Conformational Plasticity of the NNRTI-Binding Pocket in HIV-1 Reverse Transcriptase: A Fluorine Nuclear Magnetic Resonance Study

Abstract

HIV-1 reverse transcriptase (RT) is a major drug target in the treatment of HIV-1 infection. RT inhibitors currently in use include non-nucleoside, allosteric RT inhibitors (NNRTIs), which bind to a hydrophobic pocket, distinct from the enzyme's active site. We investigated RT-NNRTI interactions by solution (19)F nuclear magnetic resonance (NMR), using singly (19)F-labeled RT proteins. Comparison of (19)F chemical shifts of fluorinated RT and drug-resistant variants revealed that the fluorine resonance is a sensitive probe for identifying mutation-induced changes in the enzyme. Our data show that in the unliganded enzyme, the NNRTI-binding pocket is highly plastic and not locked into a single conformation. Upon inhibitor binding, the binding pocket becomes rigidified. In the inhibitor-bound state, the (19)F signal of RT is similar to that of drug-resistant mutant enzymes, distinct from what is observed for the free state. Our results demonstrate the power of (19)F NMR spectroscopy to characterize conformational properties using selectively (19)F-labeled protein.

Figure 1

General description of RT structure, and comparison of apo and EFV-bound crystal structures of RT. (A) Tube representation of apo-RT (PDB: 1DLO), with the fingers, palm, thumb, connection, and RNase H domains in the p66 subunit colored in blue, pink, green, yellow and orange, respectively. The p51 subunit is colored grey. (B) Structural differences between apo-RT (left, PDB: 1DLO) and EFV-bound RT (right, PDB: 1FK9). A large conformational change, including the separation of the thumb and fingers domains (indicated by the arrow), is seen in the drug-bound structure. Tyrosine residues 127, 146 and 181 are depicted in ball and stick representation and encircled. (C) Details of the binding site in apo RT and the EFV-bound RT complex, illustrating the rotation of the Y181 (black arrow) and Y188 (grey arrow) side chains out of the binding pocket. The bound EFV molecule is shown in green and pertinent distances between the benzoxazin-2-one and the backbone carbonyl oxygen of K101 (2.8 Å), and the carbonyl group of the bexzoxazine-2-one and the backbone nitrogen of atom K101 (3.2 Å) are indicated.

Figure 2

1D ¹⁹F NMR spectra of RT with 4-trifluoromethyl-phenylalanines substituted for tyrosine residues at several positions in the p66 subunit, in the absence (black) and presence of EFV at 1:1 and 1:5 molar ratios (light and dark green, respectively). ¹⁹F spectra of (A) RT127tfmF, (B) RT146tfmF, and (C) RT181tfmF at 27°C are shown.

Figure 3

1D ¹⁹F NMR spectra of RT181tfmF and several RT mutants at 27°C. (A) Superposition of the fluorine resonances of RT181tfmF (black), RT181tfmF-V108I (red), RT181tfmF-E138K(p51) (green), and RT181tfmF-K103N (purple). (B) Superposition of the fluorine resonances of RT146tfmF (black), RT146tfmF-V108I (red), and RT146tfmF-K103N (purple). All RT181tfmF and RT146tfmF variants contain amino acid changes in both the p51 and p66 domains, except for RT181tfmF-E138K in which the E138K change is only present in the p51 subunit.

Figure 4

1D ¹⁹F NMR spectra of RT181tfmF and several RT181tfmF mutants in the absence (black) and presence of NVP (pink), EFV (green), ETR (blue) and RPV (orange). (A) Superposition of the ¹⁹F spectra of apo-RT181tfmF and the ¹⁹F spectra of RT181tfmF in the presence of each NNRTI, (B) Superposition of the ¹⁹F spectra of apo-RT181tfmF-V108I and the ¹⁹F spectra of RT181tfmF in the presence of each NNRTI, (C) Superposition of the ¹⁹F spectra of apo-RT181tfmF-K103N and the ¹⁹F spectra of RT181tfmF-K103N in the presence of each NNRTI, (D) Superposition of the ¹⁹F spectra of apo-RT181tfmF-E138K(p51) and the ¹⁹F spectra of RT181tfmF-E138K(p51) in the presence of each NNRTI. The ¹⁹F spectra in the presence of each NNRTI at 1:1 and 1:5 molar ratios are shown in light and dark colors, respectively. Chemical formulae for each inhibitor are depicted in the individual panels.

Figure 5

Superposition of 1D ¹⁹F NMR spectra of (A) RT146tfmF, (B) RT146tfmF-V108I, and (C) RT146tfmF-K103N, in the absence (black) and presence of NVP (pink) or EFV (green). The ¹⁹F spectra in the presence of each NNRTI at 1:1 and 1:5 molar ratios are shown in light and dark colors, respectively.

Figure 6

Plots of (A) and (C) linewidths and (B) and (D) chemical shifts of the signals in the ¹⁹F spectra of RT181tfmF and RT146tfmF and the drug-resistant variants, respectively, in the absence and presence of each NNRTI. In (A) and (B), plots are shown for apo- and NNRTI-bound signals of RT181tfmF (o), RT181tfmF-K103N (x), RT181tfmF-V108I (□), and RT181tfmF-E138K(p51) (△). In (C) and (D), plots are shown for apo- and NNRTI-bound signals of RT146tfmF (o), RT146tfmF-K103N (x), and RT146tfmF-V108I (□). Note that since the spectra of RT146tfmF and mutants thereof comprise two resonances at approximately at −62 and −60 ppm, two sets of points are contained in the plots presented in (C) and (D). Values were obtained from the spectra provided in Figures 4 and 5.