Autoprocessing of HIV-1 protease is tightly coupled to protein folding

In the Gag-Pol polyprotein of HIV-1, the 99-amino acid protease is flanked at its N-terminus by a transframe region (TFR) composed of the transframe o ctapeptide (TFP) and 48 amino acids of the p6(pol), separated by a protease cleavage site. The intact precursor (TFP-p6(pol)-PR) has very low dimer st ability relative to that of the mature enzyme and exhibits negligible level s of stable tertiary structure. Thus, the TFR functions by destabilizing th e native structure, unlike proregions found in zymogen forms of monomeric a spartic proteases. Cleavage at the p6(pol)-PR site to release a free N-term inus of protease is concomitant with the appearance of enzymatic activity a nd formation of a stable tertiary structure that is characteristic of the m ature protease as demonstrated by nuclear magnetic resonance. The release o f the mature protease from the precursor can either occur in two steps at p H values of 4 to 6 or in a single step above pH 6. The mature protease form s a dimer through a four-stranded beta-sheet at the interface. Residues 1-4 of the mature protease from each subunit constitute the outer strands of t he beta-sheet, and are essential for maintaining the stability of the free protease but are not a prerequisite for the formation of tertiary structure and catalytic activity. Our experimental results provide the basis for the model proposed here for the regulation of the HIV-1 protease in the viral replication cycle.