Crystal structures of a multidrug-resistant human immunodeficiency virus type 1 protease reveal an expanded active-site cavity

Abstract

The goal of this study was to use X-ray crystallography to investigate the structural basis of resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors. We overexpressed, purified, and crystallized a multidrug-resistant (MDR) HIV-1 protease enzyme derived from a patient failing on several protease inhibitor-containing regimens. This HIV-1 variant contained codon mutations at positions 10, 36, 46, 54, 63, 71, 82, 84, and 90 that confer drug resistance to protease inhibitors. The 1.8-angstrom (A) crystal structure of this MDR patient isolate reveals an expanded active-site cavity. The active-site expansion includes position 82 and 84 mutations due to the alterations in the amino acid side chains from longer to shorter (e.g., V82A and I84V). The MDR isolate 769 protease "flaps" stay open wider, and the difference in the flap tip distances in the MDR 769 variant is 12 A. The MDR 769 protease crystal complexes with lopinavir and DMP450 reveal completely different binding modes. The network of interactions between the ligands and the MDR 769 protease is completely different from that seen with the wild-type protease-ligand complexes. The water molecule-forming hydrogen bonds bridging between the two flaps and either the substrate or the peptide-based inhibitor are lacking in the MDR 769 clinical isolate. The S1, S1', S3, and S3' pockets show expansion and conformational change. Surface plasmon resonance measurements with the MDR 769 protease indicate higher k(off) rates, resulting in a change of binding affinity. Surface plasmon resonance measurements provide k(on) and k(off) data (K(d) = k(off)/k(on)) to measure binding of the multidrug-resistant protease to various ligands. This MDR 769 protease represents a new antiviral target, presenting the possibility of designing novel inhibitors with activity against the open and expanded protease forms.

FIG. 1.

Structural comparison of HIV-1 wild-type protease and the MDR isolate 769 clinical variant. C_α superposition of the wild-type HIV-1 protease (green) and the MDR 769 HIV-1 protease (red). The green and red side chains represent the catalytic residue at position 25. The blue spheres represent changes in MDR 769 from the wild type.

FIG. 2.

Close-up view of MDR isolate 769 and wild-type HIV-1 protease active sites. (A) The ribbon diagram of the wild-type protease (green) and MDR 769 protease (red) indicates the side chain difference between the active-site region by the V82A and I84V mutations in the wild type and the MDR 769 protease variant. The volume in the active site has increased not only as a result of dimer flap movements but also as a result of side chain shrinkage in the individual monomers. (B) Stereo diagram of the 2F_o-F_c electron density map for the residue range 81 through 85 in the MDR 769 protease. This electron density map is representative throughout the molecule.

FIG. 3.

HIV-1 protease structural comparison of MDR isolate 769 and the wild type complexed with DMP450. C_α traces of the wild-type HIV-1 protease and the DMP450 molecule are in green, and the MDR 769 and its cognate DMP450 are shown in red.

FIG. 4.

DMP450 electron density map. A 2F_o-F_c electron density map for the inhibitor DMP450 bound to MDR isolate 769 at the protease active site is shown. Colors for the ball-and-stick model are as follows: carbon is shown in gray, nitrogen is shown in black, and oxygen is shown in red.

FIG. 5.

HIV-1 protease structural comparison of MDR isolate 769 and the wild type complexed with lopinavir. C_α traces of the wild-type HIV-1 protease and the lopinavir molecule are shown in green. The MDR 769 protease variant and its respective lopinavir molecule are shown in red.

FIG. 6.

Lopinavir electron density map. A 2F_o-F_c electron density map for the inhibitor lopinavir bound to the MDR isolate 769 protease is shown. Colors for the ball-and-stick model are as follows: carbon is shown in gray, nitrogen is shown in black, and oxygen is shown in red.

FIG. 7.

Molecular recognition of lopinavir by the MDR isolate 769 protease variant. The van der Waals contacts of the protease with lopinavir are represented by the purple residue side chains, while the gold residue side chain represents the hydrogen bond to the inhibitor. The lopinavir molecule in the protease active site is shown in blue.

FIG. 8.

SPR measurements of DMP450 binding to the wild-type (A) and MDR 769 (B) HIV-1 proteases. The proteases were coupled to a CM5 sensor chip surface, and binding of DMP450 at concentrations of 1 nM (lowest curve), 10 nM, 100 nM, and 1,000 nM (highest curve) was examined. Nonspecific binding to a control (uncoupled) sensor surface was determined for each concentration and subtracted from the data presented. The x axis represents time in seconds, and the y axis represents response units. The start and end times of DMP450 injection are indicated.

FIG. 9.

A comparison of electrostatic potential surface diagrams of the wild-type HIV-1 protease monomer (A) and the MDR isolate 769 HIV-1 protease monomer (B). Red represents van der Waals surface regions that are negatively charged, while blue represents van der Waals surface regions that are positively charged. In panel B there is a marked increase in distance between amino acid residues 50 and 81.