virus Polymerase PA Subunit

“…In particular, Arg84 is bound to a well-ordered sulfate ion in the crystal structure of PA-Nter (6). This sulfate is in the same position as one of the phosphates of the EcoRV restriction enzyme product complex (16). Tyr130 and Lys137 coordinate water molecules that bind to the monophosphate group in a complex of PA-Nter with nucleoside monophosphates (38).…”

Three Metal Ions Participate in the Reaction Catalyzed by T5 Flap Endonuclease

“…For phosphate diesterases that contain three active site zinc ions, such as nuclease P1 (34), phospholipase C (35,36), and endonuclease IV (37,38), all three metals are suggested to act in chemical catalysis, but different mechanisms involving these three metal ions have been proposed. Several magnesiumdependent nucleases with three metal ion binding sites may also exist, such as the group I intron (39), EcoRV (40), and 3Ј-5Ј exonuclease (41). Even the prototype two-metal-ion mechanism phosphoryl transferase, the phosphate monoesterase alkaline phosphatase, has an active site composed of two zinc ions plus one magnesium ion, and magnesium ions stimulate activity (2).…”