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.

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The structure of porcine pepsinogen at pH 6.1 has been refined to an R-factor of 0.173 for data extending to 1.65 A. The final model contains 180 solvent molecules and lacks density for residues 157-161. The structure of this aspartic proteinase zymogen possesses many of the characteristics of pepsin, the mature enzyme. The secondary structure of the zymogen consists predominantly of beta-sheet, with an approximate 2-fold axis of symmetry. The activation peptide packs into the active site cleft, and the N-terminus (1P-9P) occupies the position of the mature N-terminus (1-9). Thus changes upon activation include excision of the activation peptide and proper relocation of the mature N-terminus. The activation peptide or residues of the displaced mature N-terminus make specific interactions with the substrate binding subsites. The active site of pepsinogen is intact; thus the lack of activity of pepsinogen is not due to a deformation of the active site. Nine ion pairs in pepsinogen may be important in the advent of activation and involve the activation peptide or regions of the mature N-terminus which are relocated in the mature enzyme. The activation peptide-pepsin junction, 44P-1, is characterized by high thermal parameters and weak density, indicating a flexible structure which would be accessible to cleavage. Pepsinogen is an appropriate model for the structures of other zymogens in the aspartic proteinase family.