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

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Abstract

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 octapeptide (TFP) and 48 amino acids of the p6pol, separated by a protease cleavage site. The intact precursor (TFP-p6pol-PR) has very low dimer stability relative to that of the mature enzyme and exhibits negligible levels of stable tertiary structure. Thus, the TFR functions by destabilizing the native structure, unlike proregions found in zymogen forms of monomeric aspartic proteases. Cleavage at the p6pol-PR site to release a free N-terminus of protease is concomitant with the appearance of enzymatic activity and formation of a stable tertiary structure that is characteristic of the mature protease as demonstrated by nuclear magnetic resonance. The release of the mature protease from the precursor can either occur in two steps at pH values of 4 to 6 or in a single step above pH 6. The mature protease forms a dimer through a four-stranded β-sheet at the interface. Residues 1–4 of the mature protease from each subunit constitute the outer strands of the β-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.

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Figure 1: Structural organization of Gag-Pol polyprotein in HIV-1.
Figure 2: Protease precursor constructs and expression in E. coli.
Figure 3: Urea denaturation curves for wild type mature protease (□), PR (▪), PRQ () and ΔPR () as determined by measuring changes in enzymatic activity in buffer A at 25 °C.
Figure 4: Time course of the autocatalytic maturation of the precursor a , at pH 4. 0 in 50 mM sodium formate and 0.5 M urea, c, atpH 5.0 in 50 mM sodium acetate and 0.5 M urea and d, at pH 6.5 in 25 mM sodium phosphate buffers at 25 °C.
Figure 5: A proposed model for the steps involved in the maturation of HIV-1 protease.
Figure 6: Inhibition of precursor maturation by RPB inhibitor.
Figure 7: Specific activity as a function of the dimeric enzyme concentration of precursor () and mature PR (▪).
Figure 8: Inhibition of precursorQ maturation.
Figure 9: 1H-15N correlation spectra of a, precursorQ, b, ΔPR and c, ΔPR and mature PRQ in complex with DMP323 (red and blue contours, respectively), in 50 mM sodium acetate buffer, pH 5.2, at 25 °C.

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Acknowledgements

We wish to thank J. Hung for technical assistance, N.T. Nashed for discussions, L.K. Pannell for mass spectroscopic analyses, and H.R. Parikh for contract services. DMP323 was a generous gift from N. Hodge, DuPont Merck Pharmaceutical Company. This research was supported by the Intramural AIDS Targeted Program of the Office of the Director of the National Institutes of Health.

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Correspondence to Angela M. Gronenborn.

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