The DNA cleavage chemistry of a series of metallopeptides based on the amino-terminal Cu and Ni (ATCUN) binding motif of proteins has been studied. Specifically, the impact of the positioning of charged Lys side chains and their stereochemistry on metal reduction potentials and DNA cleavage reactivity have been quantitatively evaluated. Both Cu and Ni metallopeptides show a general increase in reactivity toward DNA with an increasing number of Lys residues, while a corresponding decrease in complex reduction potential reflects the enhanced sigma-donor character of the Lys side chain relative to that of Gly. Placement of Lys at the first position in the tripeptide ligand sequence resulted in a greater increase in DNA cleavage reactivity, relative to placement at the second position, while a switch from an l-Lys to a d-Lys typically resulted in enhanced reactivity, as well as perturbations of reduction potential. In the case of Cu peptides, reactivity was enhanced with both increasing positive charge density on the peptide and stabilization of the Cu3+ state. However, for Ni peptides, while the general trends are the same, the correlation with redox behavior was less pronounced. Most likely these differences in specific trends for the Cu and Ni complexes reflect the distinct coordination preferences for Cu3+/2+ and Ni3+/2+ oxidation states, and the consequent distinct positioning of metal-associated reactive oxygen species, as well as the orientation of the DNA-associated complex. Thus, the amino acid composition and stereochemistry of ATCUN metallopeptides can tune the intrinsic reactivities of these systems (their ability to promote formation and activity of metal-associated ROS) as well as their overall structural features, and both of these aspects appear to influence their reactivity and efficiency of DNA strand scission.

Structural and thermodynamic aspects of alkaline earth metal dication (Mg(2+), Ca(2+), Sr(2+), Ba(2+)) binding to E. coli ribonuclease H1 (RNase H1) have been investigated using both experimental and theoretical methods. The various metal-binding modes of the enzyme were explored using classical molecular dynamics simulations, and relative binding free energies were subsequently evaluated by free energy simulations. The trends in the free energies of model systems based on the simulation structures were subsequently verified using a combination of density functional theory and continuum dielectric methods. The calculations provide a physical basis for the experimental results and suggest plausible role(s) for the metal cation and the catalytically important acidic residues in protein function. Magnesium ion indirectly activates water attack of the phosphorus atom by freeing one of the active site carboxylate residues, D70, to act as a general base through its four first-shell water molecules, which prevent D70 from binding directly to Mg(2+). Calcium ion, on the other hand, inhibits enzyme activity by preventing D70 from acting as a general base through bidentate interactions with both carboxylate oxygen atoms of D70. These additional interactions to D70, in addition to the D10 and E48 monodentate interactions found for Mg(2+), enable Ca(2+) to bind tighter than the other divalent ions. However, a bare Mg(2+) ion with two or less water molecules in the first shell could bind directly to the three active-site carboxylates, in particular D70, thus inhibiting enzymatic activity. The present analyses and results could be generalized to other members of the RNase H family that possess the same structural fold and show similar metal-binding site and Mg(2+)-dependent activity.

Aminoglycosides are an important class of antibiotic that selectively target RNA structural motifs. Recently we have demonstrated copper derivatives of amino-glycosides to be efficient cleavage agents for cognate RNA motifs. To fully develop their potential as pharmaceutical agents it is necessary to understand both the structural mechanisms used by aminoglycosides to target RNA, and the relative contributions of hydrogen bonding and electrostatic interactions to recognition selectivity. Herein we report results from a calorimetric analysis of a stem-loop 23mer RNA aptamer complexed to the aminoglycoside neomycin B. Key thermodynamic parameters for complex formation have been determined by isothermal titration calorimetry, and from the metal-ion dependence of these binding parameters the relative contributions of electrostatics and hydrogen bonding toward binding affinity have been assessed. The principal mechanism for recognition and binding of neomycin B to the RNA major groove is mediated by hydrogen bonding.