Drug resistance in HIV-1 protease: flexibility-assisted mechanism of compensatory mutations

The emergence of drug-resistant variants is a serious side effect assocd. with acquired immune deficiency syndrome therapies based on inhibition of human immunodeficiency virus type 1 protease (HIV-1 PR). In these variants, compensatory mutations, usually located far from the active site, are able to affect the enzymic activity via mol. mechanisms that have been related to differences in the conformational flexibility, although the detailed mechanistic aspects have not been clarified so far. Here, we perform multinanosecond mol. dynamics simulations on L63P HIV-1 PR, corresponding to the wild type, and one of its most frequently occurring compensatory mutations, M46I, complexed with the substrate and an enzymic intermediate. The quality of the calcns. is established by comparison with the available NMR data. Our calcns. indicate that the dynamical fluctuations of the mutated enzyme differ from those in the wild type. These differences in the dynamic properties of the adducts with the substrate and with the gem-diol intermediate might be directly related to variations in the enzymic activity and therefore offer an explanation of the obsd. changes in catalytic rate between wild type and mutated enzyme. We anticipate that this "flexibility-assisted" mechanism might be effective in the vast majority of compensatory mutations, which do not change the electrostatic properties of the enzyme. [on SciFinder (R)]

A comparative study of galactose oxidase and active site analogs based on QM/MM Car-Parrinello simulations

Paolo Carloni, Michele Parrinello, Ursula Röthlisberger

A parallel study of the radical copper enzyme galactose oxidase (GOase) and a low mol. wt. analog of the active site was performed with dynamic d. functional and mixed quantum-classical calcns. This combined approach enables a direct comparison of the properties of the biomimetic and the natural systems throughout the course of the catalytic reaction. In both cases, five essential forms of the catalytic cycle have been investigated: the resting state in its semi-reduced (catalytically inactive) and its oxidized (catalytically active) form, Asemi and Aox, resp.; a protonated intermediate B; the transition state for the rate-detg. hydrogen abstraction step C, and its product D. For A and B the electronic properties of the biomimetic compd. are qual. very similar to the ones of the natural target. However, in agreement with the exptl. obsd. difference in catalytic activity, the calcd. activation energy for the hydrogen abstraction step is distinctly lower for GOase (16 kcal/mol) than for the mimetic compd. (21 kcal/mol). The enzymic transition state is stabilized by a delocalization of the unpaired spin d. over the sulfur-modified equatorial tyrosine Tyr272, an effect that for geometric reasons is essentially absent in the biomimetic compd. Further differences between the mimic and its natural target concern the structure of the product of the abstraction step, which is characterized by a weakly coordinated aldehyde complex for the latter and a tightly bound linear complex for the former. [on SciFinder (R)]

2000

Understanding the Effect of Magnesium Ion Concentration on the Catalytic Activity of Ribonuclease H through Computation: Does a Third Metal Binding Site Modulate Endonuclease Catalysis?

Matteo Dal Peraro

Ribonuclease H (RNase H) belongs to the nucleotidyl-transferase superfamily and hydrolyzes the phosphodiester linkage on the RNA strand of a DNA/RNA hybrid duplex. Due to its activity in HIV reverse transcription, it represents a promising target for anti-HIV drug design. While crystallographic data have located two ions in the catalytic site, there is ongoing debate concerning just how many metal ions bound at the active site are optimal for catalysis. Indeed, experiments have shown a dependency of the catalytic activity on the Mg2+ concentration. Moreover, in RNase H, the glutamate residue E188 has been shown to be essential for full enzymatic activation, regardless of the Mg2+ concentration. The catalytic center is known to contain two Mg2+ ions, and E188 is not one of the primary metal ligands. Herein, classical molecular dynamics (MD) simulations are employed to study the metal-ligand coordination in RNase H at different concentration of Mg2+. Importantly, the presence of a third Mg2+ ion, bound to the second-shell ligand E188, is a persistent feature of the MD simulations. Free energy calculations have identified two distinct conformations, depending on the concentration of Mg2+. At standard concentration, a third Mg2+ is found in the catalytic pocket, but it does not perturb the optimal RNase H active conformation. However, at higher concentration, the third Mg2+ ion heavily perturbs the nucleophilic water and thereby influences the catalytic efficiency of RNase H. In addition, the E188A mutant shows no ability to engage additional Mg2+ ions near the catalytic pocket. This finding likely explains the decrease in catalytic activity of E188A and also supports the key role of E188 in localizing the third Mg2+ ion at the active site. Glutamate residues are commonly found surrounding the metal center in the endonuclease family, which suggests that this structural motif may be an important feature to enhance catalytic activity. The present MD calculations support the hypothesis that RNase H can accommodate three divalent metal ions in its catalytic pocket and provide an in-depth understanding of their dynamic role for catalysis.

2010

Understanding the Effect of Magnesium Ion Concentration on the Catalytic Activity of Ribonuclease H through Computation: Does a Third Metal Binding Site Modulate Endonuclease Catalysis?

Matteo Dal Peraro

2010