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Visualizing transient events in amino-terminal autoprocessing of HIV-1 protease
Chun Tang, John M. Louis1, Annie Aniana, Jeong-Yong Suh & G. Marius Clore
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 
-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.
