Crystal Structure of an Engineered Subtilisin Inhibitor Complexed With
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Proc. Nati. Acad. Sci. USA Vol. 89, pp. 4407-4411, May 1992 Biochemistry Crystal structure of an engineered subtilisin inhibitor complexed with bovine trypsin (protein engneering/protein-protein interaction) YASUO TAKEUCHI*, TAKAMASA NONAKAt, KAzuo T. NAKAMURAt, SHUICHI KOJIMAU§, KIN-ICHIRO MIURAt§, AND YUKIO MITSUIt¶ *Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., Morooka, Kohoku, Yokohama 222, Japan; tFaculty of Engineering, Nagaoka University of Technology, Nagaoka, Niigata 940-21, Japan; and tFaculty of Engineering, University of Tokyo, Hongo, Tokyo 113, Japan Communicated by Frederic M. Richards, January 10, 1992 (received for review June 18, 1991) ABSTRACT Proteinase specificity of a proteinaceous in- inhibitor and proteinase surfaces is almost always found in hibitor of subtilisin (SSI; Streptomyces subtilisin inhibitor) can the crystallographically established structures of the inhibi- be altered so as to strongly inhibit trypsin simply by replacing tor-proteinase complexes (8-14) and thus such good com- P1 methionine with lysine (with or without concomitant change plementarity seems essential for the tight binding (15), and of the P4 residue) through site-directed nutagenesis. Now the yet (ii) the surface geometry of the active sites ofproteinases crystal structure of one such engineered SSI (P1 methionine is considerably varied even among enzymes of the trypsin converted to lysine and P4 methionine converted to glycine) family, much less between enzymes of the trypsin family and complexed with bovine trypsin has been solved at 2.6 A those of the subtilisin family having a completely different resolution and refined to a crystallographic R factor of 0.173. polypeptide chain folding. In this respect, the chance to Comparing this structure with the previously established struc- compare the three-dimensional structure of a proteinaceous ture of the native SSI complexed with subtilisin BPN', it was inhibitor complexed with its natural target proteinase and that found that (i) P1 lysine of the mutant SSI is accommodated in of a variant of the inhibitor complexed with a distinct target the Si pocket of trypsin as usual, and (ii) upon complex enzyme has long been awaited. Here we report the crystal formation, considerable conformation change occurs to the structure of an engineered SSI, in which P1 methionine and reactive site loop of the mutant SSI. Thus, in this case, P4 methionine were genetically converted to lysine and flexibility of the reactive site loop seems important for success- glycine, respectively, complexed with bovine trypsin. Com- fully changing the proteinase specificity through mere replace- paring this structure with the previously established structure ment of the P1 residue. of the native SSI complexed with subtilisin BPN' (16, 17), many intriguing points as to the structural mechanisms ofthe Hypervariability of reactive site amino acid residues during change in proteinase specificity have emerged. evolution of proteinaceous inhibitors of serine proteinases is SSI is one of the few well-characterized (for a detailed one of the major mysteries of protein evolution (1). In review, see ref. 6) microbial protein proteinase inhibitors and contrast, in most other biologically active proteins, the is a stable dimer (I2) composed oftwo identical subunits, each replacement of active site residues, even by closely related of Mr 11,500 (18). It strongly inhibits a microbial serine ones, leads to a complete loss of activity or a dramatic proteinase subtilisin BPN' (E), (K; 10-11 M) forming an decrease in activity. Among the reactive site residues of E2-2 complex. SSI virtually does not inhibit trypsin (Ki serine proteinase inhibitors, the P1 residue [after the 10-4 M; see ref. 19 for a review). The crystal structure offree Schechter and Berger notation (2)] most significantly affects SSI was solved (20) and refined at 2.05 A resolution (Y.T. and proteinase specificity. For example, strong trypsin inhibitors Y.M., unpublished data). The crystal structure of the SSI- most frequently have lysine or arginine at P1, while strong subtilisin complex was also solved (16, 20) and refined at 1.8 chymotrypsin inhibitors have tyrosine, phenylalanine, leu- A resolution (17). Compared with other similar complexes cine, or methionine at P1 (1). Thus, the hypervariability ofP1 containing serine proteinases ofthe trypsin family (8-14, 21), residues inevitably causes a frequent change in proteinase the SSI-subtilisin complex is unique in several respects (16): specificity during gradual evolution of the same series of (i) In addition to the usual intermolecular antiparallel P-sheet has interaction involving P1-P3 residues of the inhibitor, there is inhibitor proteins. It been known that one can also invoke another antiparallel p-sheet formed between P4-P6 residues such a dramatic change in specificity by semisynthetic re- and a previously unnoticed chain segment [the "S4-6 site" placement of the P1 residue (3, 4) or by recombinant