Memapsin 2 (beta-secretase) is a membrane-associated aspartic protease involved in the production of beta-amyloid peptide in Alzheimer's disease and is a major target for drug design. We determined the crystal structure of the protease domain of human memapsin 2 complexed to an eight-residue inhibitor at 1.9 angstrom resolution. The active site of memapsin 2 is more open and less hydrophobic than that of other human aspartic proteases. The subsite locations from S4 to S2' are well defined. A kink of the inhibitor chain at P2' and the change of chain direction of P3' and P4' may be mimicked to provide inhibitor selectivity.

Saquinavir is a widely used HIV-1 protease inhibitor drug for AIDS therapy. Its effectiveness, however, has been hindered by the emergence of resistant mutations, a common problem for inhibitor drugs that target HIV-1 viral enzymes. Three HIV-1 protease mutant species, G48V, L90M, and G48V0L90M double mutant, are associated in vivo with saquinavir resistance by the enzyme~Jacobsen et al., 1996!. Kinetic studies on these mutants demonstrate a 13.5-, 3-, and 419-fold increase in K i values, respectively, compared to the wild-type enzyme~Ermolieff J, Lin X, Tang J, 1997, Biochemistry 36:12364-12370!. To gain an understanding of how these mutations modulate inhibitor binding, we have solved the HIV-1 protease crystal structure of the G48V0L90M double mutant in complex with saquinavir at 2.6 Å resolution. This mutant complex is compared with that of the wild-type enzyme bound to the same inhibitor~Krohn A, Redshaw S, Richie JC, Graves BJ, Hatada MH, 1991, J Med Chem 34:3340-3342!. Our analysis shows that to accommodate a valine side chain at position 48, the inhibitor moves away from the protease, resulting in the formation of larger gaps between the inhibitor P3 subsite and the flap region of the enzyme. Other subsites also demonstrate reduced inhibitor interaction due to an overall change of inhibitor conformation. The new methionine side chain at position 90 has van der Waals interactions with main-chain atoms of the active site residues resulting in a decrease in the volume and the structural flexibility of S10S19 substrate binding pockets. Indirect interactions between the mutant methionine side chain and the substrate scissile bond or the isostere part of the inhibitor may differ from those of the wild-type enzyme and therefore may facilitate catalysis by the resistant mutant.