The HIV-1 protease clinical isolate (MDR 769) was overexpressed, purified, and crystallized, and the crystallographic structure solved to 1.86-Å resolution. The MDR 769 crystal structure is the first uncomplexed MDR HIV-1 protease clinical isolate to be characterized, and the sequence contains 10 mutations (distributed throughout the entire molecule) relative to the wild-type sequence. The MDR protease represents a novel drug target for antiretroviral therapy.
Note that the active-site expansion data reported for the MDR isolate 769 are consistent with both the substrate envelope hypothesis (37
; M. Prabu-Jeyabalan, N. M. King, E. Nalivaika, W. R. P. Scott, and C. A. Schiffer, abstract from the abstract from the XIth International HIV Drug Resistance Workshop 2002, Antivir. Ther. 7:
S36, 2002) and thermodynamic measurements of ligand binding (18
). Regarding the substrate envelope hypothesis, Schiffer and coworkers have determined a series of crystal structures of HIV protease-substrate complexes. The authors propose a hypothesis according to which inhibitors that fit within the substrate envelope of HIV-1 protease might be more effective and less susceptible to drug resistance mutations. Microcalorimetric measurements by Freire et al. indicate that drug-resistant mutants lower the affinity of the licensed inhibitors by 2 or 3 orders of magnitude (22
). From the thermodynamic standpoint, the combined effects of the drug-resistant mutations 82 and 84 (in combination with other mutations) involve significant alterations in the enthalpy and entropy changes for inhibitor binding (28
; A. Velazquez-Campoy, V. Sonia, and E. Freire, Abstr. 17th Annu. Meet. Groups Studying Struct. AIDS-Related Systems. Their Application Targeted Drug Design, p. 7, 2003).
The MDR isolate 769 crystal structure reveals several major differences relative to the wild-type structure. First, the V82A and I84V mutations lead to an expansion of the active-site cavity. The loss of a sigma carbon-carbon bond results in an approximate change of 1.5 Å in each of amino acid residues 82, 182, 84, and 184. Since these four residues are in opposite corners of the active sites, there is an approximately 3.0-Å expansion inside the active-site cavity. These are frequently occurring drug resistance mutations, involving a codon change from a longer to a shorter amino acid side chain. The V82A mutation confers resistance to five (indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir) of the six licensed inhibitors, and the I84V mutation confers resistance to six licensed protease inhibitors (including amprenavir). A second difference is the distance increase between residues 50 and 81, as illustrated by the side view of the thicker MDR 769 variant (Fig. ). The widened gap between residues 50 and 81 is in concert with the 82 and 84 mutations and further contributes to active-site expansion of the protease. A third structural difference regards the open flaps of the MDR 769 protease. The 8.44 Å difference between the wild-type structure and the variant structure contributes to a significant widening of the already open flaps of the uncomplexed HIV-1 protease.
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 (more ...)
Our X-ray crystallographic analysis of the two inhibitor complexes of the MDR HIV-1 protease indicates structural changes in several areas. Neither lopinavir nor DMP450 binds to MDR isolate 769 HIV-1 protease in the same way as these two protease inhibitors bind to the wild-type HIV-1 protease (19
) (Fig. and ). There is active-site expansion in the S1 and S1′ pockets due to mutations from longer to shorter amino acid side chains at residues 82 and 84. There are conformational changes in the S2 and S2′ pockets, and there is active-site expansion in the S3 and S3′ pockets. Finally, the water molecule that forms (bridging hydrogen bonds between the flap amino acid residues I50 and I150 and the ligand) is lost in the MDR HIV-1 protease.
The active-site expansion is associated with the in vitro susceptibility data, indicating an up to 100-fold increase in resistance to licensed inhibitors and experimental compounds (33
). For example, the resistance to indinavir of the MDR isolate 769 is increased 63-fold and the sequence contains the indinavir resistance mutations L10I, M46L, I54V, A71V, V82A, I84V, and L90 M. The enlargements at the 82 and 84 regions, 50 and 81 regions, and 50 and 150 regions all contribute to the expansion of the active-site cavity. While other MDR isolates have a phenotype similar to that of the MDR isolate 769 (33
), the active-site expansion for these proteases remains to be examined.
Compensatory mutations from smaller to larger amino acid side chains in the substrate cleavage sites have been reported (8
). For example, the HIV-1 protease I84V mutant contains the compensatory mutation of leucine to phenylalanine at the P1′ position of the p1/p6 cleavage site. Dauber et al. reported an alanine-to-valine change at position P2 of the NC/p1 cleavage site in drug-resistant patient isolates (6
). The authors propose that these mutations might represent a mechanism by which severely compromised, drug-resistant viral strains can increase the fitness levels of these HIV strains. While the protease active site is expanding, the substrate cleavage sites mutate to fill the expanded active-site cavity. These observations are consistent with the active-site expansion model.
To correlate binding of inhibitors to the altered HIV-1 protease, we measured the binding of a water-soluble inhibitor, DMP450, to the MDR HIV-1 protease. SPR (BIACORE) measurements indicate that DMP450 does not bind as tightly to MDR 769 variant as the wild-type protein.
These MDR HIV-1 protease crystal structures will be useful in HIV-AIDS research in a number of ways. For example, the structure could be used for increasingly powerful docking experiments to identify novel protease inhibitors. DOCK software (11
) is designed to find favorable orientations of a ligand in a receptor and has already been used successfully for the design of protease inhibitors with activity against the wild-type HIV-1 protease (7
). The crystal structure of MDR HIV-1 protease will be useful as a template for homology modeling experiments to predict the structures of numerous other MDR HIV-1 variants deposited in the Stanford HIV RT and Protease Sequence Database (16
), in the Los Alamos HIV Drug Resistance Database (34
), and in other HIV sequence databases.
Another possible application of the MDR HIV-1 protease crystal structure is the examination of flap movement by molecular dynamics simulation. Scott and Schiffer reported curling of the flap tips in HIV-1 protease as a mechanism for substrate entry and tolerance of drug resistance (41
). The authors reported that the flaps of HIV-1 protease opened completely during a 10-ns solvated molecular dynamics simulation starting from the unliganded crystal structure. The molecular dynamic simulation experiment with the MDR isolate 769 HIV-1 crystal structure would be very interesting, due in particular to the fact that the 48-G-G-I-G-G-52 sequences in the two monomers of the MDR 769 variant are much further apart compared to the distance in the wild-type flap tips. Hoffman et al. (14a
) reported covariation among positions in HIV-1 protease sequences for 536 protease inhibitor-treated persons. Two different statistical tests indicated linkage between 25 pairs of sites, eight of which involved position 10. Crystal structures such as that of the MDR 769 protease may provide elements for experimental validation of the observation that numerous pairs could interaction within a local environment.
In summary, the MDR isolate 769 structure is the first crystal structure of an uncomplexed HIV-1 MDR protease to be described and is the highest-resolution crystal structure of an uncomplexed HIV-1 protease. The structure reveals an expanded active-site cavity representing an altered receptor. The crystal structures of the MDR 769 protease-inhibitor complexes reveal unexpected binding modes which provide a structural basis for protease inhibitor resistance. Therefore, these crystal structures identify a target for a new class of protease inhibitors designed specifically to inhibit the MDR form of the enzyme.