Visualizing transient events in amino-terminal autoprocessing of HIV-1 protease

Abstract

HIV-1 protease processes the Gag and Gag-Pol polyproteins into mature structural and functional proteins, including itself, and is therefore indispensable for viral maturation. The mature protease is active only as a dimer with each subunit contributing catalytic residues. The full-length transframe region protease precursor appears to be monomeric yet undergoes maturation via intramolecular cleavage of a putative precursor dimer, concomitant with the appearance of mature-like catalytic activity. How such intramolecular cleavage can occur when the amino and carboxy termini of the mature protease are part of an intersubunit beta-sheet located distal from the active site is unclear. Here we visualize the early events in N-terminal autoprocessing using an inactive mini-precursor with a four-residue N-terminal extension that mimics the transframe region protease precursor. Using paramagnetic relaxation enhancement, a technique that is exquisitely sensitive to the presence of minor species, we show that the mini-precursor forms highly transient, lowly populated (3-5%) dimeric encounter complexes that involve the mature dimer interface but occupy a wide range of subunit orientations relative to the mature dimer. Furthermore, the occupancy of the mature dimer configuration constitutes a very small fraction of the self-associated species (accounting for the very low enzymatic activity of the protease precursor), and the N-terminal extension makes transient intra- and intersubunit contacts with the substrate binding site and is therefore available for autocleavage when the correct dimer orientation is sampled within the encounter complex ensemble.

Figure 1. Intermolecular PRE profiles

a, Organization of the Gag-Pol polyprotein,. b–d, Intermolecular PREs observed on U-[²H/¹³C/ ¹⁵N]-labelled ^SFNFPR(D25N) originating from a spin label conjugated to T12C (b), E34C (c) and V82C (d) of ^SFNFPR(D25N) at natural isotopic abundance. Residues broadened beyond detection are denoted by open bars. Error bars represent 1 s.d. Γ₂ rates back-calculated from the structure of the mature dimer (for the core residues 10–94) at populations of 1% and 2% are shown as blue and green lines, respectively. Average Γ₂ rates derived from the top 20 structures of the N_e = 4 simulated annealing calculations at a population of 5% heterodimer are shown as black lines. Grey shaded areas delineate residues that are buried at the dimer interface in the mature protease. e, f, Observed intermolecular PREs originating from the spin label attached to T12C (e) and V82C (f) colour-coded on a ribbon diagram of the mature dimer (spin label attached to the blue subunit). Atomic probability density maps (plotted at a threshold of 10% of maximum) showing the distribution of the spin-label oxygen radicals are shown as red meshes.

Figure 2. Ensemble simulated annealing and the protease mini-precursor encounter complex ensemble

a, PRE Q-factor as a function of ensemble size and population of heterodimer. Dashed line denotes the expected Q-factor when agreement between observed and calculated Γ₂ rates is comparable to the experimental error in the measurements. b, Correlation between observed and calculated Γ₂ rates for N_e = 4 and a heterodimer population of 5%. Q_ee is the ensemble of ensembles average PRE Q-factor for the 20 calculated N_e = 4 ensembles and r the correlation coefficient. Error bars in a and b represent 1 s.d. c, Atomic probability density map (grey mesh, plotted at a threshold of 20% of maximum) showing the distribution of the spin-labelled subunit relative to the isotopically labelled subunit (red ribbon) in the ^SFNFPR(D25N) encounter complexes. The location of the second subunit in the mature dimer is shown as a blue ribbon. d, Orientations in spherical coordinates of the vector joining the centre of masses of the two interacting molecules in the encounter complexes relative to the coordinate system shown in c with the z axis corresponding to the C₂ symmetry axis of the mature dimer. The ϕ,θ angles for the mature dimer are located at the crosshair. e–g, Representative encounter complexes (labelled and denoted by red dots in d) corresponding to the structures with the closest spherical angles (e), the smallest d.r.m.s. (f) and the smallest atomic r.m.s. displacement (g) relative to the mature dimer. The Cα atom of Gly 51 at the tip of the flap is shown as a sphere to guide the eye. The isotopically labelled and spin-labelled subunits are shown in red and grey, respectively; the blue subunit corresponds to the orientation relative to the red subunit seen in the mature dimer. h, Histogram of the d.r.m.s. metric for the N_e = 4 structures (total of 20 × 4 = 80 conformers) at a population of 5% heterodimer.

Figure 3. PRE profiles with spin labels attached at the N- and C termini of the ^SFNFPR(D25N) mini-precursor

a, Intermolecular PREs (red) observed for a 1:1 mixture (0.2 mM each) of N-terminal spin-labelled ^S(C)FNFPR(D25N) at natural isotopic abundance and U-[²H/¹³C/¹⁵N]–^SFNFPR(D25N), and the sum of the inter- and intramolecular PREs (blue) observed for 0.2 mM N-terminal spin-labelled U-[²H/¹³C/¹⁵N]–^S(C)FNFPR(D25N). Residues broadened beyond detection are denoted by open bars. b, Intermolecular PREs observed for a 1:1 mixture (0.2 mM each) of U-[²H/¹³C/¹⁵N]–^SFNFPR(D25N) and C-terminal spin-labelled (at N98C) ^SFNFPR(D25N) at natural isotopic abundance. Grey shaded areas in a and b delineate residues that are buried at the dimer interface in the mature protease. Error bars in a and b represent 1 s.d. c, d, Inter- and intramolecular PREs with Γ₂ rates >10 s⁻¹ colour-coded in red and blue, respectively, onto the molecular surface of the mature protease dimer originating from the N-terminal (c) and the C-terminal (d) spin labels. The intramolecular PRE rates are given by the difference in PRE rates between the blue and red profiles in a. Cartoons of modelled N-terminal (residues −4 to 9) and C-terminal (residues 95–99) regions bearing the spin labels are included in c and d, respectively.