Unfolding the HIV-1 reverse transcriptase RNase H domain--how to lose a molecular tug-of-war

Abstract

Formation of the mature HIV-1 reverse transcriptase (RT) p66/p51 heterodimer requires subunit-specific processing of the p66/p66' homodimer precursor. Since the ribonuclease H (RH) domain contains an occult cleavage site located near its center, cleavage must occur either prior to folding or subsequent to unfolding. Recent NMR studies have identified a slow, subunit-specific RH domain unfolding process proposed to result from a residue tug-of-war between the polymerase and RH domains on the functionally inactive, p66' subunit. Here, we describe a structural comparison of the isolated RH domain with a domain swapped RH dimer that reveals several intrinsically destabilizing characteristics of the isolated domain that facilitate excursions of Tyr427 from its binding pocket and separation of helices B and D. These studies provide independent support for the subunit-selective RH domain unfolding pathway in which instability of the Tyr427 binding pocket facilitates its release followed by domain transfer, acting as a trigger for further RH domain destabilization and subsequent unfolding. As further support for this pathway, NMR studies demonstrate that addition of an RH active site-directed isoquinolone ligand retards the subunit-selective RH' domain unfolding behavior of the p66/p66' homodimer. This study demonstrates the feasibility of directly targeting RT maturation with therapeutics.

Figure 1.

Stability of RH monomer and dimer. (A) Incubation of purified RHmnel monomer for a period of ∼20 h at 25°C yielded only the monomer (red curve); incubation of purified RHmnel monomer at 37°C over a similar period yielded a monomer – dimer mixture (blue curve). The position of the purified dimer corresponds to the yellow curve. Fractions were separated on a Superdex 200 10/300 column. (B) Purified samples of RHΔNT monomer (gold curve) or dimer (green) rapidly reloaded on the Superdex 200 column HiLoad 26/60, eluted as monomer-dimer mixtures. Chromatogrpahic separations were run at 4°C using 50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA as the running buffer.

Figure 2.

Crystal structure of domain swapped Rnase H (RH). (A) The unit cell contains two pairs of domain swapped dimers: RH1-RH2 and RH3-RH4, in an approximately perpendicular orientation (color coding: green+yellow; cyan+magenta). Each globular structure contains elements β1-β2-β3-αA-β4-αB derived from a single peptide chain, and αD and β5 derived from its domain swapped partner. The circled regions correspond to the β-sheet formed from Trp535 residues that link the two domain swapped dimers. (B) Expanded ribbon representation of a domain swapped dimer (green, yellow) overlaid with an RH domain monomer structure (gray, pdb: 3K2P, 41). The β1-β2-β3-αA-β4 from molecule RH1 (green) and the αD-β5 structural elements from molecule RH2 (yellow) agree closely with the monomer, however, αB exhibits a relative change in orientation. (C) Expanded view of the monomer-dimer structural variation in the Tyr427 binding pocket. The Tyr427 sidechain remains H-bonded to Ile506 in both structures, however in RH2, αB is tilted even further so that this H-bond interaction is not present.

Figure 3.

¹H-¹³C HMQC spectra of [¹³CH₃-Ile]RH constructs. (A) ¹H-¹³C HMQC spectra of [¹³CH₃-Ile]RHmnel monomer (black) overlaid with ¹H-¹³C HMQC spectra of [¹³CH₃-Ile]RHmnel dimer (red). (B) ¹H-¹³C HMQC spectra of [¹³CH₃-Ile]RHΔNT monomer (black) overlaid with ¹H-¹³C HMQC spectra of [¹³CH₃-Ile]RHΔNT dimer (red). As a consequence of faster monomer-dimer interconversion rate of RHΔNT, purified monomer or dimer samples show some contamination with the dimer and monomer resonances, respectively. As a result of the absence of Tyr427, the Ile526 and Ile522 resonances are both shifted to lower δ¹³C and δ¹H values, so that the Ile526 resonances overlap the Ile482 monomer resonance, and the Ile556 resonances partially overlap the Ile522 monomer peak. The spectra of [¹³CH₃-Ile]RHΔNT contain several additional small resonances indicated with an asterisk that have not been assigned. Spectra were obtained at 25°C and samples were in Tris-HCl-d11 in D₂O (pD 7.5), 100 mM KCl.

