Proc. Natl. Acad. Sci. USA Vol. 96, pp. 10968–10975, September 1999 Colloquium Paper This paper was presented at the National Academy of Sciences colloquium ‘‘Proteolytic Processing and Physiological Regulation,’’ held February 20–21, 1999, at the Arnold and Mabel Beckman Center in Irvine, CA. Structural aspects of activation pathways of aspartic protease zymogens and viral 3C protease precursors AMIR R. KHAN*, NINA KHAZANOVICH-BERNSTEIN,ERNST M. BERGMANN, AND MICHAEL N. G. JAMES† Medical Research Council Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada ABSTRACT The three-dimensional structures of the in- about the conversion (5). In a similar fashion, the zymogens of active protein precursors (zymogens) of the serine, cysteine, the papain-like cysteine proteases are converted to the active aspartic, and metalloprotease classes of proteolytic enzymes enzymes in a pH-regulated fashion. The in vitro activation of are known. Comparisons of these structures with those of the propapain is consistent with an initial intramolecular cleavage mature, active proteases reveal that, in general, the pre- event (6). The conversion of procarboxypeptidase is initiated formed, active conformations of the residues involved in by trypsin cleavage of the Arg-99p–Ala-1 bond at the proseg- catalysis are rendered sterically inaccessible to substrates by ment to mature enzyme junction (7). Prostromelysin-1 can be the residues of the zymogens’ N-terminal extensions or pro- converted to the active form by other proteolytic enzymes, segments. The prosegments interact in nonsubstrate-like heat, or the presence of organomercurial agents (8). There are some generalities regarding zymogen conversion fashions with the residues of the active sites in most of the that one can make in light of the three-dimensional structures cases. The gastric aspartic proteases have a well-character- of both the zymogens and the respective active enzymes (3). ized zymogen conversion pathway. Structures of human pro- First, the residues that constitute the active sites of the gastricsin, the inactive intermediate 2, and active human protease portions of the zymogens have virtually identical pepsin are known and have been used to define the conversion conformations to those of the mature, active proteases. The pathway. The structure of the zymogen precursor of plasmep- major exceptions are the serine proteases of the chymotrypsin sin II, the malarial aspartic protease, shows a new twist on the family. The activation process involves the formation of an ion mode of inactivation used by the gastric zymogens. The pair between the newly formed N-terminal residue Ile-16 ϩ ␤ prosegment of proplasmepsin disrupts the active conforma- NH3 and the -carboxylate of Asp-194 (9), which triggers the tion of the two catalytic aspartic acid residues by inducing a conformational changes that form the oxyanion binding major reorientation of the two domains of the mature pro- pocket and the active conformation of the S1 specificity pocket tease. The picornaviral 2A and 3C proteases have a chymo- [the nomenclature of Schechter and Berger (10) is used trypsin-like tertiary structure but with a cysteine nucleophile. throughout this manuscript]. These enzymes cleave themselves from the viral polyprotein in Second, the preformed active sites of the protease portions cis (intramolecular cleavage) and carry out trans cleavages of of zymogens are generally not accessible to substrates because other scissile peptides important for the virus life cycle. residues of the prosegments sterically block the approach to the active sites. This statement does not hold for the chymo- Although the structure of the precursor viral polyprotein is trypsin-like serine proteases as the active sites of these zymo- unknown, it probably resembles the organization of the proen- ␣ gens are able to bind protein inhibitors that induce confor- zymes of the bacterial serine proteases, subtilisin, and -lytic mational changes that form the oxyanion hole in spite of the protease. Cleavage of the prosegment is known to occur in cis ion pair involving Asp-194 and Ile-16 being absent (11). for these precursor molecules. Proteolysis of the portion of the prosegments that interact with the active site residues is prevented in several different Zymogens of proteolytic enzymes consist of the intact protease ways. In prostromelysin the prosegment passes through the with an N-terminal extension. Conversion of the inactive active site in the reverse polypeptide direction (N3C) relative zymogen to the mature, active protease requires limited pro- to substrates or transition state mimics (12). A reverse orien- teolysis usually of a single peptide bond (1). Molecular rear- tation of the prosegment blocking the active site in the cysteine rangements accompany the proteolytic removal of the proseg- protease zymogens also has been observed in the structures of ment of the