title: ESR spectroscopy identifies inhibitory Cu2+ sites in a DNA-modifying enzyme to reveal determinants of catalytic specificity.
abstract: The relationship between DNA sequence recognition and catalytic specificity in a DNA-modifying enzyme was explored using paramagnetic Cu(2+) ions as probes for ESR spectroscopic and biochemical studies. Electron spin echo envelope modulation spectroscopy establishes that Cu(2+) coordinates to histidine residues in the EcoRI endonuclease homodimer bound to its specific DNA recognition site. The coordinated His residues were identified by a unique use of Cu(2+)-ion based long-range distance constraints. Double electron-electron resonance data yield Cu(2+)-Cu(2+) and Cu(2+)-nitroxide distances that are uniquely consistent with one Cu(2+) bound to His114 in each subunit. Isothermal titration calorimetry confirms that two Cu(2+) ions bind per complex. Unexpectedly, Mg(2+)-catalyzed DNA cleavage by EcoRI is profoundly inhibited by Cu(2+) binding at these hitherto unknown sites, 13 Å away from the Mg(2+) positions in the catalytic centers. Molecular dynamics simulations suggest a model for inhibition of catalysis, whereby the Cu(2+) ions alter critical protein-DNA interactions and water molecule positions in the catalytic sites. In the absence of Cu(2+), the Mg(2+)-dependence of EcoRI catalysis shows positive cooperativity, which would enhance EcoRI inactivation of foreign DNA by irreparable double-strand cuts, in preference to readily repaired single-strand nicks. Nonlinear Poisson-Boltzmann calculations suggest that this cooperativity arises because the binding of Mg(2+) in one catalytic site makes the surface electrostatic potential in the distal catalytic site more negative, thus enhancing binding of the second Mg(2+). Taken together, our results shed light on the structural and electrostatic factors that affect site-specific catalysis by this class of endonucleases.
authors.affiliation: Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA.
authors.forename: Zhongyu
authors.initials: Z
authors.lastname: Yang
authors.forename: Michael R
authors.initials: MR
authors.lastname: Kurpiewski
authors.forename: Ming
authors.initials: M
authors.lastname: Ji
authors.forename: Jacque E
authors.initials: JE
authors.lastname: Townsend
authors.forename: Preeti
authors.initials: P
authors.lastname: Mehta
authors.forename: Linda
authors.initials: L
authors.lastname: Jen-Jacobson
authors.forename: Sunil
authors.initials: S
authors.lastname: Saxena
mesh_terms: D055162:Biocatalysis; D020134:Catalytic Domain; D003300:Copper / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D015254:DNA Modification Methylases / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006639:Histidine / Q000378:metabolism; D056004:Molecular Dynamics Simulation
chemical_list: D006639:Histidine; D003300:Copper; D004247:DNA; D015254:DNA Modification Methylases