Structural basis of the allosteric inhibitor interaction on the HIV-1 reverse transcriptase RNase H domain

Abstract

HIV-1 reverse transcriptase (RT) has been an attractive target for the development of antiretroviral agents. Although this enzyme is bi-functional, having both DNA polymerase and ribonuclease H (RNH) activities, there is no clinically approved inhibitor of the RNH activity. Here, we characterize the structural basis and molecular interaction of an allosteric site inhibitor, BHMP07, with the wild-type (WT) RNH fragment. Solution NMR experiments for inhibitor titration on WT RNH showed relatively wide chemical shift perturbations, suggesting a long-range conformational effect on the inhibitor interaction. Comparisons of the inhibitor-induced NMR chemical shift changes of RNH with those of RNH dimer, in the presence and absence of Mg(2+) , were performed to determine and verify the interaction site. The NMR results, with assistance of molecular docking, indicate that BHMP07 preferentially binds to a site that is located between the RNH active site and the region encompassing helices B and D (the 'substrate-handle region'). The interaction site is consistent with the previous proposed site, identified using a chimeric RNH (p15-EC) [Gong et al. (2011) Chem Biol Drug Des 77, 39-47], but with slight differences that reflect the characteristics of the amino acid sequences in p15-EC compared to the WT RNH.

Figure 1

Isolation of dimer and monomer fractions of RNH in solution. Overlay of elution profiles of the MALS for the WT RNH Fraction #1 (gray) and Fraction #2 (black). The molecular masses calculated from the refractive index were 22.47 kDa and 14.4 kDa for Fractions #1 and #2, respectively. An electrophoretic native gel for the two fractions is inserted in the graph, showing the different migration patterns in the two fractions. All experiments were performed in the absence of Mg²⁺.

Figure 2

Difference in NMR chemical shifts between dimer and monomer fractions of RNH. (A) Overlay of ¹H-¹⁵N HSQC spectra of WT RNH dimer (Fraction #1, red) and the monomer (Fraction #2, black). Both experiments were performed in the absence of Mg²⁺. (B) Normalized quadratically weighed ¹H, ¹⁵N backbone amide resonance shift differences (Δδ, cf. Eq. 1) between the monomer and dimer forms are shown relative to sequence residue. Prolines (P) and unassigned/undetected backbone NH amide groups (*) in either form are indicated; the “substrate-handle region” is denoted by a solid arrow (residues 80 to 103). A schema of secondary structure elements is included for reference [helix C is an insert that occurs only in E. Coli RNAse H (26)]. Data were recorded at 600 MHz (¹H), at 20°C and pH 7.0.

Figure 3

Dimer interface mapped onto RNH crystal structures. Two RNH chains (PDB: 3K2P) are shown in ribbon representation and colored, respectively, in green and cyan. Residues exhibiting significant chemical shift changes ($(\Delta \delta \ast \ast \ast \overline{\Delta \delta }+\sigma$, i.e. greater than one standard deviation from the average response in (Figure 2) between the dimer and the monomer are highlighted in orange; undetected/unassigned residues are shaded in gray. Purple spheres, shown in dots, delineate the position of manganese ions in the active site; the side chains of the metal coordinating residues (D20, E55, D74, and D126) are indicated by yellow sticks. (A) Interface mapped onto one RNH subunit, the substrate-handle region (residues from 80 to 103) is outlined (red dashed circle). (B) Illustration of the kinetically trapped dimer interface via a coinciding (see text) crystallographic dimer. Relative to panel (A), the view in (B) is rotated by −50° around the z-axis (see inset). Note that although the crystal structure contains manganese ions as coordinating ions, magnesium was used for NMR experiments to avoid paramagnetic effects by manganese.

Figure 4

NMR chemical shift perturbation induced by BHMP07 interaction with the RNH monomer in the absence (A) or presence (B) of 20 mM Mg²⁺; and with the RNH dimer in the absence (C) or presence (D) of 20 mM Mg²⁺. Normalized, quadratically weighed ¹H, ¹⁵N backbone amide resonance shifts induced by BHMP07 (Δδ, cf. Eq. 1) are shown relative to sequence residue. Prolines (P) and backbone NH amide groups whose chemical shift changes could not be tracked (*) are indicated. A schema of secondary structure elements (26) is included for easy reference; the “substrate-handle region” (58) is denoted by a solid arrow (residues 80 to 103). Data were recorded at 600 MHz (¹H), at 20°C and pH 7.0.

Figure 5

Interaction of BHMP07 with RNH monomer mapped onto the crystallographic structure (PDB code, 3K2P, chain A). (A) Ten BHMP07 conformers with the highest docking scores at Site I, Site II, and Site III (see text). RNH residues (ribbon cartoon) that exhibit significant NMR chemical shift changes upon addition of 1.5× excess of BHMP07 (Figure 4A) are highlighted: $\Delta \delta \ast \ast \ast \overline{\Delta \delta }+\sigma \left(\text{red}\right)$; $\Delta \delta \ast \ast \ast \overline{\Delta \delta }\left(\text{orange}\right)$; $\Delta \delta \ast \ast \ast \ast \overline{\Delta \delta }\left(\text{green}\right)$; undetected/unassigned residues are colored gray. The sampled BHMP07 poses (blue sticks, nonpolar hydrogens are omitted) were predicted via NMR-based molecular docking in the absence of divalent ions (see text). (B) Minimum energy conformer of BHMP07 docked at Site II on RNH (gray ribbon). The inhibitor pose is presented as the skeletal formula with superposed van der Waals contact surface, colored by partial atomic charges from −0.4 a.u. (red) to +0.4 a.u. (blue). Interacting RNH residues (Figure S7) are labeled and shown in stick representation with carbon, nitrogen and oxygen atoms denoted in gray, blue and red, respectively; predicted intermolecular hydrogen bonds are indicated (cyan dashes). Neighboring (< 11 Å) RNH backbone H and N atoms that exhibit significant chemical shift changes ($(\Delta \delta \ast \ast \ast \overline{\Delta \delta })$) are shown (yellow van der Waals spheres). Relative to panel (A), the view in (B) is rotated by 15°, −10° and −25° about the x, y and z-axes, respectively (see inset).