Wild-Type HIV-2
Protease (PDB: 3S45) from Human Immunodeficiency Virus 2
Created by: Olivia
Williams
Acquired immunodeficiency syndrome
(AIDS) currently affects more than 33 million people worldwide (1). The deadly
disease is caused by either type 1 or 2 human immunodeficiency virus (HIV)
infections, which have few effective treatment options due to rapid mutations
and drug resistance. This is especially true of HIV-2 infections, since these
drug-resistant mutations render existing HIV-1 treatments practically useless
(2). As a result, biochemists are especially interested in developing new drugs
that can specifically treat HIV-2 infections, particularly through examining
the binding activity of HIV-2 protease (PDB ID 3S45).
HIV-2 protease (PR-2) is member of
the aspartyl protease class of enzymes, which are responsible for proteolytic
activity via the carboxyl groups at aspartyl residues in the binding cavity (3,
4). In particular, PR-2 cleaves Gag and Gag-Pol polyproteins, which contribute
to the formation of mature virions (3). PR-2 is therefore essential for the
propagation of HIV-2 virions, which suggests that inhibiting this enzyme could
be a useful treatment method of HIV-2 infections.
PR-2 is a relatively small protein,
weighing in at 21438.8 Da with an isoelectric point of 5.29 (5). This
relatively low isoelectric point—the pH at which the protein carries no net
electrical charge—indicates that the primary structure consists of mostly acidic and neutral (hydrophobic) side chains. The overall structure consists of two identical subunits, each with 99 amino acid residues (4). Within each
subunit, the secondary structure consists of a series of nine β strands and one α helix, all connected by
random coil. The hydrophobic core of the resulting homodimer contains the
active site of the protein, which is flanked by two β-hairpins that ultimately
control access to the core of the molecule. The active site consists of three
residues: Asp-25, Thr-26, and Gly-27, which are stabilized by hydrogen bonds
within the core (4). These hydrogen bonds maintain the structural integrity of
the active site and thereby facilitate strong interactions between the protease
and the substrate. When a substrate binds to the active site, a conformational change occurs in the β-hairpin regions (residues 45-55 and 45’-55’), such that
the loops form a “gate” above the active site (3, 4). If the active site is
free, these loops adopt a more open confirmation and allow substrates to
approach and bind to the active site. Upon binding, the loops “close,” such
that the β-hairpins ultimately block access to the active site until the
enzymatic activity is complete (4).
The mechanism of the actual
proteolysis involves the action of the aspartyl residues in both subunits
(Asp-25 and Asp-25’). The cleavage of the Gag and Gag-Pol polyproteins proceeds
by a typical amide bond hydrolysis. Initially, one of the aspartyl residues
activates a water molecule by deprotonation, at which point the hydroxide anion
attacks the electrophilic carbonyl carbon; the result is a tetrahedral
intermediate and a negatively charged oxygen at the scissile bond. The next
step involves reforming the carbonyl group and then protonating the amide
nitrogen by deprotonating the aspartyl residue that initially activated the
water. The result is cleavage of the amide bond (6), as shown in Figure 1.
Since the efficacy of PR-2 depends
on the ability of these aspartyl residues to hydrolyze large proteins, the
enzymatic activity can be substantially reduced through introducing an
effective inhibitor. By binding to the active site of PR-2, inhibitors prevent
the enzyme from cleaving the Gag and Gag-Pol polyproteins. The formation of
mature viruses depends on this cleavage, so binding an inhibitor to the active
site prevents the overall propagation of the virus. Amprenavir (Figure 2) is a
particular inhibitor that has shown decreased inhibition in PR-2 as compared to
HIV-1 protease (PR-1). In comparing how amprenavir binds to PR-1 and PR-2, and
the general structural differences between the two, biochemists can understand
the specific features of PR-2 that contribute to its resistance to antiviral
drugs.
