Targeting Divalent Metal Ions at the Active Site of the HIV-1 RNase H Domain: NMR Studies on the Interactions of Divalent Metal Ions with RNase H and Its Inhibitors

Abstract

HIV-1 reverse transcriptase (RT) RNase H (HIV-RH) is a key target of anti-AIDS drugs. Metal-chelating compounds are an important class of chemicals in pharmacological drug discovery, especially in relation to HIV-RT and the highly-related HIV-integrase. The correlation between the metal-chelating properties and enzyme activities of the metal chelators is always of high scientific interest, as an understanding of this may accelerate the rational optimization of this class of inhibitors. Our NMR data show that Mg 2+ and Ca 2+ bind specifically to the active site of the RNase H domain and two Mg 2+ ions sequentially bind one molecule of RNase H. We also demonstrate here, using saturated and unsaturated tricyclic N-hydroxypyridones designed to block the active site, that the primary binding sites and affinities of divalent metal ions are correlated with the structures of the chelating motifs. Chemical shift perturbation studies of protein/metal-ion/compound ternary complexes also indicate that divalent metal ions play important roles for the specific interaction of the compounds with the RNase H active site.

Entire-Dataset Analysis of NMR Fast-Exchange Titration Spectra: A Mg2+ Titration Analysis for HIV-1 Ribonuclease H Domain

“…This observation is consistent with previous NMR titration data. 35 Although titration profiles for the active-site residues should be analyzed to clarify whether the observed rate event is due to binding of the second Mg 2+ , sufficient data were not obtained due to severe signal broadening for the active-site residues. Only two of the active-site residues, E478 and D498, exhibited partial titration profiles (Figure S3).…”

“…34,35 The slightly larger K D s in our data, compared to others, are reasonably explained by a difference in the experimental pH: our experiments were performed at pH 7.2, whereas the experiments by the London and Maegley groups were carried out at pH 6.8 and pH 6.5, respectively. 34,35 The observed K D values are higher than those obtained for the RNH domain in the full-length RT (K D , ∼0.1 mM 36 ). There are two possible reasons for it.…”

“…36 Furthermore, although Mg 2+ ion interaction with the isolated RNH domain in solution is qualitatively explained with a single Mg 2+ -binding model (K D , ∼10 mM), the previous titration data themselves are better expressed with a two-Mg 2+ interaction model (K D , ∼3 and 35 mM). 34,35 As a next step of the studies, we endeavored to answer a biophysical question about whether Mg 2+ ions nonspecifically interact with RNH in solution and how such nonspecific interaction affects titration data.…”

Determinants of Active-Site Inhibitor Interaction with HIV-1 RNase H

“…The high concentration of Mg 2+ was used due to its known weak interaction with RNH (dissociation constant >1 mM). 47−50 In addition, because the Mg 2+ interaction with RNH in solution can be explained with either a single Mg 2+ -binding model or a two Mg 2+ -binding model, 45,47,48 different numbers of bound Mg 2+ possibly influence the two binding modes. For this reason, Mg 2+ titration was also performed, in which Mg 2+ was varied from 1 to 20 mM with a constant 1:1 inhibitor/RNH ratio.…”

Structural integrity of the ribonuclease H domain in HIV-1 reverse transcriptase

“…WT spectrum exhibits folded RNH signals similar to those published previously. 14,20,43,44 In contrast to the WT spectrum, most of the resonances in the 1 H-15 N HSQC spectra of RNH F440A and RNH E438N were observed at the random coil region, that is, at a narrow 1 H chemical shift ranges (8.0-8.5 ppm), indicating that these proteins are disordered, most likely unfolded, in solution [red spectra in Fig. 2(B,C)].…”