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. 2015 Aug;83(8):1526-38.
doi: 10.1002/prot.24843. Epub 2015 Jul 1.

Structural Integrity of the Ribonuclease H Domain in HIV-1 Reverse Transcriptase

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Free PMC article

Structural Integrity of the Ribonuclease H Domain in HIV-1 Reverse Transcriptase

Ryan L Slack et al. Proteins. .
Free PMC article

Abstract

The mature form of reverse transcriptase (RT) is a heterodimer comprising the intact 66-kDa subunit (p66) and a smaller 51-kDa subunit (p51) that is generated by removal of most of the RNase H (RNH) domain from a p66 subunit by proteolytic cleavage between residues 440 and 441. Viral infectivity is eliminated by mutations such as F440A and E438N in the proteolytic cleavage sequence, while normal processing and virus infectivity are restored by a compensatory mutation, T477A, that is located more than 10 Å away from the processing site. The molecular basis for this compensatory effect has remained unclear. We therefore investigated structural characteristics of RNH mutants using computational and experimental approaches. Our Nuclear Magnetic Resonance and Differential Scanning Fluorimetry results show that both F440A and E438N mutations disrupt RNH folding. Addition of the T477A mutation restores correct folding of the RNH domain despite the presence of the F440A or E438N mutations. Molecular dynamics simulations suggest that the T477A mutation affects the processing site by altering relative orientations of secondary structure elements. Predictions of sequence tolerance suggest that phenylalanine and tyrosine are structurally preferred at residues 440 and 441, respectively, which are the P1 and P1' substrate residues known to require bulky side chains for substrate specificity. Interestingly, our study demonstrates that the processing site residues, which are critical for protease substrate specificity and must be exposed to the solvent for efficient processing, also function to maintain proper RNH folding in the p66/p51 heterodimer.

Keywords: HIV; NMR; enzyme; maturation; protein; proteolysis; virus.

Figures

Figure 1
Figure 1
(A) Domain organization of RT, illustrating p66 (below) and p51 (above); the location of the protease processing site in p66 is indicated in yellow. Ribbon representation of the structures of (B) the RNH domain and (C) the part of the RNH domain, highlighting α-helix A and the β-sheet that includes β−strand 1, 2, and 3. In (B) and (C), the p51-RNH processing site is shown by yellow ribbon, and side chains of F440 and T477 are shown by pink color sticks.
Figure 2
Figure 2
1H-15N HSQC spectra of the RNH Wild Type (WT) and mutants. (A) The RNH WT spectrum exhibits well disperse and sharp cross-peaks, characteristic of a well folded protein in solution (blue). (B) Superimposition of the RNHF440A mutant (red) and the RNHF440A/T477A mutant (black). (C) Superimposition of the RNHE438N mutant (red) and the RNHE438N/T477A mutant (black). All the spectra were obtained on a Bruker AVANCE 600 Spectrometer at 20 °C.
Figure 3
Figure 3
Gel electrophoresis profiles in (A) denatured and (B) native conditions, and (C) SEC-MALS UV profiles (solid) and molecular mass profiles (dashed) of RNHE438N (red) and RNHE438N/T477A (black). In (A), L indicates a molecular weight size marker. In (A) and (B), Lane 1: WT RNH, Lane 2: RNHF440A, Lane 3: RNHF440A/T477A, Lane 4: RNHE438N, Lane 5: RNHE438N/T477A. In (C) the average molecular mass of the eluted RNHE438N peak was determined to be 22.1 kDa, and those of the eluted RNHE438N/T477A peaks at 10.5 and 12.1 ml were determined to be 14.88 kDa and 27.25 kDa, respectively.
Figure 4
Figure 4
Global assessment of sampling and structural diversity via MD simulations. Histograms of the backbone RMSD (Å) of structures obtained in the 1–100 ns (solid line) and of 100–200 ns (symbols) MD simulations for (A) WT, (B) RNHF440A, and (C) RNHE438N (blue line, for WT; red lines, F440A and E438N mutants without T477A; black lines, with T477 mutation).
Figure 5
Figure 5
Hydrogen bonding networks observed in MD simulations. (A) Side chain orientations in the WT RNH around (A) residue 440 (pink), and residue 477 in (B) the WT and (C) the RNHT477A. In (A), dashed lines indicate salt bridge network that involves E438. In (B) and (C), black lines indicate hydrogen bonds that are frequently observed in the WT RNH and the RNHT477A, respectively (see Table 4).
Figure 6
Figure 6
Structural geometry for investigating crosstalk between sites 440 and 477 via MD. The orientation of α-helix A (Cα atoms) relative to the inertial axes of the beta sheet (β strands 1–3, backbone atoms listed in Table 4 is characterized by angles (A) θ and (B) φ. The average θ angle resulting from simulation of (C) RNHWT was 7.7 ± 1.9°. (D) The angle θ decreased slightly in the RNHF440A and RNHE438N mutants (red arrow) under MD simulation. (E) By contrast, RNHF440A/T477A and RNHE438N/T477A resulted in increased θ values. In (C) – (E), schematically, the thick black arrow represents the position of the β sheet. The blue solid and dashed arrows represent the position of α-helix A and the position of the β sheet inertial axis, respectively, for the RNHWT simulation. In (D) and (E), the red arrows indicate the position of α-helix A for the indicated simulations. In (D), because the simulation was not converged, the change of the angle is likely a transient effect of the conformational change within the 200 ns simulation. In (E), because the size of residue 477 decreases, the starting position of the helix is drawn in the cartoon differently from that in (D).
Figure 7
Figure 7
Prediction of sequence tolerance for the protein folding at residues (A) 438, (B) 440, (C) 441 and (D) 477 for RNH WT (filled bars) and RNHT477A (open bars) coordinates (see the Materials and Methods).

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