Expression, purification and preliminary X-ray crystallographic studies of the human immunodeficiency virus 1 subtype C protease

Abstract

Crystals of the human immunodeficiency virus 1 (HIV-1) subtype C protease (PR) complexed with the clinically used inhibitors indinavir (IDV) and nelfinavir (NFV) have been grown in the monoclinic space group P2(1), with mean unit-cell parameters a = 46.7 (+/-0.1), b = 59.8 (+/-0.3), c = 87.0 (+/-0.4) A, beta = 95.2 (+/-0.5) degrees. The crystals of both complexes have been shown to diffract X-rays to 2.3 A resolution. The diffraction data for the subtype C PR complexes with IDV and NFV were subsequently processed and reduced, with overall R(sym) values of 8.4 and 11.4%, respectively. Based on the unit-cell volumes, molecular-replacement results and packing considerations, there are two protease homodimers per crystallographic asymmetric unit in each of the complexes. The data were initially phased using a model based on the crystal structure of HIV-1 subtype B PR; the structures have been determined and further refinement and analysis are in progress. These structures and subsequent studies with other inhibitors will greatly aid in correlating the amino-acid variation between the different HIV PRs and understanding their differential sensitivity and resistance to current drug therapy.

Figure 1

(a) Pairwise sequence alignment of HIV-1 subtype B PR LAI strain (designated B) and subtype C PR (designated C). (b) Cartoon representation of HIV-1 subtype B and C PRs. The naturally occurring polymorphisms in subtype C PR, represented as red spheres, are superimposed onto the crystal structure of HIV-1 subtype B PR (black ribbon). The mutations that block the self-cleavage sites are shown in gray. Amino-acid positions are as numbered.

Figure 2

HIV-1 subtype C PR ribbon packing diagram in the P2₁ crystal lattice viewed down the c axis. There are four subtype C PR homodimers in the unit cell (shown in red and light red and in green and light green). The box depicts the unit cell. The model shown is based on data from subtype C PR complexed with IDV and is the same for the NFV complex.