DNA (16)] of subtilisin. (ii) Unlike various trypsin inhibitors, the techniques. Thus, the replacement of P1 methionine in a,- P4-P9 residues of SSI are very flexible in the free state (22) antitrypsin (a potent inhibitor of neutrophil elastase) with and they undergo extensive rigidification and transconfor- arginine converted the inhibitor into an efficient thrombin mation upon complex formation. The SSI gene has been inhibitor (5). The replacement of P1 methionine in a protein- cloned (23) and the expression system has been established aceous inhibitor [SSI; Streptomyces subtilisin inhibitor (6), (24). Genetic conversion of P1 Met-73 to lysine (or arginine an almost exclusive subtilisin inhibitor] with lysine or argi- but no other amino acid residues) proved to convert SSI into nine converted it to a potent inhibitor of both trypsin and a potent inhibitor of both trypsin (Ki = 4.4 x 10-9 M) and subtilisin BPN' (7). subtilisin BPN' (K; = 6 x 10-11 M) (25). Later, additional Despite rather abundant observations of the dramatic conversion of P4 Met-70 to glycine was found to enhance the change in proteinase specificity, however, the structural = 2.1 x 10-9 mechanisms of these phenomena are by no means obvious inhibitory effect against trypsin (Ki M; against considering that (i) very good complementarity between the Abbreviations: BPTI, bovine pancreatic trypsin inhibitor; SSI, Streptomyces subtilisin inhibitor. The publication costs of this article were defrayed in part by page charge §Present address: Institute for Biomolecular Science, Gakushuin payment. This article must therefore be hereby marked "advertisement" University, Mejiro, Tokyo, Japan. in accordance with 18 U.S.C. §1734 solely to indicate this fact. ITo whom reprint requests should be addressed. 4407. Downloaded by guest on September 26, 2021 4408 Biochemistry: Takeuchi et al. Proc. Natl. Acad. Sci. USA 89 (1992) subtilisin BPN', K- 10-11 M) (26), providing the best candidate for isolation of the complex with trypsin. Hereafter the (Met-73 to lysine, Met-70 to glycine) double mutant of SSI is referred to simply as "mutant SSI." MATERIALS AND METHODS Crystallization and Data Colection. Site-directed mutagen- esis, DNA construction, and purification of the mutant SSI were performed as described (25). Bovine trypsin was pur- chased from Worthington and was used without further puri- fication. The method of complex formation is similar to that described (27). Bovine trypsin and mutant SSI were dissolved separately in 0.05 M phosphate buffer (pH 7.2). The enzyme solution was added to the inhibitor solution in a 2:1.2 ratio (mol/mol) and the mixture was stirred gently at 370C for 30 min. For isolation of the complex, the reaction mixture was applied to a Biogel P-200 column previously equilibrated with 0.05 M phosphate buffer (pH 7.2) and eluted with the same buffer. The fractions containing the complex were concen- trated by ultrafiltration on a Millipore C3-TK. Hanging drop- lets of this complex solution (3 Al) in 0.05 M Tris HCl buffer (pH 7.0) containing 40%o saturated MgSO4 were equilibrated with the reservoir solutions of0.05 M Tris HCl buffer (pH 7.0) containing 80%o saturated MgSO4. Crystals, including a single exceptionally large one (0.2 x 0.2 x 0.6 mm), were grown for afew months. The crystals belong to the space group I222 with the unit cell dimensions a = 110.9 A, b = 116.8 A, c = 64.3 A. One asymmetric unit contains half the complex molecule (E-I). Hereafter, the mutant SSI-bovine trypsin complex is N terminus For data referred to simply as "SSI-trypsin complex." x-ray FIG. 1. Schematic drawing of one SSI subunit showing the collection at =15'C, a macromolecule-oriented Weissenberg P-strands (arrow plates), a-helices (solid bonds), the flexible loop camera (28) installed at the synchrotron radiation source at the (shaded bonds), S-S bridges (zigzag line), scissible peptide bond (tri- National Laboratory for High Energy Physics (Tsukuba) was angle), N andC termini. Notations (P1, Pi, etc.)ofSchechterand Berger used. The wavelength was set to 1.04 A. The single excep- (2) are indicated for some residues along the reactive site loop [or the tionally large crystal mentioned above was used with the c axis primary contact region (20)]. Similar notations (Q1, Qi, etc.) of Hirono as the rotation axis. The data sets recorded on a pack of Fuji et al. (16) are also indicated for some residues along the secondary imaging plate were processed as described (17). The Rsym contact region (20) having some direct contacts with the surface of subtilisin BPN'. For of solid see 4. (l hu7,jl) - Ijj/11h(I)) was 6.3% for 23,106 observations up to explanation arrows, Fig. > were reduced to 7579 2.6 A resolution, I 2a (I), which complex (Fig. 2). The corresponding whole views of the two independent reflections. The intermolecular Structure Determination and Refinement. The structure complex molecules are shown in Fig. 3. contacts found in the SSI-trypsin complex are summarized in was determined by the molecular replacement method (29) between making use of(i) the known structure ofbovine trypsin in the Table 1.