Structure of a Serine Protease Poised to Resynthesize a Peptide Bond
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Structure of a serine protease poised to resynthesize a peptide bond Elena Zakharova, Martin P. Horvath, and David P. Goldenberg1 Department of Biology, University of Utah, Salt Lake City, UT 84112-0840 Edited by Brian W. Matthews, University of Oregon, Eugene, OR, and approved May 11, 2009 (received for review March 6, 2009) P1' O The serine proteases are among the most thoroughly studied en- O O OH2 P1' P1' zymes, and numerous crystal structures representing the enzyme– ++ N OH H N substrate complex and intermediates in the hydrolysis reactions have H O H2N 2 P1 been reported. Some aspects of the catalytic mechanism remain P1 OH P1 E OH controversial, however, especially the role of conformational changes E E in the reaction. We describe here a high-resolution (1.46 Å) crystal structure of a complex formed between a cleaved form of bovine Scheme 1. Two-step mechanism of serine protease action. pancreatic trypsin inhibitor (BPTI) and a catalytically inactive trypsin variant with the BPTI cleavage site ideally positioned in the active site we are aware, no structure of a bound P1Ј product has been for resynthesis of the peptide bond. This structure defines the posi- reported, probably because this product usually diffuses away tions of the newly generated amino and carboxyl groups following rapidly. Precisely locating the newly generated termini in an the 2 steps in the hydrolytic reaction. Comparison of this structure appropriate complex would provide insights into both steps with those representing other intermediates in the reaction demon- of the mechanism, because the P1Ј amino nitrogen atom is strates that the residues of the catalytic triad are positioned to the leaving group for the first nucleophilic substitution, and the promote each step of both the forward and reverse reaction with position of the P1 carboxyl group defines the endpoint in the remarkably little motion and with conservation of hydrogen-bonding trajectory of the deacylation step. interactions. The results also provide insights into the mechanism by Although rebinding of the hydrolysis products is disfavored for BIOCHEMISTRY which inhibitors like BPTI normally resist hydrolysis when bound to most substrates, an important exception was discovered by their target proteases. Finkenstadt and Laskowski (21), who showed that a natural ͉ ͉ serine protease inhibitor, soybean trypsin inhibitor (SBTI), was trypsin bovine pancreatic trypsin inhibitor enzyme mechanisms slowly hydrolyzed when bound to trypsin and that the peptide bond could be resynthesized. Since then, it has been found that erine proteases are found throughout all 3 domains of SBTI is one of a very large number of inhibitors, described as Scellular life and function in a wide range of physiological ‘‘standard mechanism’’ or ‘‘Laskowski mechanism’’ inhibitors, processes, including digestion, protein maturation and turnover, that act by binding tightly to the active sites of their targets as a hemostasis, and immune responses (1). Approximately 0.6% of substrate would, but resist hydrolysis for many hours or longer human protein-encoding genes are predicted to specify serine (22–25). Because the products of hydrolysis remain physically proteases, and this family is even more prevalent in other associated, the resynthesis of these inhibitors is an intramolec- organisms, notably the arthropods (2, 3). A large body of ular reaction and is, therefore, much more favorable than the biochemical and structural data have established a 2-step mech- equivalent intermolecular reaction at modest reactant concen- anism for hydrolysis of peptide bonds by this class of proteases trations. Despite the prevalence of Laskowski inhibitors (25), the (4), as shown in Scheme 1. mechanisms by which they resist proteolysis remain poorly The first step of the reaction is a nucleophilic attack by the understood (26–29). catalytic serine residue (Ser-195 in trypsin and other members of To gain structural insights into the mechanisms of both serine the chymotrypsin, or S1, family) on the carbonyl carbon atom of proteases and their inhibitors, we have determined the crystal the residue labeled P1, generating a covalent acyl-enzyme structure of the complex formed between the cleaved form of a Ј intermediate and a new peptide amino terminus, on the P1 Laskowski-mechanism inhibitor, bovine pancreatic trypsin in- residue. A second nucleophilic attack, by a water molecule, leads hibitor (BPTI), and a trypsin variant with the catalytic Ser to hydrolysis of the acyl-enzyme, releasing the new carboxyl residue (Ser-195) replaced with Ala, rat anionic trypsin S195A. group and restoring the catalytic Ser residue to its initial state. BPTI is one of the most extensively studied protease inhibitors