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The conserved active-site residues (Asp25, Thr26, and Gly27 from both monomers) form a symmetrical
and highly hydrogen-bonded arrangement virtually identical to that described for pepsin [17]. The two
aspartates are nearly coplanar with the “inner” carboxylate oxygens hydrogen bonded to the amide
hydrogens of Gly27/27'. This designation (e.g. Gly 27/27') will be used throughout this text to indicate
equivalent residues of the dimer. The two threonines are inaccessible to solvent and are hydrogenbonded
to the main-chain amide groups of the other monomer, forming a rigid network called a
“fireman's grip” [17]. As in the case of the structures of eukaryotic pepsins, there is electron density for
a water molecule bound between the two carboxylates of the active-site aspartates.
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In the structure of the apo-form of HIV PR, the flaps from both monomers are related by
crystallographic two-fold symmetry and can be considered as being in an open conformation. In the
structures of related proteases from Rous Sarcoma Virus and HIV-2, the flaps are either
crystallographically disordered or in a partly closed conformation [18]. This suggests that, in solution, in
the absence of ligands, the flaps are rather flexible and that the stable conformation of the flaps observed
in the crystal structure of the apo-enzyme of HIV PR could be considered to result from kinetic trapping
during the crystallization process.
In the apo-form of HIV PR, the active site residues are located at the bottom of a rather shallow groove.
Upon binding an inhibitor, the protease undergoes significant structural changes, particularly apparent in
the flap region. As a result, a tunnel-like site is formed, which runs diagonally across the dimer
interface. The tunnel has a volume of approximately 1140 Å 3 and is 23 Å long. Because of the dimeric
nature of HIV PR, the active site has approximate two-fold symmetry with the dyad axis intersecting the
plane of the catalytic aspartates. Along the active site tunnel, starting from the central aspartates, there
are distinct subsites S1, S2, S3, and S4, and corresponding symmetry related subsites S1', S2' S3', and
S4' (Figure 2). It should be noted that in this chapter, the convention of Schechter and Burger [19] will
be used to describe enzyme specificity subsites (S1, S1', etc.) and the corresponding side chains of
inhibitors (P1, P1', etc.). The boundaries of the subsites are formed by residues from both monomers of
HIV PR. All subsites, with the exception of S4/S4', which are exposed to solvent, are bounded by mostly
aliphatic side chains and have hydrophobic character. The borders of the S1/S1' subsites are formed by
the side chains of Ile23/23', Ile50/50', Ile84/84', Pro81/81', the γ carbon of Thr80/80', carboxylates of the
active site Asp25/25', and the carbonyl oxygens of Gly27/27'. The S2/S2' subsites are bounded by
Val32/32', Ile50/50', Ile47/47', Leu76/76', Ala28/28', and the carboxylates of Asp30/30'. The S3/S3',
subsites are partly exposed to solvent and are bordered by the side chains of Leu23/23', Val82/82',
Pro81/81', and the guanidinium groups of Arg8/8', which form a salt bridge with the carboxylates of
Asp29/29'. Most of
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