family of proteases present before species divergence. Not surprising, but a nice clean tree.

Protease inhibitors are key components in the chemotherapy of HIV-1 infection. However, despite their success, the appearance of viral mutations routinely compromises their clinical efficacy, creating a constant need for new and better inhibitors. An ideal inhibitor should be able to neutralize the wild type as well as mutants associated with drug resistance with high potency and high selectivity towards human targets, thus minimizing side effects. The evolution of protease inhibitors since 1995 has revealed the basis for extremely high affinity as well as different mechanisms by which inhibitors can escape the deleterious effects of mutations.

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 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 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.