In addition to the Ser residue, the mechanism of most, but not and also one of the most effective: The dissociation constant for all, serine proteases depends on the side chains of a His and an the complex formed with bovine trypsin is Ϸ10Ϫ13 M, and the Asp residue (His-57 and Asp-102 in the chymotrypsin family) half-time for hydrolysis has been estimated to be several years (5). The His side chain serves as a base for activating the (30). A ribbon representation of the structure of intact BPTI nucleophilic species and as an acid that transfers a proton to the bound to rat anionic trypsin is shown in Fig. 1A. As described leaving group in each step of the reaction. The Asp side chain is below, the structure of the complex with the cleaved inhibitor generally believed to stabilize the charge on the protonated His residue, but the details of the interaction between these 2 residues are not fully agreed on (6, 7). Author contributions: E.Z. and D.P.G. designed research; E.Z. and M.P.H. performed re- Crystallographic studies of serine proteases with bound sub- search; E.Z., M.P.H., and D.P.G. analyzed data; and E.Z., M.P.H., and D.P.G. wrote the paper. strates and inhibitors have provided detailed structural infor- The authors declare no conflict of interest. mation about the enzyme–substrate complex (8, 9), the acyl- This article is a PNAS Direct Submission. enzyme (10–13), and the high-energy tetrahedral intermediates Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, that separate these species (14–17). Crystal structures with www.pdb.org (PDB ID codes 3FP6 and 3FP7). bound P1 products have also been reported (18–20), but at 1To whom correspondence should be addressed. E-mail: [email protected]. relatively low resolution or in forms that appear to be mixtures This article contains supporting information online at www.pnas.org/cgi/content/full/ of the carboxyl, tetrahedral, and acyl-enzyme species. As far as 0902463106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902463106 PNAS Early Edition ͉ 1of6 Downloaded by guest on October 2, 2021 determined by the method of molecular replacement, using the A structure of the complex of rat trypsin and intact BPTI deter- mined at 1.8-Å resolution (35). Refinement yielded R values of C38 18.7% and 17.5% for complexes of trypsin with BPTI and C14 D102 BPTI*, respectively. Refinement and data collection statistics H57 are provided in SI Table 1. The structure of the active-site regions of the enzyme and S195 inhibitor are shown in Fig. 2, for both the intact inhibitor bound to wild-type trypsin (A) and the cleaved inhibitor bound to S195A trypsin (B). As observed for other enzyme-bound Las- kowski inhibitors (29), the scissile amide group of the intact inhibitor displayed planar geometry, and the carbonyl carbon was ideally positioned for attack by the O␥ atom of Ser-195 (shown in red on the surface representation of the enzyme in Fig. 2). The electron density map calculated for the complex con- B taining cleaved BPTI showed the newly generated amino and carboxyl groups in well-defined positions in the enzyme active site. Hydrolysis of the peptide bond was accommodated by a small change in the conformation of Lys-15 and a displacement BPTI BPTI* iv. of Ϸ1 Å in the position of the nitrogen atom of Ala-16. The electron density maps in the region of Cys-38 of the iii. cleaved inhibitor indicated the presence of 2 side-chain confor- ii. mations, one nearly identical to that seen in the intact inhibitor, and the other differing by rotation of the 1 dihedral angle by i. Ϫ100°, thereby changing the chirality of the Cys-14–Cys-38 disulfide bond. The occupancy of the altered conformation was 45 55 65 75 estimated to be Ϸ20%. This disulfide isomerization has been HPLC Elution time (min) detected at a very low level (Ϸ5%) by NMR spectroscopy in free intact BPTI (36, 37) and in a crystal structure of a BPTI mutant Fig. 1. Structure of the BPTI–trypsin complex and resynthesis of the scissile with 3 amino acid replacements in the trypsin-binding loop (38, bond of cleaved BPTI by rat trypsin. (A) Ribbon diagram representation of the 39). The alternate isomer is accommodated within the con- complex formed between rat trypsin and the intact form of BPTI, drawn from straints of the complex with essentially no perturbation of Cys-14 the atomic coordinates determined in this study and deposited as ID code 3FP6 in the PDB. The backbone structures of BPTI and trypsin are shown as blue and or the backbone of either Cys residue. Beyond the active-site green ribbons, respectively. The side chains of the 3 disulfides of BPTI are region, the structures of the enzyme and inhibitor were essen- shown as balls and sticks, and the catalytic triad of trypsin is shown as sticks. tially identical in the 2 complexes. The carbonyl carbon of the BPTI scissile bond is shown as a black sphere.