Figure 4.

Shortening of the B-D hinge loop. (A) Ribbon representations of the αB-αD regions of one molecule of the dimer (green) and the RH monomer (gray) in which the αD helices have been aligned. As is apparent from the figure, several residues that are part of the B-D loop in the monomer are incorporated into an extended αD in the dimer. The reduction in length of the dimer B-D linker is also apparent in Figure 2B. (B) Ribbon diagram overlay of the RH monomer (gray, pdb: 3K2P) with the domain swapped dimer illustrating the change in length of αD and the B-D linker.

Figure 5.

H/D exchange behavior of RH and RHΔNT. (A) ¹H-¹⁵N HSQC spectrum of RHmnel obtained during the first 40-min period after replacement of the H₂O buffer with D₂O buffer (black spectrum), overlaid with a spectrum obtained during the 40-min period starting at 6.6 h after buffer replacement (red spectrum). (B) ¹H-¹⁵N HSQC spectra of RHΔNT obtained during the first 40-min accumulation period after buffer exchange (black spectrum) and the second 40-min period (green spectrum). The amides protons of the cleavage site residues F440 and Y441 in RHmnel are readily observed after 6.6 h, while in RHΔNT they have been fully exchanged. Exchange studies were performed at 25 °C in 25 mM Tris-HCl-d11 in D₂O (pD 7.5), 100 mM KCl.

Figure 6.

Monomer-dimer transition state diagram. (A) The monomer-dimer interconversion is suggested to proceed via a transition state that loosely resembles either molecule of the dimer. As outlined in the text, the monomer stability is proposed to be slightly compromised by intrinsic structural features, particularly the lack of constraints on αB orientation and the truncation of αD. Loss of several N-terminal residues resulting from the inter-domain tug-of-war further destabilizes the monomer. (B) Overlaid ribbon diagrams for residues 427–506 (i.e. showing β1- β2- β3-αA-β4-αB) in the monomer (gray) and each molecule of the domain swapped dimer (green, yellow). The molecules were aligned from residues 427–495 to illustrate the variable positioning of helix αB.

Figure 7.

Effect of an RH active site-directed ligand on the H/D exchange kinetics of RHΔNT and on the maturation behavior of the p66 homodimer. (A) ¹H-¹⁵N HSQC spectra of RHΔNT in the presence of 5 mM MgCl₂ and 1.5 mM HIQ after an H₂O/D₂O buffer change, obtained during the time periods indicated. The red spectrum in the lower right hand panel was obtained over the 760–800 min accumulation period after changing to the D₂O buffer. Other conditions as in Figure 5B. (B) Time dependent changes of connection′ (Ile329 and Ile375) and RH′ domain (Ile434) Ile resonances in the [¹³CH₃-Ile,U-²H]p66/[¹³CH₃-Ile,U-²H]p66′ homodimer in the presence of a Mg-HIQ RH-directed ligand. The ‘E’ and ‘C’ resonance labels indicate assignments to the extended and compact polymerase conformations that are present in the p66 and p66′ subunits, respectively. Conditions favoring dimerization were introduced at t = 0, and the samples also contained 6 mM MgCl₂, 1 mM HIQ in 25 mM Tris HCl-d11, pD 7.5, 100 mM KCl and 0.02% NaN₃. Each spectrum corresponds to a sequential 5.5 h accumulation period. Samples were run on an Agilent 800 MHz NMR spectrometer at 35°C, under conditions identical to those reported previously without the addition of Mg-HIQ (23).