Two-metal ion mechanism of RNA cleavage by HIV RNase H and mechanism-based design of selective HIV RNase H inhibitors - PubMed (original) (raw)

Two-metal ion mechanism of RNA cleavage by HIV RNase H and mechanism-based design of selective HIV RNase H inhibitors

Klaus Klumpp et al. Nucleic Acids Res. 2003.

Abstract

Human immunodeficiency virus (HIV) RNase H activity is essential for the synthesis of viral DNA by HIV reverse transcriptase (HIV-RT). RNA cleavage by RNase H requires the presence of divalent metal ions, but the role of metal ions in the mechanism of RNA cleavage has not been resolved. We measured HIV RNase H activity associated with HIV-RT protein in the presence of different concentrations of either Mg2+, Mn2+, Co2+ or a combination of these divalent metal ions. Polymerase-independent HIV RNase H was similar to or more active with Mn2+ and Co2+ compared with Mg2+. Activation of RNase H by these metal ions followed sigmoidal dose-response curves suggesting cooperative metal ion binding. Titration of Mg2+-bound HIV RNase H with Mn2+ or Co2+ ions generated bell-shaped activity dose-response curves. Higher activity could be achieved through simultaneous binding of more than one divalent metal ion at intermediate Mn2+ and Co2+ concentrations, and complete replacement of Mg2+ occurred at higher Mn2+ or Co2+ concentrations. These results are consistent with a two-metal ion mechanism of RNA cleavage as previously suggested for a number of polymerase-associated nucleases. In contrast, the structurally highly homologous RNase HI from Escherichia coli is most strongly activated by Mg2+, is significantly inhibited by submillimolar concentrations of Mn2+ and most probably cleaves RNA via a one-metal ion mechanism. Based on this difference in active site structure, a series of small molecule N-hydroxyimides was identified with significant enzyme inhibitory potency and selectivity for HIV RNase H.

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Figures

Figure 1

Figure 1

Polymerase-independent HIV RNase H activity. Comparative analysis of 5′-phosphorylated and 5′-capped RNA substrates at different concentrations of KCl as indicated. RNase H products were analysed by denaturing acrylamide gel electrophoresis. HIV RNase H reactions were incubated for 0, 0.5, 1, 2, 4, 6, 8 and 10 min with 1 mM MgCl2. T, U, RNA sequencing reactions. T, RNase T1; U, RNase U2; C, annealed substrate incubated for 10 min. The positions of the 20mer RNA substrate and the major 18mer and 9mer cleavage products are indicated. Sequencing reactions with RNases U2 and T1 generate products with 3′-phosphate groups, which migrate slightly faster than the corresponding products of HIV RNase H, which generates 3′-OH ends.

Figure 2

Figure 2

Cleavage pattern differences between HIV and E.coli RNases H. RNase H products were analysed by denaturing acrylamide gel electrophoresis. HIV RNase H reactions were incubated for 0, 0.5, 1, 2, 4, 6, 8, 10, 15 and 30 min with substrate CGK1 and 1 mM MgCl2 or 0.1 mM MnCl2 at pH 7 as indicated. The positions of the 20mer RNA substrate, the major 18mer and 9mer cleavage products and the 11mer and 12mer size marker are indicated on the left of the gel. HIV, HIV RNase H; EC, E.coli RNase H.

Figure 3

Figure 3

Activation of HIV RNase H by divalent metal ions. (a) Time courses of 9mer formation from CGK1 substrate were determined in the presence of 0.02 mM MnCl2 (filled circles), 0.2 mM MgCl2 (open circles) or 0.02 mM MnCl2 plus 0.2 mM MgCl2 (filled squares). (b) Time courses of 9mer formation from CGK1 substrate were determined in the presence of 0.02 mM CoCl2 (filled circles), 0.2 mM MgCl2 (open circles) or 0.02 mM CoCl2 plus 0.2 mM MgCl2 (filled squares). (c) Rates of 9mer formation were determined from time courses in the presence of increasing concentrations of MnCl2 alone (filled squares) or 0.2 mM MgCl2 (open circles). The dose–response curve of RNA cleavage activation by MnCl2 (filled squares) fitted to a sigmoidal equation with a Hill coefficient of _n_H = 3.1. (d) As (c) in the presence of increasing concentrations of CoCl2 alone (filled squares) or 0.2 mM MgCl2 (open circles). The dose–response curve of RNA cleavage activation by CoCl2 (filled squares) fitted to a sigmoidal equation with a Hill coefficient of _n_H = 1.6. (e) Rates of 9mer formation were determined from time courses in the presence of increasing concentrations of MgCl2. The dose–response curve of RNA cleavage activation by MgCl2 (filled squares) fitted to a sigmoidal equation with a Hill coefficient of _n_H = 2.0. (f) Hill plot of 9mer formation kinetics in the presence of MgCl2. Determination of the slope provided a Hill coefficient of _n_H = 1.8. All reactions were performed as described in Materials and Methods and at pH 8.

Figure 4

Figure 4

Design and inhibitory activity of a chemical probe to interact with two-metal ion active sites. (a) _N_-hydroxyimides present three oxygen atoms at positions compatible with the interaction of two divalent metal ions at a distance of 4 Å. Each metal ion can undergo two liganding interactions with oxygen atoms at equal distances of 2.1 Å. (b) Structures of the _N_-hydroxyimides described. Inhibition of enzyme activity was determined and the results are shown in Table 1. (c) Compound 1 inhibited the formation of 9mer from substrate CGK1 in a dose-dependent manner in the presence of 1 mM MgCl2. Polyacrylamide gel analysis of HIV RNase H activity. Lanes 1 and 2, control reactions in the absence of protein; lanes 3–10, RNase H reactions in the presence of 0, 0.01, 0.05, 0.1, 0.5, 1, 5 and 10 µM compound 1. (d) The dose–response curve obtained with compound 1 on the gel-based assay was fitted to a hyperbolic, single site interaction equation. IC50 = 0.6 µM. (e) Compound 1 inhibited HIV RNase H-directed degradation of poly(dC:rG) in the presence of 1 mM MgCl2. IC50 = 1.0 µM (filled circles). Escherichia coli RNase H activity was not affected under identical conditions (filled squares).

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