zymogen, eventually leading to the mature pro- rat procathepsin B (13) and human procathepsin L (14). The region of the prosegment of the gastric aspartic proteases tease. The prosegments of the zymogens range in size from two interacting with the catalytic residues Asp-32 and Asp-215 residues for some of the granzymes to more than 150 residues ␣ (porcine pepsin numbering) most intimately, includes a highly for -lytic protease, a bacterial serine protease (2). conserved lysine at position 36p (the residue numbers of the The conversion of zymogens to the respective active en- ␧ ϩ prosegment are followed by p). The NH3 group of Lys-36p zymes is achieved by several different mechanisms (3). The forms an ion pair with each of the two active site carboxylate active serine proteases of the chymotrypsin family result from groups (15). limited proteolysis of the zymogens by convertases. For ex- ample, the cascade of the blood-clotting enzymes (4) involves Conversion of Gastric Aspartic Protease Zymogens the conversion of inactive forms (e.g., prothrombin) to active forms of the enzyme (thrombin) by a highly specific catalytic The molecular structures of human progastricsin (16), activa- cleavage by another of the clotting enzymes (factor Xa). On the tion intermediate 2 of human gastricsin (17), and a structural other hand, simply changing the pH of the solution in which the Ϸ gastric aspartic protease zymogens are dissolved from 6.5 to Abbreviation: HPV, human polio virus. ϩ 3.0 (an increase in [H ]ofϷ3,100-fold) is sufficient to bring *Present address: Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138. †To whom reprint requests should be addressed. E-mail: michael. PNAS is available online at www.pnas.org. [email protected]. 10968 Downloaded by guest on September 26, 2021 Colloquium Paper: Khan et al. Proc. Natl. Acad. Sci. USA 96 (1999) 10969 homolog to mature, human gastricsin, human pepsin 3 (18) each of these three molecular structures. Fig. 2 shows a allow one to construct a reasonably detailed view of the diagrammatic view of the conversion pathway. This pathway is pathway followed in the conversion of the inactive zymogen to a general pathway for the gastric aspartic proteases but the the active protease. Fig. 1 shows stereo ribbon diagrams of individual enzymes differ in detail. A FIG. 1. Structures on the conver- sion pathway of the aspartic protease B zymogen progastricsin. The structure of human gastricsin is not known; the human pepsin structure therefore has been used as a model for gastricsin. This figure, as well as Figs. 3–6 have been prepared with BOBSCRIPT (19) and RASTER 3D (20). (A) The structure of human progastricsin (16) repre- sented in stereo. The residues of the prosegment (Ala-1p to Leu-43p) are in green, those of the gastricsin portion of the zymogen are in blue except for those regions that undergo large con- formational changes, Ser-1 to Ala-13, Phe-71 to Thr-81 and Tyr-125 to Ala- 136, which are represented in mauve. The promature junction is Leu-43p– Ser-1, the peptide bond cleaved in- tramolecularly is Phe-26p to Leu-27p. The side chains of Asp-32 and Asp-217 are represented in red. (B) Stereo view of the molecular structure of interme- diate 2 on the activation pathway of human gastricsin (17). The color C scheme used is the same as in A. The residues missing on this figure, Leu- 22p to Phe-26p and Ser-1, are disor- dered in the structure, and there is no interpretable electron density for them on the maps. The water molecule bound between the two carboxyl groups of Asp-32 and Asp-217 is shown as a red sphere. The final step in the conversion involves the dissocia- tion of the peptide Ala-1p to Phe-26p from gastricsin with the N-terminal residues of gastricsin, Ser-1 (N-ter) to Ala-13, replacing the N-terminal ␤-strand of the prosegment. (C) The structure of human pepsin (18) shown as a model of human gastricsin. The regions of gastricsin that undergo large conformational changes from their po- sitions in progastricsin are shown in pink, and the active site aspartates with the bound catalytic H2O molecule are colored red. Reproduced with permis- sion from ref. 3. Downloaded by guest on September 26, 2021 10970 Colloquium Paper: Khan et al. Proc. Natl. Acad. Sci. USA 96 (1999) FIG. 2. A diagrammatic representation of the conversion pathway of progastricsin to gastricsin. The prosegment of progastricsin (I), A1p to L43p, has three helical segments and a net positive charge forming several ion pair interactions and electrostatic stabilization with the mature portion of the zymogen. The highly conserved K37p interacts directly with the catalytic aspartates Asp-32 and Asp-217. Lowering the pH of the solution below 4.0 converts progastricsin into intermediate 1 (26) represented by II. Refolding of the prosegment in the vicinity of the active gastricsin brings the first scissile bond Phe-26p–Leu-27p to the exposed active site aspartates (III).