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

The HIV-1 protease enzyme is indispensable for viral maturation, making it a major potential target of anti-HIV therapy. Its role is to split newly formed Gag and Gag-Pol polyproteins to produced finished structural and functional proteins — itself included. The early events in the autoprocessing of HIV-1 protease, in which a precursor dimer is thought to undergo intramolecular cleavage, have now been visualized using NMR spectroscopy and paramagnetic relaxation enhancement. This reveals that although primarily monomeric, the protease is also present as transient encounter complexes that occupy a wide range of orientations relative to the mature dimer. The N-terminal region makes transient intra- and intersubunit contacts with the substrate binding site, allowing autocleavage to occur when the correct dimer orientation is sampled by the encounter complex. HIV-1 protease processes the Gag and Gag-Pol polyproteins into mature structural and functional proteins. The mature protease is only active as a dimer, with catalytic residues contributed by each subunit. The precursor of the active protease undergoes 'maturation' via the intramolecular cleavage of a dimeric species, but it is not clear how this cleavage reaction occurs. The early events in N-terminal auto-processing were visualized using NMR spectroscopy, and it was determined that the precursor forms transient, lowly populated dimeric encounter complexes that occupy a wide range of orientations relative to the mature dimer. The N-terminal region makes transient intra- and intersubunit contacts with the substrate binding site, enabling auto-cleavage to occur when the correct dimer orientation is sampled by the encounter complex ensemble. HIV-1 protease processes the Gag and Gag-Pol polyproteins into mature structural and functional proteins, including itself, and is therefore indispensable for viral maturation1,2. The mature protease is active only as a dimer3,4,5 with each subunit contributing catalytic residues6. The full-length transframe region protease precursor appears to be monomeric yet undergoes maturation via intramolecular cleavage of a putative precursor dimer5,7,8,9,10,11, concomitant with the appearance of mature-like catalytic activity7,9. How such intramolecular cleavage can occur when the amino and carboxy termini of the mature protease are part of an intersubunit β-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 precursor5,12. Using paramagnetic relaxation enhancement, a technique that is exquisitely sensitive to the presence of minor species13,14,15,16, 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.