1. Laboratory of Chemical Physics, Building 5, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA
2. Present address: Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, USA.

Correspondence to: G. Marius Clore¹ Correspondence and requests for materials should be addressed to G.M.C. (Email: mariusc@mail.nih.gov).

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^{1, 2}. The mature protease is active only as a dimer^{3, 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 dimer^{5, 7, 8, 9, 10, 11}, concomitant with the appearance of mature-like catalytic activity^{7, 9}. How such intramolecular cleavage can occur when the amino and carboxy termini of the mature protease are part of an intersubunit -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^{5, 12}. Using paramagnetic relaxation enhancement, a technique that is exquisitely sensitive to the presence of minor species^{13, 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.

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