Mutational and Structural Studies Aimed at Characterizing the Monomer of HIV-1 Protease and Its Precursor

Rieko Ishima,Dennis A Torchia,John M Louis

doi:10.1074/jbc.m701304200

Abstract

An experimental protocol for folding the mature human immunodeficiency virus-1 (HIV-1) protease is presented that facilitates NMR studies at a low protein concentration of approximately 20 micoM. Under these conditions, NMR spectra show that the mature protease lacking its terminal beta-sheet residues 1-4 and 96-99 (PR(5-95)) exhibits a stable monomer fold spanning the region 10-90 that is similar to that of the single subunit of the wild-type dimer and the dimer bearing a D25N mutation (PR(D25N)). Urea-induced unfolding monitored both by changes in (1)H-(15)N heteronuclear single quantum correlation spectra and by protein fluorescence indicates that although PR(5-95) monomer displays a transition profile similar to that of the PR(D25N) dimer (50% unfolded (U(50)) = approximately 1.9 M), extending the protease with 4 residues (SFNF) of its N-terminally flanking sequence in the Gag-Pol precursor ((SFNF)PR(D25N)) decreases the stability of the fold (U(50) = approximately 1.5 M). Assigned backbone chemical shifts were used to elucidate differences in the stability of the PR(T26A) (U(50) = 2.5 M) and (SFNF)PR(D25N) monomers and compared with PR(D25N/T26A) monomer. Discernible differences in the backbone chemical shifts were observed for N-terminal protease residues 3-6 of (SFNF)PR(D25N) that may relate to the increase in the equilibrium dissociation constant (K(d)) and the very low catalytic activity of the protease prior to its autoprocessing at its N terminus from the Gag-Pol precursor.

Highlights

The HIV-12 genome encodes a protease as part of the large Gag-Pol precursor
Previous studies using a mini-precursor, in which the protease is flanked by sequences corresponding to the cleavage sites at its termini, showed that the processing at the N terminus of the protease, which is concomitant with the appearance of catalytic activity, precedes the C-terminal cleavage (8 –10)
Solution NMR studies of the uninhibited dimeric protease are feasible for a limited period of time only by using an optimized PR construct, to limit autoproteolysis, and by conducting experiments at pH 5.8, which is above the optimal pH for catalytic activity

Summary

Introduction

The HIV-12 genome encodes a protease as part of the large Gag-Pol precursor. Like all retroviral proteases, HIV-1 protease is active only as a homodimer. NMR spectra show that the mature protease lacking its terminal ␤-sheet residues 1– 4 and 96 –99 (PR5–95) exhibits a stable monomer fold spanning the region 10 –90 that is similar to that of the single subunit of the wildtype dimer and the dimer bearing a D25N mutation (PRD25N).

Results

Conclusion