The structure of PR-1 complexed with
darunavir (PDB ID 3GGU), another antiviral drug that works as a protease
inhibitor, can provide additional information about the binding pockets of HIV
proteases. Darunavir (Figure 3) and amprenavir share many structural features,
like hydrophobic phenyl rings and nitrogenous groups that are capable of
hydrogen bonding. The primary intermolecular interactions at the active site of
PR-2 is hydrogen bonding and hydrophobic interactions with Asp-25, Thr-26, and
Gly-27, such that the inhibitors are able to interact favorably with these key
residues (7). PR-1 and PR-2 are also very similar, sharing upwards of 48% of
their primary structures according to queries conducted by the PSI-BLAST
program, which assigns an E-score based on similarities in the protein sequence.
PSI-BLAST increases the E score when there are “gaps” in the sequence of the
subject protein as compared to the queried protein’s sequence. As a result, an
E score of 0.0 indicates a 100% match; the E score for PR-1 as compared to PR-2
is 8e-51, indicating a very close match in amino acid sequence (8). The secondary structures of these two proteases are also comparable according to the Dali
server, which assigns a Z score based on resemblances in the secondary
structure of a subject protein as compared to the queried protein. When using the Dali server, a higher Z score corresponds to a closer structural match;
PR-1 scores a 19.5 out of a possible 22.3, indicating a fairly analogous secondary structure (9). Overall, PR-1
and PR-2 are similar enough to be considered homologous proteins, indicating
functional semblances as well as structural parity.
PR-1 and PR-2 both cleave
polyproteins to form mature viruses, so it is not surprising that their
structures, both primary and secondary, are so similar. The active site of each
protease is flanked by the β-hairpin “flaps,” which change confirmation when a
substrate binds to the enzyme. In addition, both proteases contain a
hydrophobic core that serves as the binding pocket—these hydrophobic residues
create favorable hydrophobic interactions when in the proximity of the Gag and
Gag-Pol polyproteins. These favorable interactions lead to strong binding
between the protease and polyprotein substrate, thereby ensuring effective
catalysis in the proteolytic cycle. Although there are three substitutions
within the active site (Val-32, 47, and 82 in PR-1, Ile-32, 82 in PR-2), the
binding pocket remains distinctly hydrophobic overall (10, 11).
Amprenavir was designed to inhibit
the function of PR-1, therefore its structure is uniquely suited to block the
active site of HIV-1 protease. Amprenavir’s backbone is a hydroxyethylamine
derivative, which mimics the transition state of HIV proteases during enzymatic
activity. This design element ensures maximum stability of the
inhibitor-protease complex, such that there is preferential binding to the
inhibitor rather than to the target substrate. In addition, amprenavir has very
limited polar, hydrophilic regions; this results in amprenavir preferentially
binding to the hydrophobic active site rather than the outer hydrophilic
regions (4). This preference defines amprenavir’s efficacy as a PR-1 inhibitor—the
strong hydrophobic interactions between the drug and the active site of the
protease ultimately prevents catalytic proteolysis of the target substrate and
the propagation of HIV-1 viral units. While amprenavir has proven useful in
treatment of HIV-1 infections, it is overall less effective in the treatment of
HIV-2 due to slight conformational changes (2).
Amprenavir blocks the active site of PR-2 through a combination of hydrophobic interactions and hydrogen bonds. Hydrophobic
interactions occur at three main positions: Ile-32, Ile-47, and Ile-82. The
aliphatic side chain in Ile-32 interacts favorably with the π electrons of the
aniline ring in amprenavir, while the side chains in Ile-47 and Ile-82 display
more general hydrophobic interactions with the aliphatic portions of the
amprenavir inhibitor. The difference in amprenavir’s binding efficacy to PR-1
and PR-2 lies in the hydrogen bond interactions at Asp-30. In PR-2, the
hydrogen bond between Asp-30 and the NH2 of the aniline ring is
about 0.5 Å longer than what is typically observed for a strong hydrogen
bond—like the bond length observed when amprenavir binds to the same position
in PR-1. This elongated hydrogen bond is likely what contributes to the 19-fold
increase in amprenavir’s ability to inhibit PR-1 versus PR-2; since an
increased distance effectively decreases the attractive, stabilizing force of
the hydrogen bond, there is an overall weaker amprenavir-protease interaction (2).
Biochemists can thus use this knowledge to construct more effective inhibitors
for treatment of HIV-2 infections.