Structural Basis for the Inhibition of RNase H Activity of HIV-1 Reverse Transcriptase by RNase H Active Site-Directed Inhibitors ▿
- Hua-Poo Su1,*,
- Youwei Yan1,
- G. Sridhar Prasad1,
- Robert F. Smith1,
- Christopher L. Daniels1,
- Pravien D. Abeywickrema1,
- John C. Reid1,
- H. Marie Loughran2,
- Maria Kornienko1,
- Sujata Sharma1,
- Jay A. Grobler3,
- Bei Xu1,
- Vinod Sardana1,
- Timothy J. Allison1,
- Peter D. Williams2,
- Paul L. Darke1,
- Daria J. Hazuda3 and
- Sanjeev Munshi1
-
FIG. 1.Structures of compounds MK1 and MK2 bound to the RNase H active site of HIV-1 RT. (A) The structure of RT is shown as a ribbon diagram, with the p66 subunit colored blue and the RNase H domain colored a lighter shade. The p51 subunit is colored orange. The inhibitor, MK1, is shown as a space-filling model in magenta, bound to the RNase H active site. On the opposite side of RT is the NNRTI pocket, which contains the NNRTI, nevirapine (yellow), required for crystallizing RT. (B) MK1 is shown on the left, bound to the RNase H active site. The electron density (sigmaA weighted 2Fo-Fc map [33] contoured at 1σ) is shown in purple. The naphthyridine rings of the compound coordinate two Mn2+ ions (green) with the catalytic site residues. (C) The structure of the inhibitor, MK2 (cyan), binds to the active site in a similar pose to MK1. The electron density (sigmaA weighted 2Fo-Fc map contoured at 1σ) is shown in purple and a two-dimensional image of the inhibitor is shown on the right.
-
FIG. 2.Binding of MK2 to the RNase H active site of RT. Close-up view of MK2 (cyan) bound to the active site. The two Mn2+ ions are shown in green, and bonds to the Mn2+ ions are shown in blue. One Mn2+ is coordinated by carbonyl oxygens from D443 and D549 and two oxygens of the naphthyridinone. The other Mn2+ is coordinated by the other carbonyl oxygen of D443, E478, D498, and the naphthyridine moiety of the compound. In addition to contacts with the metal ions, the inhibitor also interacts with a loop in RT containing A538 and H539. Select protein 1:1 inhibitor bonds are shown in black.
-
FIG. 3.Binding of MK3 to a site close to the NNRTI site. (A) The full-length RT is shown as ribbons and colored with the p66 subunit in blue and the p51 subunit in orange. Two Mn2+ ions (green) are present in the active site of the RNase H domain (green). The inhibitor, MK3 (orange), is shown as a space-filling model binding away from the RNase H domain. The binding of MK3 displaces the nevirapine used for cocrystallization. (B) The interactions of MK3 are mostly made by hydrophobic interactions and are metal independent. The electron density is shown in dark blue (sigmaA weighted 2Fo-Fc map contoured at 1σ).
-
FIG. 4.ThermoFluor analysis of metal dependence on inhibitor binding to full-length RT. Fluorescence-based thermal denaturation temperatures for RT in the absence (DMSO control) or presence of inhibitor. Experiments are conducted in the absence of metal (blue), the presence of Mg2+ (yellow), or the presence of Mn2+ (purple). There is a metal-dependent increase in thermal stability for all three inhibitors.
-
FIG. 5.The structure of MK3 bound to the active site of the isolated RNase H domain of RT. (A) MK3 is shown in orange as a space-filling model bound to the active site of RNase H (ribbons). For reference, the two-dimensional representation for the inhibitor is shown on the right. (B) The electron density map (sigmaA weighted 2Fo-Fc map contoured at 1σ) for MK3 bound to RNase H is shown in purple on the left. Binding without the electron density map is shown on the right. The Mn2+ ions are shown in green. (C) MK3 from the RNase H domain crystals is overlaid on top of the inhibitors MK1 and MK2 from the full-length RT crystals based on structural alignment of the RNase H domain. The two Mn2+ ions, which bind each inhibitor, do not move significantly. The metal-coordinating naphthyridine of MK3 is rotated (left) and tilted out of the plane of MK1 and MK2 (right) due to interactions mediated by the additional benzyl ring.
-
FIG. 6.Comparison of the binding of MK3 and DHBNH away from the RNase H active site. (A) The structure of full-length RT is shown as a ribbon diagram (p66 subunit in blue and p51 subunit in orange) bound to MK3 (orange) on the left and DHBNH (green) on the right(16). (B) The two inhibitors are overlaid with MK3 in orange and DHBNH in green, showing a common binding site for both inhibitors. A rotated perspective of the binding site is shown on the right.
-
FIG. 7.Comparison between the binding of naphthyridinones with other RNase H binding inhibitors. (A) Overlay of MK2 (cyan) with the pyrimidal carboxylic acid inhibitor (yellow) (PDB code: 3HYF) (18). On the right is a rotated view showing the overlap of the planes of the metal-coordinating scaffold. (B) Overlay of MK2 (cyan) with beta-thujaplicinol (green) (PDB code: 3IG1) (15) and a rotated view illustrating the overlap of the metal-coordinating scaffold. The Mn2+ ions from the MK2 structure are shown in green; the Mn2+ ions from the other compared structures are shown in gray.
- American Society for Microbiology
