Although the majority of HIV-1 patients are infected with non-B forms of the virus, molecular studies have been carried out predominantly with clade B variants. The AE protease has several polymorphisms that are associated with inhibitor resistance in clade B. AE also shows altered patterns of drug resistance to NFV. We have performed detailed studies to determine the effects of sequence polymorphisms on enzyme structure, activity, and inhibitor binding. These analyses led to a structural rationalization for the altered pathways for drug resistance.
AE-WT protease has an inherently weaker affinity for NFV and DRV than that of B-WT, as is evident from the thermodynamic data (Table ). The weaker affinity observed for NFV is consistent with previously published data for another AE protease variant (3
), as well as for clade A protease (42
), which is closely related. The inherent weaker affinity for NFV likely allows the AE protease to gain resistance to NFV through a single nonactive-site substitution, N88S. The clade B protease, in contrast, which has a relatively stronger affinity for NFV, requires a combination of an active-site mutation (D30N) and a nonactive-site mutation (N88D) to gain NFV resistance. The ability of the AE-N88S protease to maintain affinity for substrates is evident from our enzyme kinetics data (Table ), in which the Km
value for AE-N88S was comparable to that of AE-WT and B-WT protease. The Km
value for clade B-D30N/N88D, on the other hand, was significantly worse than that of the B-WT, likely reflecting the effect of the altered active site.
As an active-site residue, Asp30 plays a key role in substrate recognition by interacting with substrates through side chain-mediated hydrogen bonds with the MA-CA, CA-p2, p1-p6, and p2-NC cleavage sites (36
). Therefore, as is evident from our enzyme kinetics data, the D30N/N88D mutations in clade B will likely affect substrate binding and processing. Several studies have observed substrate coevolution in instances in which the protease mutates active-site residues in order to confer inhibitor resistance (22
). However, since the AE-N88S protease variant has no active-site mutations, the enzyme retains the ability to effectively recognize substrates while conferring NFV resistance. Therefore, the presence of the N88S substitution in AE protease is unlikely to induce coevolution of the viral substrates in order to maintain effective enzymatic activity.
Despite having Km
values that were comparable to that of B-WT protease, both AE-WT and AE-N88S had significantly lower catalytic turnover rates (kcat
) than that of the B-WT protease (Table ). As a result, the catalytic efficiency of the AE variants is lower than that of the B-WT protease. The lower turnover rates of the AE variants could be a direct result of the reduced flexibility of the flap hinge (residues 33 to 39) and core regions (residues 16 to 22) of the protein. Molecular dynamics studies have revealed that hydrophobic sliding of the core region facilitates substrate binding through the opening of the active site (9
). The unique hydrogen bonds observed between the flap hinge and the core in the AE variants alter movement of the core, thus impacting the ability of the active site to open up for substrate binding and product release. Based on our enzyme kinetics data, this altered flexibility of the flap hinges in the AE variants has little effect on substrate binding but rather affects the catalytic step of the reaction by slowing down product release.
The higher vitality value observed for AE-WT with NFV provides supporting evidence for the reduction in the efficacy of NFV against the AE protease compared with that of clade B (Table ). This result is consistent with previous vitality calculations for the clade A protease (42
). In addition, these results further highlight the idea that background polymorphic sequence variations in the AE protease can affect the potency of NFV. The suboptimal efficacy of NFV against the AE-WT protease likely permits a nonactive-site variant, AE-N88S, to emerge over variants with active-site mutations to effectively confer resistance to NFV.
The impact on other inhibitors, however, is complex. APV and DRV are chemically very closely related compounds, and similar susceptibility and resistance patterns have been observed for these two inhibitors (31
). However, this pattern is not evident for this series of resistant variants. Both the N88S mutation in the AE and the D30N/N88D mutations in the clade B proteases result in hypersusceptibility to APV. Similar results have been observed also for a B-N88S protease variant (24
). In contrast, the same substitutions in the protease give rise to even greater resistance to DRV. However, since DRV presents a greater genetic barrier to resistance than APV (33
), the in vivo
implications of weaker affinity for DRV in the AE variants are likely negligible. Indeed, our calculated vitality values indicate that DRV maintains its potency against the AE variants despite having a weaker affinity for AE-WT and AE-N88S relative to clade B protease.
A close look at the NFVB-WT
protease complex reveals an important interaction between the Asp30 residue side chain and the inhibitor bound in the active site. (PDB code 3EKX) (Fig. ). One of the side chain oxygen atoms of Asp30 forms a direct hydrogen bond with the O38 atom of NFV. Our crystal structures of the NFV-resistant variants show that N88S in AE and N88D in clade B have the ability to interact with residue 30 and orient it away from the active site (Fig. ) and thereby disrupt the interaction between residue 30 and the inhibitor. These structural observations are similar to interpretations made in previous molecular dynamics studies involving NFV-protease complexes (27
). Thus, NFV resistance is likely caused in large part due to the loss of this interaction in the NFV-resistant variants.
FIG. 4. Hydrogen bond network involving residue 88. (A) Asn88 bridges the terminal helix with Asp30 from the active site and Thr74 from one of the outer beta strands. The red box indicates the region of the protease molecule highlighted in panels B to D. (B) (more ...)
Overall, mutations that emerge in response to inhibitor therapy need to have a minimal impact on protease structure and activity to maintain the enzyme's function. The D30N substitution, which is associated with NFV resistance, is one of the few drug-resistant mutations that involve a change in charge. The additional substitution of N88D likely helps preserve the net charge on the protein. In AE, resistance to NFV occurs indirectly with the N88S mutation. Likewise, the sole NFV-resistant alteration, N88S, in the AE protease does not change the overall electrostatics. Thus, in both clade B and AE, NFV resistance is attained with no change to the net charge of the enzyme. In the wild-type variants, Asn88 is one of the few internal hydrogen bonding side chains in the core of the protease monomer. The side chain of Asn88 has a key role in the protease structure bridging the terminal helix, with residues 30 and 31 coming from the active site to the backbone of Thr74 in the center of one of the outer beta strands (Fig. ). With the substitutions of Asp in clade B and Ser in AE for Asn at position 88 in the NFV-resistant protease variants, the hydrogen bonding network is preserved through the coordination of some key water molecules in the core of the protease monomer (Fig. ). Thus, mutations confer resistance to NFV through a series of interdependent changes that preserve the structural and electrostatic properties of HIV-1 protease.
In conclusion, protease activity and the response to protease inhibitors can be affected by clade-specific sequence differences. Our findings likely extend beyond HIV-1 protease to other drug targets within HIV and underscore the need to consider clade-specific polymorphisms when developing new drugs and formulating treatment plans. Furthermore, drug resistance pathways observed in the context of clade B viruses cannot be assumed to hold true for other HIV-1 clades.